Immobilization of enzymes onto nanoporous materials and application as immobilized biocatalyst

IMMOBILIZATION OF ENZYMES ONTO NANOPOROUS MATERIALS AND APPLICATION AS IMMOBILIZED BIOCATALYST MALIK JAMAL J (B.Tech., University of Madras) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements At the outset, I thank almighty for providing me the strength and courage to complete this thesis successfully. I wish to record my deep sense of gratitude to my research guide, Assoc. Prof. Sibudjing Kawi and Assoc. Prof. Kus Hidajat, Department of Chemical and Biomolecular Engineering, National University of Singapore for their constant support and encouragement during the course of my research work. Their guidance and suggestion was invaluable and went a long way towards the completion of this thesis. My thanks are duly acknowledged to the department and the University for their Support in the form of Graduate Student Tutorship. A very special thanks goes to Song Shiwei for getting me familiarized with all sophisticated characterization techniques. My heartfelt thanks to my friends Yang Jun, Sun Gebiao, Yong Siek Ting, Li Peng and Dr.M.Selvaraj for their untiring and continued support during my thesis work. Their timely help and friendship shall always be remembered. My heartfelt thanks to Mdm Jamie Siew, Mdm Chow Pek, Ms Novel Chew, Ms Sylvia Wan, Ms Li Feng Mei and other staffs in the department who have gone out of their way to help me with all the lab facilities and making me feel at home during my days of M.Eng Studies. The cooperation I received from other faculty members is gratefully acknowledged. This thesis could not have been completed without the endless love and blessing from my parents, family members and friends for their constant encouragement and support to go ahead, especially during difficult times. i Table of Contents 1 Introduction……………………………………………………………………………1 1.1 Biocatalysis...................................................................................................................1 1.2 Immobilization of Enzymes…………………………………………………………..3 1.3 Research Objectives…………………………………………………………………..5 1.4 Organization of Thesis………………………………………………………………..6 2 Literature Review……………………………………………………………………...7 2.1 Introduction…………………………………………………………………………...7 2.2 Nonaqueous Enzymology…………………………………………………………….7 2.2.1 Factors Affecting the Activity of Enzymes in Organic Media…………………..8 2.2.2 Enzyme Activation in Organic Media………………………………………….12 2.2.3 Enzyme Activation by Immobilization……………………………………………15 2.2.3.1 Enzyme Immobilization in Polymers………………………………………...15 2.2.3.2 Enzyme Immobilization in Inorganic Materials……………………………...17 2.3 Enzyme Immobilization in Mesoporous Silicate for Use in Aqueous Media……….19 2.4 Conclusions………………………………………………………………………….21 3 Synthesis of Mesoporous Silicate……………………………………………………22 3.1 Introduction………………………………………………………………………….22 3.2 Experimental Methods………………………………………………………………23 3.2.1 Materials………………………………………………………………………..23 3.2.2 Synthesis of Pure SBA-15……………………………………………………...23 3.2.3 Synthesis of Thiol Functionalized SBA-15…………………………………….23 3.2.4 Synthesis of Pure SBA-15 with Rod-like Morphology………………………...24 3.2.5 Characterization of Materials…………………………………………………..24 3.3 Results and Discussion………………………………………………………………25 ii 3.3.1 Characterization of Pure and Thiol Functionalized SBA-15…………………...25 3.3.2 Characterization of SBA-15 with Rod-Like Morphology……………………...28 3.4 Conclusions………………………………………………………………………….30 4 Immobilization of α-chymotrypsin into Mesoporous Silicate and Its Activity in Aqueous or Organic Media……………………………………………….31 4.1 Introduction………………………………………………………………………….31 4.2 Materials and Methods………………………………………………………………33 4.2.1 Materials………………………………………………………………………..33 4.2.2 Model Reaction for Activity Studies in Aqueous and Organic Media…………33 4.2.3 Enzyme Immobilization for Activity Studies in Aqueous or Organic Media….35 4.2.4 Thermal Stability and Leaching Studies in Aqueous Media…………………...37 4.3 Results and Discussion………………………………………………………………38 4.3.1 Immobilization of α-chymotrypsin into Mesoporous Silicate and Commercial Silica Gel……………………………………………………………….38 4.3.2 Activity of Native and Immobilized α-chymotrypsin in Aqueous Media……...39 4.3.3 Thermal Stability of Native and Immobilized α-chymotrypsin in Aqueous Media……………………………………………………………………40 4.3.4 Leaching of α-chymotrypsin form Pure SBA-15 and Thiol Functionalized SBA-15………………………………………………………………41 4.3.5 Activity of Immobilized α-chymotrypsin in Organic Media…………………..42 4.4 Conclusions………………………………………………………………………….44 5 Effects of Enzyme Loading and Thermodynamic Water Activity………………..45 5.1 Introduction………………………………………………………………………….45 5.2 Materials and Methods………………………………………………………………46 5.2.1 Materials………………………………………………………………………..46 5.2.2 Activity of Immobilized α-chymotrypsin in Acetonitrile and Tetrahydrofuran…………………………………………………….46 5.2.3 Immobilization of α-chymotrypsin……………………………………………..47 iii 5.3 Results and Discussion………………………………………………………………50 5.3.1 Effect of Enzyme Loading on Activity in Acetonitrile and Tetrahydrofuran….50 5.3.2 Effect of Thermodynamic Water Activity……………………………………...53 5.4 Conclusions………………………………………………………………………….55 6 Conclusions and Future Research…………………………………………………..56 6.1 Conclusions………………………………………………………………………….56 6.2 Future Research……………………………………………………………………...58 References………………………………………………………………………………59 iv Abstract Mesoporous silicate (purely siliceous SBA-15, thiol functionalized SBA-15 and rod-like SBA-15) have been utilized as hosts for the immobilization of α-chymotrypsin and the activities of the immobilized enzymes in aqueous and organic media have been investigated. The activity of α-chymotrypsin in aqueous media decreased upon immobilization in pure and thiol functionalized SBA-15 but immobilized αchymotrypsin showed enhanced thermal stability at high temperature (70oC) compared to native α-chymotrypsin. Interestingly, α-chymotrypsin immobilized in pure and thiol functionalized SBA-15 showed minimal leaching from the support in aqueous buffer due to the strong electrostatic interaction between positively charged enzyme and negatively charged mesoporous silicate. α-chymotrypsin immobilized in pure SBA-15 and thiol functionalized SBA-15 showed higher activity in organic media compared to αchymotrypsin immobilized in the commercial silica gel at the thermodynamic water activity of 0.22. It is postulated that the higher activity of α-chymotrypsin immobilized in mesoporous silicate in organic media as compared with that on commercial silica gel is due to the smaller particle size of mesoporous silicate, which reduces the internal mass transfer limitation and hence increases the activity of the immobilized enzyme. Furthermore, α-chymotrypsin loading of 20 wt % in rod-like SBA-15 showed higher catalytic activity compared to other enzyme loading amount as well as α-chymotrypsin immobilized onto pure SBA-15 and commercial silica gel at the thermodynamic water activity of 0.22. The optimum thermodynamic water activity of α-chymotrypsin immobilized in rod like SBA-15 was found to be 0.55 in either acetonitrile or tetrahydrofuran. v List of Tables 3.1 Textural parameters of pure SBA-15, thiol functionalized SBA-15, rod-like SBA-15 and commercial silica gel…………………..………………………………29 4.1 Activity of immobilized α -chymotrypsin in dry octane, tetrahydrofuran or acetonitrile at thermodynamic water activity of 0.22………..….…………………...42 5.1 Water content required to attain selected water activities in 1-propanol, acetonitrile and tetrahydrofuran (Halling, 2002)………………….………………..47 vi List of Figures 3.1 X-ray diffraction spectra of pure SBA-15, rod-like SBA-15 and thiol functionalized SBA-15…………………..…………………………………………..27 3.2 N2 adsorption/desorption isotherm of the pure SBA-15 and thiol functionalized SBA-15………………………………..……………………………………………..28 3.3 Pore size distribution curve of Pure and thiol functionalized SBA-15……………...29 3.4 N2 adsorption/desorption isotherm of the rod-like SBA-15…………………………30 3.5 Pore size distribution curve of rod-like SBA-15…………………………………….30 3.6 FESEM images of Pure SBA-15 and Rod Like SBA-15……………………………31 4.1 Reaction scheme for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester…………...33 4.2 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester…………………….……………………………………………………...34 4.3 Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in dry octane, acetonitrile or tetrahydrofuran at thermodynamic water activity of 0.22……………………………………………………………………………….37 4.4 Thermal stability of native and immobilized α-chymotrypsin in aqueous media…...41 4.5 Leaching of α-chymotrypsin from pure and thiol functionalized SBA-15………….42 5.1 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester in acetonitrile and tetrahydrofuran……………………..………………..47 5.2 Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in either acetonitrile or tetrahydrofuran……..……………………………..49 5.3 Effect of enzyme loading on the activity of α-chymotrypsin immobilized in pure SBA-15 and rod-like SBA-15 in either acetonitrile or tetrahydrofuran at aw of 0.22………..…………………………………………………………………...50 5.4 Effect of enzyme loading on the activity of α-chymotrypsin immobilized in commercial silica gel in either acetonitrile or tetrahydrofuran at aw of 0.22………………………………………………………………………..…...51 5.5 Schematic representation of the effect of enzyme loading in porous support………52 5.6 Effect of thermodynamic water activity on the activity of α-chymotrypsin immobilized in rod-like SBA-15 in either acetonitrile or tetrahydrofuran…..……...53 vii List of Abbreviation Ǻ C angstrom ○ Degree Centigrade FESEM Field Emission Scanning Electron Microscopy XRD X-Ray Diffraction HPLC High Performance Liquid Chromatography aw Thermodynamic Water Activity Pure SBA-15 Unfunctionalized SBA-15 SH-SBA-15 Thiol functionalized SBA-15 Rod-like SBA-15 SBA-15 with rod like morphology TEOS Tetraethylorthosilicate MPTMS (3-mercaptopropyl) trimethoxy silane HCl Hydrochloric Acid KCl Potassium Chloride v Volume wt Weight APEE N-acetyl-L-phenylalanine ethyl ester BTEE N-benzoyl-L-tyrosine ethyl ester CaCl2 Calcium Chloride viii Chapter 1 Introduction Chapter 1 Introduction 1.1 Biocatalysis Biocatalysis is an important tool for the production of pharmaceutical and fine chemicals because of its high selectivity and specificity, mild reaction temperature, neutral or almost neutral pH. Moreover, it offers less pollution, hazardous and energy consumption compared to conventional metal based catalyst. Biocatalysis can also provide new routes for synthesis of high value compounds that cannot be produced by conventional metal based catalyst. The total number of biotransformation process carried out on an industrial scale increased tremendously during the past decade (Straathof et al., 2002). It is expected that there will be a huge growth on the application of enzymes in large scale production of fine chemicals (Schmid et al., 2002). If the reaction proceeds slowly under given temperature, an increase in temperature often leads to denaturation of enzymes. Moreover, enzymes are expensive and it often needs to be recovered and reused for economic viability. The soluble nature of enzymes in aqueous media requires the use of laborious techniques to separate enzyme from reaction mixture. However, these limitations can be overcome by immobilizing the enzymes. Immobilization of enzymes onto the solid support offers the most inexpensive way of removing enzymes from reaction media. Furthermore, it imparts some special features to biocatalyst such as improved operational and thermal stability, as well as 1 Chapter 1 Introduction stability against solvent induced denaturation. Even though it provides the advantages mentioned above, the major disadvantages upon immobilization are the loss of enzyme activity, diffusional limitation and increased cost. Despite of these disadvantages, immobilized enzymes are often used in pharmaceutical, food and chemical industries (KatchalskiKatzir, 1993). The most economical and preferred way to carry out biotransformation is to use aqueous media. However, most of the organic compounds are not soluble in water thereby requiring the use of organic solvent as the reaction medium. The use of organic solvent is not recommended for synthesis due to environmental concern, however it still serves as the best choice compared to the reaction performed in water due to the following reasons (Dordick, 1989): Increased solubility of substrate since most of the organic compounds are sparingly soluble in water Prevents undesirable side reaction e.g. hydrolysis Shifts thermodynamic equilibrium to favor synthesis over hydrolysis e.g. ester synthesis Easier recovery of enzymes from organic media due to its insolubility Enhances thermal stability Eliminates microbial contamination Alters the specificity of enzyme Enzymes in organic media possess very low activity due to diffusional limitation. However, this limitation can be overcome by immobilizing the enzymes onto solid support. Various factors which can affect the activity of enzymes in organic media are 2 Chapter 1 Introduction discussed in Chapter 2. It should be noted that, in the case of aqueous media, enzyme is immobilized in order to recycle the enzyme, but in the case of organic media enzyme is immobilized in order to suppress the diffusional limitation which leads to increased activity. 1.2 Immobilization of Enzymes Immobilization is the process of arresting the mobility of enzyme. Numerous methods are available in the literature for immobilization of enzymes and each method has its own efficiency and complexity. Various methods used till date for immobilization of enzymes can be divided into two main categories namely, covalent method and non-covalent method. Covalent method involves the formation of covalent linkage between amino acid side chain residues of protein with functional group on the support. The latter utilizes week forces such as ionic, hydrogen and van der Walls interaction. Enzyme entrapment in the polymer matrix, sol-gel encapsulation, adsorption of enzymes on the surface and porous materials are some of the examples of non-covalent method of immobilization. Selection of a particular method depends on the requirements to be met and the efficiency of the selected method depends on the nature of the enzyme and supports. In this study, enzyme is adsorbed onto porous support which is segregated under non-covalent method of immobilization. In some cases, the adsorption of enzymes onto solid support shows severe leaching due to weak interaction between the support and enzyme. In those cases, covalent method of immobilization is preferred over noncovalent methods. 3 Chapter 1 Introduction The supports available for the immobilization of enzymes range from simple synthetic polymers such as polypropylene, polyamide, polystyrene to inorganic materials such as zeolites, controlled pore glass, sol-gel encapsulation and silica gel. Each of these materials has its own advantages and disadvantages. Inorganic materials are preferred over polymeric materials because of its high pore volume and high surface area. One of the most widely used methods for immobilizing enzymes is encapsulation inside sol-gel silica. However, due to small pore sizes and partially closed pore structures, most of the enzymes immobilized by this procedure show much lower activity compared to the native enzymes. The invention of ordered mesoporous silicate attracted much attention as a host for enzyme immobilization due to its tunable large pore size (25 – 300 Å), very high surface area (~ 1200 m2 g−1), narrow pore size distribution and easier chemical surface modification. Porous materials are classified by International Union of Pure and Applied Chemistry (IUPAC) into three classes such as microporous (pore size < 20 Å), mesoporous (pore size 20 - 500 Å), and macroporous materials (pore size >500 Å) (Taguchi and Schuth, 2005). Zeolite is a well known member of the microporous materials but its application as a host for immobilization of enzymes is limited due to its smaller pore size (2 - 10 Å) compared to molecular dimension of the enzymes. This limitation can be overcome by utilizing mesoporous silicate which has the pore size larger than the molecular dimension of enzyme (Yiu and Wright, 2005). In 1990s, researchers at Mobil Corporation invented the first family of highly ordered mesoporous molecular sieves M41S called MCM-41 where long chain cationic surfactant was used as the template or pore forming agents during the hydrothermal 4 Chapter 1 Introduction solgel synthesis. This material possesses narrow pore size distribution in the range of 26 – 40 Å (Kresge et al., 1992). The early studies on the use of this material were focused on immobilizing organometallic compounds and very few studies were carried out in utilizing MCM-41 as a host for the immobilization of enzymes. Moreover, this material possesses low hydrothermal stability due to thin pore walls. In 1998, Stucky and coworkers invented another mesoporous material called SBA-15 with non-ionic amphiphilic triblock copolymer micelles as template under acidic reaction conditions (Zhao et al., 1998). The specific surface area and pore volumes are somewhat smaller than that of MCM-41 but it possess thicker pore walls. Therefore the mechanical and hydrothermal stability of these materials are found to be better. The pore diameters can be varied between 50 – 300 Å, which is quite large enough to accommodate macromolecules. The functionalization of SBA-15 with organic moieties in the framework was also developed, attracting enzymologist to use this material as a host for the immobilization of enzymes. 1.3 Research Objectives The objective of this work is to use unfunctionalized and thiol functionalized SBA-15 as a host for the immobilization of enzymes and to find out the activity of the immobilized enzymes in aqueous and organic media. α-chymotrypsin is chosen as the model enzyme for this study. Since the native enzyme possesses very low thermal stability in aqueous media, it is of great interest to investigate whether the enzyme immobilized in mesoporous silicate has better thermal stability compared to native enzyme. Organic solvent is the most preferred media for biotransformation. Hence, we are interested in 5 Chapter 1 Introduction finding out the efficiency of enzyme immobilization in mesoporous silicate for application in organic media. We also studied the effect of particle of size and αchymotrypsin loading on the activity in polar organic solvents. Furthermore, it is our interest to investigate whether the particle size and enzyme loading have any effect on the activity of enzyme because in most of studies reported in the literature, very little attention had been paid to this parameter. Moreover, enzyme hydration is one of the critical parameter affecting the activity of enzyme; it is also of interest to investigate how the activity of immobilized enzyme changes with enzyme hydration. 1.4 Organization of Thesis The rest of this thesis is divided into five chapters. Chapter 2 gives a brief introduction to non-aqueous enzymology, various factors affecting the activity of enzymes in organic media and the methods to increase the activity of enzymes in organic media. In Chapter 3, preparation and characterization of pure SBA-15, thiol functionalized SBA-15 and SBA-15 with rod like morphology are described. Chapter 4 is the most important chapter in this thesis as it describes the immobilization of α-chymotrypsin into pure SBA-15 and thiol functionalized SBA-15 and its activity studies in aqueous or organic media. In Chapter 5, the effects of enzyme loading and thermodynamic water activity on the reaction rate in polar organic solvents are also discussed. Conclusions and future scope of the work are presented in Chapter 6. 6 Chapter 2 Literature Review Chapter 2 Literature Review 2.1 Introduction This literature review gives the brief introduction to non-aqueous enzymology, various reasons for the low activity of enzyme in organic media as well as the ways to increase the activity of enzymes in organic media. Enzyme immobilization is one of the methods of increasing the activity of enzymes in organic media, which is the main objective of this work; hence, it is discussed in details. At first, immobilization of enzymes onto polymers for use in organic media is given followed by the enzyme immobilization in controlled pore glass, sol-gel and mesoporous silicate. Finally, enzyme immobilization in mesoporous silicate for use in aqueous media is also discussed. 2.2 Nonaqueous Enzymology Biocatalysis in organic media emerged in the nineteenth century. Scientists in the early days recognized the insolubility of organic compounds in water and tried to replace it with organic solvents. At first water miscible organic solvents such as ethanol or acetone were added to the bulk water medium. As long as high water content was retained, the enzymes were found to be catalytically active. The next phase of non-aqueous enzymology was to use the biphasic mixture in which aqueous solution of enzyme was 7 Chapter 2 Literature Review emulsified in a water immiscible organic solvent e.g. isooctane or heptane. In this system, the substrate present in the organic phase diffused into the aqueous media underwent reaction and the product diffused back to the organic media (Klibanov, 1986; Krishna, 2002). The seminal work of Zaks and Klibanov (Zaks and Klibanov, 1998a, 1998b) led to the next stage of development to employ the enzymes in nearly nonaqueous solvents. They found that the enzymes were not only catalytically active in dry organic solvents but also possess very distinct properties such as very high thermal stability, altered regio- enantio- and stereo selectivity compared to pure aqueous and aqueous-organic solvent mixtures (Klibanov, 2001; Krishna, 2002; Castro and Knubovets, 2003). However, the activity of enzymes in dry organic media was very low compared to its activity in aqueous media (Klibanov, 2001). Michels et al. (1997) reported that the kcat/Km (turnover frequency/specific binding constant) of the transesterification of N-acetyl-L-phenylalanine ethyl ester in hexane and its hydrolysis in water were 0.43 and 2800 M−1s−1 respectively. The very low activity of enzymes in organic media was a main problem and various solutions for improving the activity of enzymes in organic media had been suggested which ranged from simple addition of salts during the lyophilization (Khmelnitsky et al., 1994) to the covalent incorporation of enzymes into the polymer named ”Biocatalytic Plastics” (Wang et al., 1997). 2.2.1 Factors Affecting the Activity of Enzymes in Organic Media Several attempts had been made to elucidate the various reasons for the decrease in the activity of enzyme upon switching from aqueous to organic media. Dependence of enzyme activity on its three dimensional conformation is well known. The question, which first comes to our mind, is whether the enzyme maintains its native fold upon 8 Chapter 2 Literature Review placing in organic media. X-ray crystal structure of α-chymotrypsin soaked in hexane was found to be identical to the aqueous solution structure, which indicated that the conformational changes did not, occurred upon placing the α-chymotrypsin in organic media (Yennawar et al., 1994). Schmitke et al. (1997) studied the X-ray crystal structure of another widely studied enzyme subtilisin carlsberg soaked in acetonitrile or dioxane and compared it with aqueous solution structure which also confirmed that the conformational changes did not occurred upon soaking the crystal in organic media. Moreover, active site structure also found to be similar in all the media studied. Molecular dynamics simulation of enzymes in organic solvents also provided the evidence that the enzyme maintained its native fold in organic solvents (Soares et al., 2003; Yang et al., 2004). All these studies eliminated the dogma that the diminished activity of enzymes upon switching from aqueous to organic media was not due to conformational changes in enzymes. Fourier transform infrared (FTIR) spectroscopy is the most powerful technique for studying the secondary structure of lyophilized enzyme powders, enzymes suspensions in organic solvents and enzyme adsorbed onto solid surfaces (Griebenow et al., 1999). FTIR spectroscopic studies on several lyophilized enzyme powder had shown that lyophilization can cause severe structural damage to the enzyme but reversible upon dissolving in aqueous media (Griebenow and Klibanov, 1995). However, the secondary structure of the lyophilized enzyme powder suspended in several organic solvents remained unchanged between the solvents and to the lyophilized enzyme powder (Griebenow and Klibanov, 1997). Recently, solid-state nuclear magnetic resonance spectroscopic study of lyophilized papain also confirmed that enzyme underwent conformational changes during lyophilization and irreversible upon placing in organic 9 Chapter 2 Literature Review media (Matsubara et al., 2006). Thus, lyophilization induced conformational changes in the enzymes which was irreversible upon placing in organic media was suggested as one of the reason for the drastic decrease in the activity of enzymes upon switching from aqueous to organic media. Various methods of preventing the lyophilization induced structural changes in enzymes are discussed in Section 2.2.2. Enzymes found to be precipitate upon placing in organic media (with an exception to dimethyl sulfoxide, formamide) in contrast to the aqueous media in which the enzyme dissolves (Chin et al., 1994). Zaks and Klibanov, (1998b) reported that the decrease in the activity of enzymes in organic media was not due to the diffusional limitation because the increase in the shaking rate from 160 to 300 rpm or ultrasonication which reduced the particle size from 270 to 5 µm did not increase the activity of enzyme in organic media. Rees and Halling (2001) studied the acetylation of lyophilized and immobilized myoglobin powder in organic media by electron spray-mass spectrometry. The rate of acetylation was higher on immobilized myoglobin compared to lyophilized myoglobin powder. They concluded that, the adsorption of myoglobin onto the solid surfaces spread the myoglobin over the large surface area and thereby eliminates the mass transfer limitation that resulted in higher affinity for acetylation. However, lyophilized myoglobin power had suffered severe mass transfer limitation, which hindered the acetylation process. Persson et al. (2002a) showed that the specific activity of lipase immobilized in polypropylene was 770 times higher compared to lyophilized lipase powder and they interpreted that the higher activity of lipase upon immobilization was due to reduced mass transfer limitation. All these results clearly showed the advantages of employing immobilized enzymes in organic media, which was the main aim of this work. 10 Chapter 2 Literature Review From the origin of non-aqueous enzymology, attention had been paid to the availability of water around the enzyme molecule. Water in close proximity to the protein surface is fundamental to protein folding, stability, recognition, and activity (Phillips and Pettitt, 1995). Moreover, water also acts as a molecular lubricant, which facilitates the conformational flexibility necessary for the enzyme to perform catalysis (Klibanov, 1997). Hence, the completely dehydrated enzyme remains inactive due to the reduced flexibility and it is always necessary to have a minimum amount of water molecule in the enzyme in order to have a flexibility and thereby the activity. However, the minimum number of water molecules necessary for the enzyme to possess the catalytic activity found to be dependent on enzyme. For example, α -chymotrypsin suspended in octane possess catalytic activity with only 50 molecules of water per enzyme molecule but polyphenol oxidase requires about 3.5 ×107 molecules of water per enzyme molecule to possess the activity in chloroform (Dordick, 1989). It was found that the catalytic activity of the enzymes in organic media depend on the water molecules associated with an enzyme and did not depend on the water content in the system (Zaks and Klibanov, 1998a). In order to quantify the amount of water associated with an enzyme molecule, thermodynamic water activity was suggested as the useful parameter (Halling, 1994) because if the water equilibrated between the various phases present, they would all come to the same water activity. The enzyme molecule will tend to equilibrate in this way; hence, the quantity of water associated with the enzyme molecule will reflect the system water activity (Halling, 2004). Apart from decrease in the activity of enzyme upon transition from aqueous to organic media, it is interesting to note that the activity changes over 10000 fold within the organic media itself and the activity is higher in nonpolar solvents compared to polar 11 Chapter 2 Literature Review solvents (Zaks and Klibanov, 1998b). Removal of essential water molecules from the enzyme surface (water striping) by polar solvents was suggested as one of the reason for the low activity of enzymes in polar solvents. Because, when the enzyme was assayed in dry polar solvent, the solvent can strip the essential water molecule present in the enzyme surface and thereby lock the enzyme conformation, which led to decreases enzyme flexibility and activity. In the case of nonpolar solvent, water stripping will be minimum due to its low solubility in water, which results in low dehydration of enzymes, hence high activity (Zaks and Klibanov, 1998b; Wangikar et al., 1997; Klibanov, 2001). The water stripping by polar solvents can be clearly visualized in the molecular dynamics simulation by Yang et al. (2004). However, the water stripping in polar solvents could be eliminated by carrying out the reaction at constant thermodynamic water activity. This is because, in a non-controlled system (phases with uneven thermodynamic water activity or water content), water molecules tend to transfer between various phases until they all reach the same water activity. However, if the reaction is initiated at constant thermodynamic water activity, then the transfer of water molecules between the phases will not take place and thereby the enzyme dehydration will be prevented (Partridge et al., 1998a; Halling, 2002). 2.2.2 Enzyme Activation in Organic Media There are several methods reported in the literature in order to improve the activity of enzymes in organic media. Some methods prevent the conformational changes in enzyme during lyophilization and there by increases the activity, and other methods are specific and do not involve the lyophilization. The enzymes in organic media show pH memory i.e. its activity in organic media depends on the pH to which it was finally 12 Chapter 2 Literature Review exposed and it was found to be maximum when it is lyophilized from the optimum pH for the catalytic activity in aqueous media. For example, transesterification activity of subtilisin Carlsberg increased to about 75 times when it was lyophilized from the optimum pH compared to subtilisin Carlsberg as received from the company (Zaks and Klibanov, 1998b). Hence, the enzyme as received from the company serves as the poor control for comparing the activity of enzymes in organic media and it is a good practice to obtain enzyme from optimum pH for use in organic media. Through out this literature survey, lyophilized enzyme refers to relyophilized enzyme from optimum pH unless otherwise stated. Khmelnitsky et al. (1994) found that the lyophilization of subtilisin Carlsberg in the presence of 98 wt % potassium chloride enhanced the activity to about 4000 fold in hexane compared to lyophilized enzyme powder. By optimizing the freeze-drying time, water and salt content, the transesterification activity of subtilisin Carlsberg in hexane increased to about 20000 fold relative to lyophilized enzyme powder (Ru et al., 1999). The prevention of lyophilization induced conformational changes and the role of salt matrix as the immobilization support suggested as the reason for the increased activity of enzyme upon addition of salt during lyophilization (Griebenow and Klibanov, 1997). However, Laszlo and Compton, (2001) showed that α-chymotrypsin lyophilized in the presence of potassium chloride possessed negligible activity in acetonitrile and they concluded that the salt activation was effective only in nonpolar organic solvents. Not only salt but also addition of crown ether during lyophilization increases the activity of enzymes in organic media. Engbersen et al. (1996) found that the addition of crown ethers increases the transesterification activity of α-chymotrypsin to about 640 13 Chapter 2 Literature Review fold relative to the lyophilized α-chymotrypsin. Unen et al. (2002) reported the crown ether activation of enzymes was due to the macrocyclic interaction of crown ethers with enzyme (lysine ammonium groups) which reduced the formation of inter and intramolecular salt bridges and hence enabled the enzyme to refold into more active conformation as well as preservation of enzymes during lyophilization. Tremblay et al. (2005) found that the crown ether modified peptide shows higher activity compared to native crown ether. Several other methods such as addition of sorbitol (Debulis and Klibanov, 1993), methyl cyclodextrin (Santos et al., 1999; Griebenow et al., 1999; Montanez et al., 2002), urea (Guo and Clark, 2001) and Poly (ethylene glycol) (Debulis and Klibanov, 1993; Kwon et al., 1999 (1999); Castillo et al., 2006) during lyophilization had also been reported to increase the activity of enzymes in organic media. Cross-linked enzyme crystal (CLEC) serves as the most robust biocatalyst in either aqueous or organic media. Although there were some successful applications of CLEC in organic media (Khalaf et al., 1996 ; Wang et al. (1997)), immobilization of subtilisin carlsberg onto silica gel followed by rinsing with propanol (Patridge et al., 1998b) and lyophilization of subtilisin carlsberg in presence of methyl cyclodextrin shows higher activity in organic solvents compared to subtilisin CLEC. Moreover, CLEC is not available for some enzyme such as chymotrypsin, papin and the advantages of using CLEC compared to other methods of enzyme preparation for use in organic media remains unclear. Cross-linked enzyme aggregates (CLEA) (Cao et al., 2000), protein-coated micro crystal (PCMC) (Kreiner et al., 2001), enzyme precipitated and rinsed with propanol 14 Chapter 2 Literature Review (Roy and Gupta, 2004), three-phase partitioning (Roy et al., 2004) were some of the other methods available in the literature which increases the enzyme activity in organic solvents compared to lyophilized enzyme powders. 2.2.3 Enzyme Activation by Immobilization Immobilized enzyme employed in organic media from the origin of non-aqueous enzymology (Dordick, 1989). Organic and inorganic materials were used as an immobilization support for use in organic media. In all cases, the immobilized enzymes possess higher activity in organic media compared to lyophilized enzyme powder due to very low diffusional limitation. In this section, enzyme immobilization in polymer was discussed first followed by enzyme immobilization in controlled pore glass, sol-gel and mesoporous silicate for application in organic media. 2.2.3.1 Enzyme Immobilization in Polymers Barros et al. (1998) showed that the α-chymotrypsin immobilized in polyamide not only showed low activity for peptide synthesis but also possessed very low enzyme loading (5-10 mg g−1 of support) compared to controlled pore glass (100 mg g−1 of support). Persson et al. (2002a) reported that lipase adsorbed onto polypropylene possessed higher activity compared to lyophilized lipase. Jia et al. (2002) immobilized α -chymotrypsin in polystyrene nanofiber, which showed higher activity in organic media compared to native α-chymotrypsin, but they did not state whether the native α-chymotrypsin used was relyophilized or used as received from Sigma. Not only native polymers, functionalized polymers have also been used for the immobilization of enzymes for use 15 Chapter 2 Literature Review in organic media which showed higher activity compared to lyophilized enzyme powder (Markvicheva et al., 2005; Bacheva et al., 2005). Apart from the enzyme adsorption onto polymers, covalent incorporation of enzymes into the polymeric materials was also carried out to improve the activity of enzymes in organic media (Ito et al., 1993; Yang et al., 1995a; Yang et al., 1995b). Wang et al. (1997) incorporated acryloyl chloride modified α-chymotrypsin into the various polymers such as poly vinylalcohol, poly (methyl methacrylate), poly (styrene) and studied its activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with propanol in hexane and toluene. They observed not only less diffusional limitation even at the enzyme loading of 9.6 wt % but also high activity. Kim et al. (2001) used flash devolatilization to incorporate α-chymotrypsin into the polymer and subsequent crosslinking resulted in the enzyme with the high stability and activity relative to lyophilized α-chymotrypsin. However, they observed severe leaching of enzyme while cross-linking and the final enzyme loading was only 2 wt %. Enzymes incorporated in roomtemperature vulcanizable silicone composite had also showed high activity and stability when the reaction was carried out in non-polar solvents saturated with buffer (Gill et al., 1999; Ragheb et al., 2003). Although, the covalent incorporation of enzymes into the polymeric support has showed higher activity in organic media, it is not clear in what way it is a more advantageous to use this method than simple enzyme adsorption which also has showed higher activity in organic media (Halling, 2002) 16 Chapter 2 Literature Review 2.2.3.2 Enzyme Immobilization in Inorganic Materials Most of the studies in the immobilization of enzymes into inorganic materials use celite, controlled pore glass, sol-gel glass, and silica gel. Barros et al. (1998) showed that the activity and enzyme loading of α-chymotrypsin immobilized in controlled pore glass was higher compare to polyamide and celite. However, lipase adsorbed onto polypropylene shows higher activity compared to celite (Persson et al., 2000). Reetz et al. (1996) showed that the lipase immobilized in sol-gel possessed 157 fold higher activity in isooctane compared to lyophilized enzyme powder. Unen et al. (2001) immobilized α-chymotrypsin, subtilisin Carlsberg and trypsin into sol-gel glass, which also showed higher activity in non-polar solvents at the thermodynamic water activity of 0.7 compared to lyophilized enzyme powder. However, Persson et al., (2002b) found that lipase adsorbed onto polypropylene powder showed higher activity (400 fold) compared to crude lipase but sol-gel entrapment was also showed 340 fold higher activity compared to crude lipase. However, the other form of enzyme preparation such as salt and crown ether activation showed lower activity compared to lipase adsorbed in polypropylene but higher than the lyophilized lipase. Mesoporous silicate is a unique material for the immobilization of enzymes due to its large specific surface area, tunable pore size, narrow pore size distribution, and easier chemical modification. However, to the best of our knowledge very few studies were carried out in the application of enzyme immobilized in mesoporous silicate and its use in organic media. 17 Chapter 2 Literature Review Takahashi et al. (2000) was the first who evaluated the activity of enzyme in organic media upon immobilization in mesoporous silicate. They immobilized horseradish peroxidase in FSM-16, MCM-41 and SBA-15 of different pore size and compared its activity with horseradish peroxidase immobilized in commercial silica gel and lyophilized horseradish peroxidase as received from company. The adsorption of horseradish peroxidase were found to be high when the material pore size was larger then the molecular dimension of horseradish peroxidase, FSM-16 and MCM-41 were found to possess highest enzyme loading of 183 and 147 mg g-1 respectively. The activities of horseradish peroxidase immobilized in FSM-16 and MCM-41 were higher when pore size of the support just matches with the molecular dimension of the horseradish peroxidase. However, SBA-15 and silica gel possess not only possess low enzyme loading (24 and 49 mg g−1 respectively) but also low activities. Since the activity of the immobilized enzyme depends on the enzyme loading (Wehtje et al., 1993; Day and Legge (1995); Barros et al., 1998), it is not clear to us that the low activity of horseradish peroxidase immobilized in silica gel and SBA-15 was due to the low enzyme loading or intrinsic nature of the interaction between enzyme and support. Moreover, the reaction was not performed at constant thermodynamic water activity, which also can lead to misleading conclusion about the effect of support. Deere et al. (2003) reported the catalytic activity of cytochrome c in organic media upon immobilization in commercial kieselgel silica and MCM-41 of pore size 28 and 45 Ǻ respectively. The activity was higher in methanol, ethanol and formamide (containing 2.5% v/v water) but lower in 1-methoxy 2-propanol compared to lyophilized cytochrome C powder. Wang et al., (2001) found that immobilization of α-chymotrypsin 18 Chapter 2 Literature Review onto mesoporous silicate showed higher activity compared to lyophilized enzyme powder. As the enzyme hydration is the critical parameter affecting the activity in organic media to about 100 fold (Klibanov, 1997), none of these studies carried out the reaction at constant thermodynamic water activity which is of paramount importance in order to observed the true nature of support (Adlercretuz, 1991). Moreover, none of the studies in mesoporous silicate had paid attention to the internal mass transfer limitation commonly occurs in porous support which can also greatly affect the performance of the enzyme. This inspired us to study the importance of mesoporous silicate a host for the immobilization of enzymes for catalysis in organic media. 2.3 Enzyme Immobilization in Mesoporous Silicate for Use in Aqueous Media Application of mesoporous silicate as a host for the immobilization of enzymes commence in 1996. Initial attempt on the immobilization of enzymes onto mesoporous silicate showed that the loading efficiency of the enzyme depend on its molecular size as well as solution pH and the immobilized enzyme was also possessed high storage stability compared to native enzyme (Diaz and Balkus, 1996). He et al. (2000) showed that the incorporation of aluminium in the framework of MCM-41 not only enhanced the activity of penicillin acylase but also prevented enzyme leaching from the support. Lei et al. (2002) found that the activity of organophosphorus hydrolase immobilized on carboxylic acid functionalized SBA-15 showed higher activity and enzyme loading compared to amine functionalized SBA-15. They rationalized that positive charge on amine functionalized SBA-15 and in the enzyme caused repulsion in between them which leads to lower enzyme loading, however the negative charge on acid 19 Chapter 2 Literature Review functionalized SBA-15 with positive charge on enzyme increased the interaction between support and enzyme, which led to higher enzyme loading. The study on the effect of pore size on the activity of trypsin by Yiu et al. (2001b) showed the activity increases with increase in pore size, however the trypsin adsorbed in unfunctionalized mesoporous silicate shows severe leaching. The same group latter found that the surface functionalization can not only prevents the enzyme leaching but also influences the enzyme activity (Yiu et al., 2001). From these studies, we can infer that the presence of functional group in the framework of mesoporous silicate not only prevents the enzyme leaching but also induces conformational changes in enzyme and thereby affecting the activity. Pandya et al. (2005) found that α- amylase immobilized in MCM-41 and SBA15 in which the enzyme adsorbed only on the outer surface showed low specific activity compared to meso-cellular foams (MCF) in which the enzyme seemed to be immobilized inside the pores and exhibit high specific activity. It is also interesting to note that horseradish peroxidase immobilized in mesoporous silicate possesses high thermal stability compared to native enzyme when the support pore size matches with the molecular dimension of enzyme (Takahashi et al., 2000). Ravindra et al., 2004 found that the melting point of ribonuclease A increased dramatically (∆Tm ~ 30○C) compared to native enzyme upon immobilization in mesoporous silicate. They concluded that the increase in thermal stability of ribonuclease A upon immobilization was not only due to excluded volume effect but may also due to an increased strength of the protein in the narrow pore channel. This study confirmed that enzyme immobilization in mesoporous silicate not only provides the easier way to recover enzymes from aqueous media but also increases the thermal stability dramatically. Fan et al. (2003) invented that the rate of immobilization was found to be 20 Chapter 2 Literature Review varied depending on the morphology of mesoporous silicate; however, activity was not reported in their study. To the best of our knowledge, none of the studies had considered the effect of particle size on the activity of enzyme. 2.4 Conclusions Mesoporous silicate was found to be the excellent host for the immobilization of proteins but little attention had been paid in the use of mesoporous material as a host for the immobilization of enzymes for application in organic solvents. Although some have reported, the activities of enzymes in organic media upon, none of the studies considered the enzyme hydration, which can greatly affect the activity of enzymes in organic media. In this study, we utilized mesoporous silicate as a host for the immobilization of enzymes and found out its activity in aqueous and organic media taking enzyme hydration into account. 21 Chapter 3 Synthesis of Mesoporous Silicate Chapter 3 Synthesis of Mesoporous Silicate 3.1 Introduction Porous materials have been used as adsorbents and catalyst supports for many decades (Corma, 1997). Most of them are based on zeolites which possess pore sizes in the range of 2- 10 Ǻ. However, they are not suitable for the immobilization of enzymes due to their smaller pore sizes as the molecular dimension of enzymes are several angstroms larger than the pore size of zeolites. Hence, zeolites receive less attention as supports for the immobilization of enzymes. The discovery of highly ordered mesoporous silica materials (M41S) has opened the way to use inorganic material as a support for the immobilization of enzymes due to its tunable larger pore size (40-300 Å), narrow pore size distribution, high specific surface area (~ 1000 m2 g-1 ), highly ordered pore structure and easier chemical surface modification. This chapter describes the preparation of mesoporous silicate unfunctionalized SBA-15 (pure SBA-15), thiol functionalized SBA-15 (SH-SBA-15) as well as pure SBA-15 with rod-like morphology (rod-like SBA-15). The prepared material was well characterized by various techniques. α-chymotrypsin immobilized onto commercial silica gel was used as a control for comparing the activity of α-chymotrypsin immobilized onto mesoporous silicate in organic media (Chapter 4 and 5). Hence, surface area, pore 22 Chapter 3 Synthesis of Mesoporous Silicate volume and particle size of commercial silica gel obtained from Sigma were given in this chapter. 3.2 Experimental Methods 3.2.1 Materials Silica source tetraethylorthosilicate (TEOS) (98 %) and poly (ethylene glycol)-blockpoly (propylene glycol)-block-poly (ethylene glycol) (Pluronic P123) were purchased from Aldrich. Hydrochloric acid (HCl) (37%) and potassium chloride (KCl) were purchased from Merck. (3-mercaptopropyl)trimethoxy silane (MPTMS) (95%) was purchased from Alfa Aesar. Commercial silica gel was purchased from Sigma. 3.2.2 Synthesis of Pure SBA-15 Pure SBA-15 was prepared according to the procedure reported in the literature (Zhao et al., 1998). The molar composition of the gel was 1 mole TEOS: 0.017 mole pluronic P123:2.9 mole HCl: 202.6 mole water. In a brief protocol, about 11.4 g of hydrochloric acid was added to the solution of 4 g of pluronic P123 mixed with 138 g of water. Then 8.4 g of TEOS was added and the resulting solution was stirred for about 24 hours at 40○C. The slurry was then transferred to polypropylene bottle and heated at 100○C for about 48 hours. The solid was collected by filtration and dried at 60○C. Surfactant was removed by calcination at 550○C for about 8 hours. 3.2.3 Synthesis of Thiol Functionalized SBA-15 Synthesis of thiol functionalized SBA-15 was carried out by the method similar to the synthesis of pure SBA-15 except that MPTMS was introduced along with TEOS at 23 Chapter 3 Synthesis of Mesoporous Silicate MPTMS/TEOS ratio of 95:5 mol %. The surfactant was removed by refluxing with ethanol for about 24 hours. 3.2.4 Synthesis of Pure SBA-15 with Rod-like Morphology Pure SBA-15 with rod-like morphology was prepared according to the procedure reported in the literature (Yu et al., 2002). The molar composition of the gel was 1mole TEOS: 0.02 mole pluronic P123: 1.5 mole KCl: 6 mole HCl: 166 mole water. In a brief protocol, 2.3 g of pluronic P123 and 2.2 g of potassium chloride was dissolved in 59.8 g of water containing 4.4 g of hydrochloric acid at 38±1○C. Then 4.2 g of TEOS was added and the resulting solution was stirred vigorously for about 8 min and kept at the same temperature under static condition for 24 hours. The slurry was then maintained at 100○C for 24 hours. The solid was collected by filtration and dried at room temperature. Surfactant was removed by calcination at 550○C for about 8 hours. 3.2.5 Characterization of Materials X-ray diffraction (XRD) is a well established method for the identification of ordered phases. This is because the wavelength of X-rays is comparable to the size of the atoms. Hence it is ideally suitable for probing the structural arrangement of atoms and molecules in a wide range of materials. XRD patterns of the prepared materials were obtained using Shimadzu XRD-6000 with CuKα radiation. The X-ray tube was operated at 40 kV and 30 mA and a continuous 2θ scan was performed. Since the walls of mesoporous materials are amorphous and only the arrangement of the pores induces the regularity in the solid, hence the reflections in powder XRD were only obtained in the 2θ range between 0.5 to 10○. 24 Chapter 3 Synthesis of Mesoporous Silicate The specific surface area, pore diameter and pore volume of the pure SBA-15, SH-SBA-15 and pure SBA-15 with rod-like morphology were analyzed by N2 adsorption/ desorption measurements using Quantachrome Autosorb-1 at 77 K. Before subjecting to the analysis, about 50 - 100 mg of sample was pelletized and degassed at 170○C for pure SBA-15 and SBA-15 with rod-like morphology for about 6 hours. SHSBA-15 was degassed at 80○C for about 6 hours. The specific surface area of the samples were calculated in the relative pressure (P/P0) range of = 0.05 - 0.35. Pore size distribution was obtained from the adsorption branch of the isotherm using BarrettJoyner-Halenda method. The total pore volume was determined from the adsorption branch of the N2 isotherm at the relative pressure of P/P0 = 0.95. Particle size distributions were measured using Coulter particle size analyzer. Sulfur content in thiol-functionalized SBA-15 was determined from Perkin-Elmer Series II CHNS Analyzer 2400. Field-emission scanning electron microscopy (FESEM) images of pure SBA-15 and rod-like SBA-15 were obtained using JEOL JSM-6700F microscope. 3.3 Results and Discussion 3.3.1 Characterization of Pure and Thiol Functionalized SBA- 15 X-ray diffraction pattern of pure SBA-15 and SH-SBA-15 were given in Figure 3.1. The spectra clearly shows three well resolved peaks which can be assigned to (100), (110) and (200) reflections associated with 2D hexagonal space group (P6mm) consistent with 25 Chapter 3 Synthesis of Mesoporous Silicate the spectra reported in Zhao et al., (1998). The lower intensity of (110) and (200) reflections in the spectra of SH-SBA-15 indicates the decrease in the ordered structure of this sample due to the addition of 5 mol % functionalization agent (MPTMS) along with the silica source. The decrease in the ordered structure upon functionalization is also Intensity (Arbitary Units) 200 35000 110 100 observed by Hodgkins et al., (2005). 30000 SH-SBA-15 100 25000 20000 Rod-like SBA-15 100 15000 10000 5000 Pure SBA-15 0 0 2 4 2θ (Degree) 6 8 Figure 3.1: X-ray diffraction spectra of pure SBA-15, rod-like SBA-15 and thiol functionalized SBA-15 Figure 3.2 shows the N2 adsorption/desorption isotherms of the pure and thiol functionalized SBA-15. Pure SBA-15 exhibits the typical type IV adsorption isotherms where the volume of nitrogen adsorbed increases with increasing relative pressure with sharp rise in adsorption due to capillary condensation in the mesopores. However, SHSBA-15 sample shows less-well defined capillary condensation step and lower porosity due to the introduction of functionalization agent along with the silica source. 26 Chapter 3 Synthesis of Mesoporous Silicate Volume (cc/g) 1200 1000 800 600 400 Pure SBA-15 200 SH-SBA-15 0 0 0.2 0.4 0.6 0.8 Relative Pressure (P/Po) 1 Figure 3.2: N2 adsorption/desorption isotherm of the pure SBA-15 and thiol functionalized SBA-15 Figure 3.3 show the pore size distribution of pure SBA-15 and SH-SBA-15 which indicate the synthesized material possess narrow pore size distribution which is the hallmark of mesoporous silicate. The pore diameter, specific surface area, pore volume and particle size of pure SBA-15 and SH-SBA-15 were given in Table 3.1. The decrease in specific surface area, pore diameter and pore volume of SH-SBA-15 can be attributed to the addition of 5 mol % MPTMS in the initial synthesis mixture. Even though, the surface area and pore volume of pure SBA-15 differ from SH-SBA-15, the particle size of pure SBA-15 and SH-SBA-15 were 22 and 20 µm respectively. Particle size of the support mentioned in Table 3.1 refers to the length of the particle. Table 3.1: Textural parameters of pure SBA-15, thiol functionalized SBA-15, rod-like SBA-15 and commercial silica gel Materials Pore Diameter Surface Area Pore Volume Particle Size (Ǻ) (m2 g-1) (cm3 g-1) (µm) Pure SBA-15 83 790 1.09 22 SH-SBA-15 67 660 0.84 20 Commercial Silica Gel 56 470 0.73 64 Rod-like SBA-15 75 690 0.96 2.0 27 Adsorption Dv (d) (cm3/Ǻ/g) Chapter 3 Synthesis of Mesoporous Silicate 1.00E-01 Pure SBA-15 8.00E-02 SH-SBA-15 6.00E-02 4.00E-02 2.00E-02 0.00E+00 0 50 100 150 200 250 Pore Diameter (Å) Figure 3.3: Pore size distribution curve of Pure and thiol functionalized SBA-15 CHNS elemental analysis of SH-SBA-15 sample shows that the carbon, hydrogen, and sulfur content were 15.58, 3.38, 2.12 % respectively, confirming the presence of sulfur in the SH-SBA-15. 3.3.2 Characterization of SBA-15 with Rod-Like Morphology Figure 3.1 shows the XRD spectra of rod-like SBA-15 consisting of three well resolved peak which can be assigned to (100), (110) and (200) reflections of the 2D hexagonal space group (P6mm) which are similar to those of pure SBA-15. Figure 3.4 and 3.5 shows the N2 adsorption/desorption isotherms and pore size distribution curve of rod-like SBA-15 which are identical to that of pure SBA-15. The surface area, pore volume and pore size of rod-like SBA-15 given in 3.1 were closer to pure SBA-15 but differ only in particle size. The particle size (length of the rod) of rod like SBA-15 is 2 µm which is 10 times smaller compared to pure SBA-15 and SH-SBA-15. This smaller particle size of rod-like SBA-15 is of paramount importance in our investigation on the 28 Chapter 3 Synthesis of Mesoporous Silicate effect of particle size on the catalytic activity of immobilized α-chymotrypsin in organic media. 700 Volume (cc/g) 600 500 Rod Like SBA-15 400 300 200 100 0 0 0.2 0.4 0.6 Relative Pressure (P/Po) 0.8 1 3 Adsorption Dv(d) (cm /Å/g) Figure 3.4: N2 adsorption/desorption isotherm of the rod-like SBA-15 3.50E-02 3.00E-02 2.50E-02 2.00E-02 1.50E-02 1.00E-02 5.00E-03 0.00E+00 Rod Like SBA-15 0 50 100 150 Pore Diameter (Å) 200 250 Figure 3.5: Pore size distribution curve of rod-like SBA-15 The FESEM images shown in the Figure 3.6 also confirm that rod-like SBA-15 possesses discrete rod-like morphology with uniform length (1-2 µm) while pure SBA29 Chapter 3 Synthesis of Mesoporous Silicate 15 has a fibrous macrostructure with the length extending to 20 µm by stacking and coupling of rod-like SBA-15 (Yu et al., 2002; Fan et al., 2003). Pure SBA-15 Rod-like SBA-15 Figure 3.6: FESEM images of Pure SBA-15 and Rod-like SBA-15 3.4 Conclusions The synthesized mesoporous silicates pure SBA-15 and SBA-15 with rod-like morphology were found to possess highly ordered structure consistent with the published results in the literature. The less ordered pore structure of thiol-functionalized SBA-15 was attributed to the presence of 5 mol % MPTMS in the initial synthesis mixture. Rodlike SBA-15 possesses smaller particle size compared to pure SBA-15 and SH-SBA-15. The smaller particle size of rod-like SBA-15 is used in our study as reported in Chapter 5 to investigate the effect of particle size on the activity of immobilized α-chymotrypsin in acetonitrile or tetrahydrofuran. 30 Chapter 4 Activity Studies in Aqueous and Organic Media Chapter 4 Immobilization of α-chymotrypsin into Mesoporous Silicate and its Activity in Aqueous or Organic Media 4.1 Introduction Biocatalysis in nonaqueous media has grown rapidly since 1980s. The use of organic solvent is not recommended for synthesis due to environmental concern; however, it serves as the best choice compared to the reaction performed in water due to the increased solubility of substrate in organic solvents (Klibanov, 2001). Lyophilized enzymes in organic media possess very low activity, which hinders its potential application (Michels et al., 1997). Diffusional limitation experienced by lyophilized enzyme powder is suggested as one of the reason for low activity of enzymes in organic media (Rees and Halling, 2001). However, these limitations can be overcome by immobilizing enzymes onto solid support, which spreads the enzyme over large surface area and thereby increases the active site accessibility. Mesoporous silicate such as SBA-15 has emerged to be an excellent carrier for immobilization of enzymes due to its high specific surface area, tunable large pore size and narrow pore size distribution. Although there are several studies on the immobilization of enzymes in mesoporous silicate and its activity studies in aqueous media (discussed in section 2.3), to the best of our knowledge there are only few reports (Takahashi et al., 2000; Deere et al., 2003; Goradia et al., 2006) which have evaluated the activity of enzymes in organic media upon immobilization in mesoporous silicate. 31 Chapter 4 Activity Studies in Aqueous and Organic Media Enzyme hydration is one of the critical parameter affecting the activity of enzymes in organic media to about 100 fold (Klibanov, 1997), Adlercretuz (1991) had shown that the true nature of the support can only be revealed upon carrying out the reaction at constant thermodynamic water activity (aw). Since water adsorption depends on the nature of the support, if one uses uncontrolled support (not at constant aw) in organic media, the support may adsorb the water available for the enzyme and thereby changes the enzyme hydration, which in turn can affect reaction rate and led to misleading conclusion about the effect of support. Therefore, the reaction needs to be carried out at constant thermodynamic water activity when comparing immobilized enzymes in organic media. This chapter describes the immobilization of α-chymotrypsin onto unfunctionalized SBA-15 (pure SBA-15) and thiol functionalized SBA-15 (SH-SBA-15). The activities of α-chymotrypsin immobilized in pure SBA-15 and SH-SBA-15 in aqueous or organic media are studied and compared with native α-chymotrypsin and αchymotrypsin immobilized in commercial silica gel. The thermodynamic water activity of the immobilized α-chymotrypsin is fixed to 0.22 by rinsing it with 1-propanol containing 1.60 % water (Patridge et al., 1998b). As the reaction is carried out at constant thermodynamic water activity, the experimental results should reflect the true nature of support. 32 Chapter 4 Activity Studies in Aqueous and Organic Media 4.2 Materials and Methods 4.2.1 Materials α-chymotrypsin (EC 3.4.21.1) from bovine pancreas, N-benzoyl-L-tyrosine ethyl ester (BTEE), N-acetyl-L-phenylalanine ethyl ester (APEE), N-acetyl-L-phenylalanine and commercial silica gel were all purchased from Sigma. Tris(hydroxymethyl) aminomethane was purchased from Aldrich. 1-propanol was purchased from Merck. All the organic solvents used in this study were dried overnight by 3 Å molecular sieves before use. The mesoporous silicates used in this work were prepared as described in Chapter 3. 4.2.2 Model Reaction for Activity Studies in Aqueous and Organic Media Hydrolysis of 2 mM BTEE in 0.01 M tris buffer (pH = 7.5) containing 0.1 M NaCl, 1 mM CaCl2 and 30 % v/v ethanol was chosen as a model reaction for studying the enzyme activity in aqueous media. The reaction scheme for the hydrolysis of Nbenzoyl-L-tyrosine ethyl ester by native and immobilized α-chymotrypsin is given in figure 4.1. Tris buffer pH 7.5 with 0.1M NaCl and 1mM CaCl2 O HO H NH O O N-Benzoyl-L-tyrosine ethyl ester 30 wt % v/v Ethanol, 23°C O HO H NH OH + CH3-CH2-OH O N-Benzoyl-L-tyrosine Ethanol Figure 4.1 Reaction scheme for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester 33 Chapter 4 Activity Studies in Aqueous and Organic Media The hydrolysis reaction was followed by an auto titrator Titralab TIM 854 from Radiometer Analytical, France at room temperature equipped with magnetic stirrer (500rpm). The activity was calculated by measuring the volume of 0.02 M sodium hydroxide required for maintaining the pH of the reaction mixture at 7.5 for about 10 minutes is calculated using the formula given below. (Amount. of NaOH added per minute x Normality of NaOH x 1000) Specific Activity (Units/mg of enzyme) = ----------------------------------------------------mg of enzyme Transesterification of APEE with 1 M 1-propanol in dry octane, tetrahydrofuran or acetonitrile at the thermodynamic water activity of 0.22 was chosen as the model reaction for finding the activity of immobilized α-chymotrypsin in organic media. The reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester by immobilized α-chymotrypsin is given in figure 4.2. O H N H O + O CH3-CH2-CH2-OH 1-propanol N-acetyl-L-phenylalanine ethyl ester Water as required, 23°C Acetonitrile or Octane or Tetrahydrofuran O H N H O O N-acetyl-L-phenylalanine propyl ester + CH3-CH2-OH Ethanol Figure 4.2 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester 34 Chapter 4 Activity Studies in Aqueous and Organic Media The reaction was carried out in 5 ml scale at room temperature (23○C). At first, APEE was dissolved in dry 1-propanol and then mixed with dry octane, acetonitrile or tetrahydrofuran at the thermodynamic water activity of 0.22, followed by the addition of immobilized α-chymotrypsin. The resulting suspension was shaken at 260rpm using water bath shaker. At regular interval, 100 µl of the sample was withdrawn and the rate of formation of propyl ester (product) was analyzed by Shimazdu HPLC system equipped with Inertsil ODS-3 column and UV-detector operated at the wavelength of 254 nm. The column was eluted isocratically with mobile phase composition of 55 % water containing 0.1 % v/v trifluoroacetic acid and 45 % acetonitrile at the flow rate of 1 ml min−1. All the experiments were repeated at least twice and the error was found to be less than 15 %. The activity was calculated on the basis of the formation of propyl ester by assuming that the extinction coefficients of the product and substrate (APEE) were equal (Wang et al., 2001) using the formula given below. Specific Activity (µmole min-1.mg of enzyme-1) µmole of product formed per minute = -------------------------------------------------------mg of enzyme 4.2.3 Enzyme Immobilization for Activity Studies in Aqueous or Organic Media Enzyme immobilization for activity studies in aqueous media was carried out as follows. At first, α-chymotrypsin (4 mg ml-1) was dissolved in 25 mM tris buffer (pH = 7.8) containing 10 mM CaCl2. Then 25 mg of support was added to 1 ml of chymotrypsin solution and the mixture was shaken at 260 rpm at room temperature (23○C) for about 4 hours. The amount of α-chymotrypsin loaded onto the support was obtained by measuring the concentration of supernatant collected by centrifugation. The protein concentration was determined using Bradford assay (Bradford, 1976). The solid collected by centrifugation was dried overnight in vacuum dryer at room temperature. 35 Chapter 4 Activity Studies in Aqueous and Organic Media Enzyme immobilization for activity studies in organic media was carried out according to the procedure reported in the literature (Patridge et al., 1998b). Figure 4.3 shows the schematic representation of the preparation of immobilized α-chymotrypsin for activity studies in dry octane, tetrahydrofuran or acetonitrile at thermodynamic water activity of 0.22. Thermodynamic water activity was fixed to 0.22 in 1-propanol, acetonitrile and tetrahydrofuran by adding 1.60, 1.00 and 0.45 % water into dry 1propanol, acetonitrile and tetrahydrofuran (Halling, 2002). At first, α-chymotrypsin (4 mg ml-1) was dissolved in 25 mM tris buffer (pH = 7.8) containing 10 mM CaCl2. Then 25 mg of support was added to 1 ml of α-chymotrypsin solution and the mixture was shaken at 260 rpm at room temperature (23○C) for about 4 hours (Step 1). The amount of α-chymotrypsin loaded onto the support was obtained by measuring the concentration of supernatant collected by centrifugation. The protein concentration was determined using Bradford assay (Bradford, 1976) (Step 2). The solid collected by centrifugation was rinsed with 1-propanol containing 1.60 % water which led α-chymotrypsin immobilized onto support (either pure SBA-15 or SH-SBA-15 or commercial silica gel) with thermodynamic water activity of 0.22 which of paramount importance for determining the true nature of support (step 3). Then it is rinsed with solvent used in the reaction mixture and assayed without further delay (Step 4). 36 Chapter 4 Activity Studies in Aqueous and Organic Media Step 1 Mixing 1 ml of α-chymotrypsin (4 mg ml-1) with 25 mg of support and shaken for 4 hours Step 2 Centrifuge to separate immobilized α-chymotrypsin Step 3 Rinsing immobilized α-chymotrypsin with 1-propanol containing 1.60 % water Step 4 Addition of Immobilized α-chymotrypsin into 2.5 mM N-acetyl-L- phenylalanine ethyl ester and 1M 1-propanol solution mixed in dry Octane or Tetrahydrofuran containing 0.45 % water or Acetonitrile containing 1.00 % water Figure 4.3: Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in dry octane, acetonitrile or tetrahydrofuran at thermodynamic water activity of 0.22 The first three steps for the preparation of immobilized α-chymotrypsin for activity studies in organic media were same regardless of the solvent used in the reaction mixture. For example, to find the activity of immobilized α- chymotrypsin in dry octane, after step 3, the immobilized α-chymotrypsin was rinsed with dry octane and quickly transferred to dry octane which contains substrates APEE and 1-propanol (step 4). Then the initial rate of transesterification was followed by HPLC described in section 4.1. 4.2.4 Thermal Stability and Leaching Studies in Aqueous Media The thermal stability of both native and immobilized α-chymotrypsin was determined by placing the free enzyme (native α-chymotrypsin) and immobilized 37 Chapter 4 Activity Studies in Aqueous and Organic Media enzyme (immobilized α-chymotrypsin in either pure SBA-15 or SH-SBA-15) in tris buffer (pH 7.8, containing 10 mM CaCl2) in a water bath maintained at (70○C) under static condition. Samples were removed at the regular interval and allowed to cool down to room temperature and the residual hydrolytic activity was determined as described above. The initial activity of native and immobilized enzymes before subjected to thermal exposure was taken as 100 %. To study the leaching of α-chymotrypsin from the support, about 100 mg of immobilized enzyme was suspended in a tris buffer (pH 7.8, containing 10 mM CaCl2) solution and placed in a water bath shaker shaken at 260 rpm at room temperature. The amount of enzyme leaching from the support was determined by removing 1 ml of solution and the α-chymotrypsin concentration in the solution was determined by Bradford assay (Bradford, 1976). 4.3 Results and Discussion 4.3.1 Immobilization of α-chymotrypsin into Mesoporous Silicate and Commercial silica Gel α-chymotrypsin is a class of serine protease, which hydrolyzes ester bonds in addition to peptide bonds. Its molecular dimension and isoelectric point are 50 Å × 40 Å × 40 Å and 8.1 respectively (Zoungrana et al., 1997). Since the pore size of all the support (given in Chapter 3) utilized in this study is higher than the molecular dimension of αchymotrypsin, it is expected that the enzyme can easily diffuse into the pores. It is well known that the enzymes in organic media shows pH memory i.e. the activity of enzymes in organic media depends on the pH to which it is finally exposed and it is found to be maximum when it is brought from the optimum pH for catalytic activity in aqueous media (Zaks and Klibanov, 1998b). Since the optimum pH for the catalytic activity of α38 Chapter 4 Activity Studies in Aqueous and Organic Media chymotrypsin has been found to be 7.8 (Patridge et al., 1998b), hence this pH has been selected in this study for the immobilization of α-chymotrypsin into the mesoporous silicate SBA-15. Several studies (Barros et al., 1998; Persson et al., 2000; Persson et al., 2002a) have shown that the activity of immobilized enzyme depends on the amount of enzyme loaded onto the support; hence, the α-chymotrypsin loading in all the support (pure SBA15, SH-SBA-15 and commercial silica gel) is fixed to 8 % only. Because the commercial silica gel has lower surface area (470 m2 g-1) than mesoporous silicate (660-790 m2 g-1), hence it is expected that commercial silica gel has a lower enzyme loading capacity compared to mesoporous silicate. Patridge et al., (1998b) used only 8 wt % loading of chymotrypsin in silica gel and found that the immobilized α- chymotrypsin showed better activity compared to the lyophilized enzyme powder. Since the silica gel used in Patridge et al. (1998b) work has surface area, pore volume and pore size more close to the commercial silica gel used in our study, hence only 8 wt % enzyme loading is used our study for comparison purpose. 4.3.2 Activity of Native and Immobilized α-chymotrypsin in Aqueous Media Upon immobilization in pure SBA-15 and SH-SBA-15, α-chymotrypsin retains only about 3.35 and 3.44 % of its original activity at 23°C, indicating that there were significant changes in α-chymotrypsin conformation upon adsorption in the mesopores of SBA-15. This is not surprising as the enzyme activity depends on its three dimensional conformation and perturbation in the three-dimensional conformation often leads to changes in enzyme activity (Clark, 1993). Upon adsorption, the catalytic triad where the substrate molecule will bind may orient in a different conformation, there by limiting the 39 Chapter 4 Activity Studies in Aqueous and Organic Media substrate binding and decreasing the enzyme activity (Clark and Bailey, 2002). The decrease in the activity of α-chymotrypsin upon immobilization is not only attributed to the changes in the active site conformation but also due to diffusional limitation often encountered in immobilized enzyme. The decrease in activity of α-chymotrypsin upon immobilization is also observed in other immobilization carriers such as Ludox silica and Teflon (Zoungrana et al., 1997) and recently single walled carbon nanotube (Karajanagi et al., 2004). Lee et al. (2005) have also shown that the Michaelis constant (Km) was increased to about 7 fold and turnover number (kcat) declined to about 37 fold upon immobilization of α-chymotrypsin into hierarchically ordered mesocellular mesoporous silica materials (HMMS) relative to the native α-chymotrypsin. All these observation support our conclusion that both conformational changes and mass transfer limitation are the main factors responsible for the decrease of activity of α-chymotrypsin upon immobilization in mesoporous silicate. 4.3.3 Thermal Stability of Native and Immobilized α-chymotrypsin in Aqueous Media The slight increase of temperature in an enzymatic reaction generally leads to the increase in reaction rate, at relatively high temperature (> 55○C), most of the enzyme shows very low thermal stability, which hinders its potential application. This has prompted us to study the thermal stability of α-chymotrypsin immobilized in mesoporous silicate at 70○C. It can be seen from Figure 4.4 that α-chymotrypsin immobilized in pure SBA-15 and SH-SBA-15 losses only about 40 % of the original activity after exposing it to 70○C for about 3 hours while the native α-chymotrypsin losses all its activity ~ 98 % within 20 minutes. This increased thermal stability of α-chymotrypsin upon immobilization may be due to the effect of excluded volume and strong hydration of 40 Chapter 4 Activity Studies in Aqueous and Organic Media protein upon immobilization in the narrow pore channel, which stabilizes the enzyme against unfolding and thereby increases thermal stability (Ravindra et al., 2004). Residual Activity (%) 120 SH-SBA-15 Pure-SBA-15 Native α-chymotrypsin 100 80 60 40 20 0 0 30 60 90 120 Time in Minutes 150 180 210 Figure 4.4: Thermal stability of native and immobilized α-chymotrypsin in aqueous media 4.3.4 Leaching of α-chymotrypsin form Pure SBA-15 and Thiol Functionalized SBA-15 Figure 4.5 shows the leaching of α-chymotrypsin immobilized in pure SBA-15 and SHSBA-15 at 23°C. α-chymotrypsin immobilized in mesoporous silicate shows only about 18 % of leaching from the support even after 12 hours. Adsorption of protein onto the support is believed to be stabilized by electrostatic interaction. The isoelectric point of αchymotrypsin and SBA-15 has been reported to be 8.1 and 2.0, respectively (Zhao et al., 1998; Zoungrana et al., 1997). At pH value of 7.8, which is the pH value used in the leaching studies, α-chymotrypsin is slightly positively charged and silica surface is negatively charged. Hence, the lower leaching of α-chymotrypsin from the support can be attributed to the strong electrostatic interaction between α-chymotrypsin and mesoporous silicate. 41 % of Chymotrypsin in Support Chapter 4 Activity Studies in Aqueous and Organic Media 105 100 SH-SBA-15 95 Pure-SBA-15 90 85 80 75 70 0 120 240 360 480 600 720 Time in Minutes Figure 4.5: Leaching of α-chymotrypsin from pure and thiol functionalized SBA-15 4.3.5 Activity of Immobilized α-chymotrypsin in Organic Media Table 4.1 shows the activities of α-chymotrypsin immobilized in pure SBA-15, SHSBA-15 and commercial silica gel in dry octane and acetonitrile or tetrahydrofuran with thermodynamic water activity of 0.22 at 23°C. In the all the media taken in this study, αchymotrypsin immobilized in mesoporous silicate (pure SBA-15 and SH-SBA-15) shows higher activity compared to α-chymotrypsin immobilized in commercial silica gel. Table 4.1: Activity of immobilized α-chymotrypsin in dry octane, tetrahydrofuran or acetonitrile at thermodynamic water activity of 0.22 Supports SH-SBA-15 Pure SBA-15 Commercial Silica gel Activity (nanomole min-1 mg of enzyme-1) Octane Tetrahydrofuran Acetonitrile 212 9.5 2.75 89 6.5 2.0 75 3.8 0.6 The reason for the higher activity of α-chymotrypsin upon immobilization is mesoporous silicate (pure SBA-15 and SH-SBA-15) and the difference in activity between the solvents is rationalized as follows. 42 Chapter 4 Activity Studies in Aqueous and Organic Media Factors that can affect the activity of immobilized enzyme in organic media is suggested to be related to enzyme hydration, substrate solvation and mass transfer limitation. Since our reaction was carried out at constant thermodynamic water activity of 0.22, the difference in activity due to enzyme hydration/dehydration (water stripping in polar solvents) was eliminated (Halling, 2002). The difference in activity between the solvents is suggested to be attributed to the difference in the solubility of substrate (APEE) in the solvents employed. Because more polar solvents, such as acetonitrile and tetrahydrofuran has better solvation for the substrate (APEE) compared to octane, hence APEE is less available to the immobilized enzyme in polar solvents, resulting in lower activity of the immobilized enzyme in polar solvents than in octane (Schmitke et al., 1996; Partridge et al., 1998a). The higher activity observed in nonpolar solvents compared to polar solvents is also in agreement with the results reported for immobilized α-chymotrypsin (Suzawa et al., 1995). The higher activity of α-chymotrypsin upon immobilization in mesoporous silicate compared to commercial silica gel can be explained based on mass transfer limitation, which commonly occurs in porous supports. The distance that has to be traveled by substrate and product inside the pore depends on the particle size of the support. In the case of support with larger particle size, the substrate and product have to travel longer distance, thereby limiting the activity of the immobilized enzyme by internal mass transfer (Barros et al., 1998). Thus the higher activity of α-chymotrypsin immobilized in mesoporous silicate is due to the smaller particle size of mesoporous silicate, which results in reduced internal mass transfer and thereby led to higher activity. The increase in reaction rate upon decrease in particle size of the support is also observed in the immobilization of lipase in controlled pore glass (Bosley and Clayton, 1994). The 43 Chapter 4 Activity Studies in Aqueous and Organic Media slightly higher activity of α-chymotrypsin immobilized in SH-SBA-15 compared to pure SBA-15 may be due to the difference in the mode of binding of α-chymotrypsin in SHSBA-15 compared to pure SBA-15. 4.4 Conclusions The activity of α-chymotrypsin in aqueous and organic media has been investigated upon immobilization in pure and SH-SBA-15. The activity of α-chymotrypsin in aqueous media decreases upon immobilization, but the immobilized enzyme shows better thermal stability, which is believed to be of practical importance for relatively high temperature applications. Immobilized α-chymotrypsin in mesoporous silicate also shows less leaching from the support. α-chymotrypsin immobilized in SBA-15 also shows higher activity in organic media compared to enzyme immobilized in commercial silica gel, possibly attributed to the smaller particle size of SBA-15, which reduces the internal mass transfer limitation. However, α-chymotrypsin immobilized in SH-SBA-15 shows only minor increase in activity as compared to pure SBA-15. 44 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity 5.1 Introduction This chapter describes our results on the effect of enzyme loading and thermodynamic water activity on the activities of α-chymotrypsin immobilized in pure SBA-15, SBA-15 with rod-like morphology (rod like SBA-15), and commercial silica gel. The reaction media taken for this study were acetonitrile or tetrahydrofuran. SBA-15 with rod like morphology which has the surface area, pore diameter and pore volume closer to pure SBA-15 but differ only in the particle size (given in Chapter 3) has been used in this study in order to observe the effect of particle size on the reaction rate. The use of rodlike SBA-15 in this study is also to re-confirm our conclusion observed in Chapter 4 that the higher activity of α-chymotrypsin immobilized in mesoporous silicate was due to the reduced internal mass transfer limitation because of the smaller particle size of mesoporous silicate (pure SBA-15 and thiol functionalized SBA-15) compared to that of commercial silica gel. At first, the effect of α-chymotrypsin loading on the activities in either acetonitrile or tetrahydrofuran was studied. The combination of support and loading of enzyme that showed the highest activity was chosen to study the effect of thermodynamic water activity on the reaction rate of immobilized α-chymotrypsin in either acetonitrile or tetrahydrofuran. 45 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity 5.2 Materials and Methods 5.2.1 Materials N-acetyl-L-phenylalaine ethyl ester (APEE), N-acetyl-L-phenylalaine, α-chymotrypsin from bovine pancreas, commercial silica gel was purchased from Sigma. 1-propanol was purchased from Merck. Acetonitrile and tetrahydrofuran were purchased from Tedia. All the organic solvents used in this work were dried overnight using 3 Å molecular sieve before use. Pure SBA-15 and rod-like SBA-15 were prepared as described in the Chapter 3. 5.2.2 Activity of Immobilized α-chymotrypsin in Acetonitrile and Tetrahydrofuran The activities of the immobilized α-chymotrypsin were measured in acetonitrile or tetrahydrofuran through the initial rate of transesterification of 2.5 mM APEE with 1 M 1-propanol. The reaction was carried out in 5 ml scale at room temperature (23○C). At first, APEE was dissolved in dry 1-propanol and then mixed with acetonitrile or tetrahydrofuran containing water (the water content was varied according to the thermodynamic water activity in the reaction mixture as shown in Table 5.1), followed by the addition of immobilized α-chymotrypsin. The resulting suspension was shaken at 260 rpm using water bath shaker. At regular interval, 100 µl of the sample was withdrawn and the rate of formation of N-acetyl-L-phenylalanine propyl ester (product) was analyzed by Shimazdu HPLC system equipped with Inertsil ODS-3 column and UVdetector operated at the wavelength of 254 nm. The column was eluted isocratically with mobile phase composition of 55 % water containing 0.1 % v/v trifluoroacetic acid and 45 % acetonitrile at the flow rate of 1 ml min-1. All the experiments were repeated at least twice and the error was found to be less than 15 %. The activity was calculated on 46 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity the basis of the formation of propyl ester by assuming that the extinction coefficients of the product and substrate (APEE) were equal (Wang et al., 2001) which is given below. µmole of product formed per minute = -------------------------------------------------------mg of enzyme Specific Activity (µmole min-1.mg of enzyme-1) The reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester by immobilized α-chymotrypsin is given in figure 5.1. O HN H O + CH3-CH2-CH2-OH O 1-propanol N-acetyl-L-phenylalanine ethyl ester Acetonitrile or Tetrahydrofuran Water as required, 23°C O HN H O O N-acetyl-L-phenylalanine propyl ester + CH3-CH2-OH Ethanol Figure 5.1 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester in acetonitrile and tetrahydrofuran 5.2.3 Immobilization of α-chymotrypsin The immobilization of α-chymotrypsin onto pure SBA-15, SBA-15 with rod-like morphology and commercial silica gel was carried out as follows. At first, αchymotrypsin (4 to 9 mg ml-1) was dissolved in 25 mM tris buffer (pH = 7.8) containing 47 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity 10 mM CaCl2. Then 15 mg of support was mixed with 1 ml of α- chymotrypsin solution and shaken at 260 rpm using water bath shaker (23○C) for about 4 hours and then centrifuged to collect the immobilized α-chymotrypsin. The loading amount of αchymotrypsin into the support was obtained by measuring the concentration of supernatant collected by centrifugation. The α-chymotrypsin concentration in the supernatant was determined using Bradford assay (Bradford, 1976). The solid collected by centrifugation was rinsed with 1-propanol containing water (the water content was varied according to the thermodynamic water activity in the reaction mixture as shown in Table 5.1) for about 3 times, finally rinsed with the solvent used in the reaction mixture (acetonitrile or tetrahydrofuran) and assayed without further delay. The α-chymotrypsin loading into the support was controlled by varying the enzyme concentration from 4 to 9 mg ml-1 and the amount of support was fixed to 15 mg. Table 5.1: Water content required to attain selected water activities in 1-propanol, acetonitrile and tetrahydrofuran (Halling, 2002) Water Activity (aw) 0.22 0.44 0.55 0.76 1-propanol 1.60 3.40 4.70 9.80 Water Content v/v % Acetonitrile Tetrahydrofuran 1.00 0.45 2.70 1.20 4.50 1.60 13.00 3.60 Figure 5.2 shows schematic representation of the procedure adopted to immobilize α-chymotrypsin for carrying out the activity studies in either acetonitrile or tetrahydrofuran as the reaction media. For example, to determine the activity of immobilized α-chymotrypsin at the enzyme loading of 8 % in acetonitrile at the thermodynamic water activity of 0.22, the procedure adopted was follows. 1 ml of αchymotrypsin solution (4 mg ml-1) was mixed with 15 mg of support and shaken at room temperature for about 4 hours and then centrifuged. The supernatant was removed 48 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity carefully and then the solid was rinsed with 1-propanol containing 1.60 % water (aw = 0.22). Then the immobilized α- chymotrypsin was transferred to acetonitrile solution containing 1.00 v/v % water (aw = 0.22) and substrates. The product formation was determined by HPLC as described in the above paragraph. The α-chymotrypsin loading onto the support was varied by changing the solution concentration in step 1. The water content in 1-propanol at step 3 as well as in reaction medium (acetonitrile and tetrahydrofuran) at step 4 was varied according to the thermodynamic water activity required in the reaction mixture. The amount of water content to be added into the dry organic solvent to achieve the required thermodynamic water activity is given in Table 5.1. Step 1 Mixing 1 ml of α-chymotrypsina with 15 mg of support and shaken for 4 hours Step 2 Centrifuge to separate immobilized α-chymotrypsin Step 3 Rinsing Immobilized α-chymotrypsin with 1-propanol containing waterb Step 4 Addition of Immobilized α-chymotrypsin into 2.5 mM N-acetyl-L-phenylalanine ethyl ester and 1M 1-propanol solution mixed in tetrahydrofuran or acetonitrile containing waterb a b Concentration varied from 4 to 9 mg ml-1 to vary the enzyme loading Water content varied according to thermodynamic water activity required Figure 5.2: Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in either acetonitrile or tetrahydrofuran 49 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity 5.3 Results and Discussion 5.3.1 Effect of Enzyme Loading on Activity in Acetonitrile and Tetrahydrofuran Figure 5.3 and 5.4 show the effect of α-chymotrypsin loading on the activities in acetonitrile or tetrahydrofuran with thermodynamic water activity of 0.22 at 23°C. Since the reaction is carried out at constant thermodynamic water (aw) of 0.22, the difference in activity between the supports cannot be attributed to the enzyme hydration, but due to the intrinsic nature of the support. It is seen that the activity of immobilized α-chymotrypsin increases with increase of enzyme loading up to the certain limit (20 wt % in mesoporous silicate and 13 wt % in commercial silica gel) and then decreases irrespective of nature of the support, which indicates that this phenomenon is common for all the support Activity (nanomole/min.mg of enzyme) utilized in this study. 30 Tetrahydrofuran 25 Rod-like SBA-15 Pure SBA-15 Rod-like SBA-15 Pure-SBA-15 Acetonitrile 20 15 10 5 0 50 100 150 200 250 300 Enzyme Loading (mg/g of support) Figure 5.3: Effect of enzyme loading on the activity of α-chymotrypsin immobilized in pure SBA-15 and rod-like SBA-15 in either acetonitrile or tetrahydrofuran at aw of 0.22 50 Activity (nanomole/min.mg of enzyme) Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity Acetonitrile 10 Tetrahydrofuran 8 6 4 2 0 70 80 90 100 110 120 130 140 150 160 170 Enzyme Loading (mg/g of support) Figure 5.4: Effect of enzyme loading on the activity of α-chymotrypsin immobilized in commercial silica gel in either acetonitrile or tetrahydrofuran at aw of 0.22 Figure 5.5 shows the schematic representation of the effect of enzyme loading on changes in the microenvironment of the immobilized enzyme. At low enzyme loading, the enzyme molecule has a higher tendency to unfold/spread on the support surface which results in lowering the catalytic activity. As the loading increases, enzyme unfolding at the support surface gets restricted by adjacent enzyme molecules which lad to increases in the catalytic activity of immobilized enzyme. However, as the loading increases further, the problem of mass transfer limitation arise which causes the drop in the catalytic activity of immobilized enzyme. Hence, increase in the activity of immobilized αchymotrypsin upon increase in enzyme loading in all the support can be attributed to the suppression of enzyme unfolding at the support surface by enzyme-enzyme interaction. However, the decrease in activity at higher loading can be attributed to mass transfer limitation which is quite common on porous support. 51 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity Solid Support Low Enzyme Loading Pore Opening Increasing Enzyme Loading High Enzyme Loading Enzyme Molecule Figure 5.5: Schematic representation of the effect of enzyme loading in porous supports It is seen from Figure 5.3 that mesoporous silicate not only possess high enzyme loading capacity but also shows high enzyme activity compared to commercial silica gel (Figure 5.4). At loading amount we tested, α-chymotrypsin immobilized in mesoporous silicate shows higher catalytic activity compared to commercial silica gel which can be attributed to the smaller particle size of mesoporous silicate compared to commercial silica gel which reduces the internal mass transfer limitation and thereby increases the activity. The enzyme loading at which α-chymotrypsin immobilized in mesoporous silicate shows higher catalytic activity is around 20 wt %, which is higher compared to commercial silica gel in which the maximum activity can be attained at only 13 wt % enzyme loading. The higher loading capacity of mesoporous silicate compared to commercial silica gel can be attributed to higher surface area and pore volume of mesoporous silicate compared to commercial silica gel (given in Chapter 3). The increase in enzyme activity with an increase in enzyme loading is also observed in many 52 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity studies (Zoungrana et al., 1997, Barros et al., 1998 and Persson et al., 2000). It is interesting to note that the enzyme immobilized in rod-like SBA-15 shows higher activity compared to pure SBA-15 which has the same physico-chemical properties with an exception to particle size. The higher activity of α-chymotrypsin immobilized in rodlike SBA-15 can be attributed to the smaller particle size (2 µm) compared to pure-SBA15 (22 µm) which greatly reduces the internal mass transfer limitation and leads to higher activity. These results also support our conclusion in Chapter 4 that the internal mass transfer limitation was responsible for increased activity of α-chymotrypsin upon immobilization in mesoporous silicate compared to commercial silica gel. 5.3.2 Effect of Thermodynamic Water Activity Figure 5.6 shows the effect of thermodynamic water activity on the reaction rate either in acetonitrile or tetrahydrofuran as the reaction medium at 23°C. Rod-like SBA-15 with an enzyme loading of 20 wt % has been selected for determining the effect of thermodynamic water activity due to its higher activity compared to other supports and Activity (nanomole/min.mg of enzyme) enzyme loading. Acetonitrile 60 Tetrahydrofuran 50 40 30 20 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Thermodynamic Water Activity (a w) Figure 5.6: Effect of thermodynamic water activity on the activity of α-chymotrypsin immobilized in rod-like SBA-15 in either acetonitrile or tetrahydrofuran 53 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity The activity increases with increasing thermodynamic water activity up to about 0.55 and then decreases upon further increase in thermodynamic water activity. There are a number of possible explanations on this effect of thermodynamic water activity on the reaction rate. One possible reason is that, enzyme hydration which can greatly affect the flexibility of the enzyme molecule necessary for the catalysis. At lower water activity, there will be less number of water molecules present on the enzyme surface which reduces the enzyme flexibility and thereby the activity. However, as the thermodynamic water activity increases, the number of water molecules on the enzyme surface increases, thus increasing the flexibility and thereby the activity (Partridge et al., 1998a). However, enzyme activity decreases above the thermodynamic water activity of 0.55 may be due to the competitive inhibition by water molecule. At higher thermodynamic water activity, the water content in the system is higher which can compete well with the propanol, which results in the favor of hydrolysis instead of synthesis. In other words, the equilibrium has been shifted thermodynamically to hydrolysis instead of synthesis. The other possible reason may be the conformational changes. It should be noted that the enzyme denatures in aqueous-organic mixture but not in pure organic solvents due to the availability of molecular lubricant (water) in the former which can aid in the unfolding of enzyme. Hence the higher water content at higher thermodynamic water activity can also aid in the unfolding of enzyme which will results in diminishing the activity. The higher activity of α-chymotrypsin immobilized in mesoporous silicate in tetrahydrofuran compared to acetonitrile can be attributed to the difference in substrate solvation (Schmitke et al., 1996). This is because acetonitrile has higher solvation capability compared to tetrahydrofuran, thus affecting the substrate binding and reducing the activity of the immobilized enzyme. 54 Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity 5.4 Conclusions α-chymotrypsin immobilized in rod-like SBA-15 shows higher activity compared to pure-SBA-15 and commercial silica gel, which can be attributed to the smaller particle size, which reduces the internal mass transfer limitation and increases the activity. The activity was found to be increased with increase in thermodynamic water activity due to increased flexibility of the enzyme and the optimum thermodynamic water activity was found to be 0.55 in both tetrahydrofuran and acetonitrile. 55 Chapter 6 Conclusions and Future Research Chapter 6 Conclusions and Future Research 6.1 Conclusions Mesoporous silicate was found to be an excellent carrier for the immobilization of enzymes for application in aqueous and organic media. The activity of α-chymotrypsin decreased upon immobilization as compared to the native α-chymotrypsin possibly due to the active site orientation and diffusional limitation. However, thermal stability studies carried out at 70○C in aqueous buffer showed that the immobilized α-chymotrypsin losses only 40 % of original activity after 3 hours of exposure but the native αchymotrypsin losses almost all of its activity within 20 minutes. Interestingly, the immobilized α-chymotrypsin also showed low leaching from the support in aqueous buffer due to strong electrostatic interaction between positively charged enzyme and negatively charged support. The combination of enhanced thermal stability and low leaching of α- chymotrypsin immobilized in mesoporous silicate may find application in industry even though the immobilized enzyme possess low activity. α-chymotrypsin immobilized in mesoporous silicate (pure SBA-15 and SH-SBA15) possess higher activity in octane, tetrahydrofuran and acetonitrile compared to αchymotrypsin immobilized in commercial silica gel, possibly due to the smaller particle size of mesoporous silicate compared to commercial silica gel which reduces the internal 56 Chapter 6 Conclusions and Future Research mass transfer limitation and thereby increases the activity. Since the reaction was carried out at constant thermodynamic water activity of 0.22, the difference in activity between the three supports cannot be attributed to the difference in enzyme hydration. The higher activity of α-chymotrypsin immobilized in thiol functionalized SBA-15 may be due to the difference in the mode of binding compared to pure SBA-15. The study on the effect of α-chymotrypsin loading on the activity of immobilized enzyme in pure SBA-15, rod-like SBA-15 and commercial silica gel shows that the activity increases with increase in α-chymotrypsin loading up to certain level and then decreases. The enzyme loading at which the catalytic activity maximum was found to be around 20 wt % in rod-SBA-15 and pure SBA-15 which can be attributed to the high pore volume and surface area of mesoporous silicate compared to commercial silica gel. The activity of α-chymotrypsin immobilized in rod-like SBA-15 in acetonitrile and tetrahydrofuran was found to be higher compared to other support which can be attributed to the reduced internal mass transfer limitation in rod-like SBA-15 due to its smaller particle size (2 µm) compared to pure SBA-15 (22 µm) and commercial silica gel (64 µm). The study on the effect of thermodynamic water activity on the reaction rate of α-chymotrypsin immobilized in rod-like SBA-15 shows that the activity increases with increase in thermodynamic water activity and optimum attained up to 0.55 in acetonitrile and tetrahydrofuran and the catalytic activity of the immobilized α-chymotrypsin reached a maximum before it decreased upon further increase in thermodynamic water activity. 57 Chapter 6 Conclusions and Future Research 6.2 Future Research The future work on this study involves the effect of various functional groups on the activity of α-chymotrypsin in aqueous and organic media. Since, the enzyme loading can be influenced by solution pH at the time of immobilization, the effect of functional group together with initial solution pH and enzyme loading can give more information on the interaction between mesoporous silicate and α-chymotrypsin. The study on the effect of thermodynamic water activity on the reaction rate of α-chymotrypsin immobilized in functionalized mesoporous silicate in non-polar organic solvents can provide information on the effect of functional group on the activity in nonpolar organic solvents. 58 References References Adlercretuz, P. (1991), ‘On the importance of the support material for enzymatic synthesis in organic media support effects at controlled water activity’, European Journal of Biochemistry 199, 609-614. Bacheva, A. V., Belyaeva, A. V., Lysogorskaya, E. N., Oksenoit, E. S., Lozinsky, V. I. and Filippova, I. Y. 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(1997), ‘Structure, stability and activity of adsorbed enzymes’, Journal of Colloids and Interface Science 190, 437- 448. 65 [...]... media as well as the ways to increase the activity of enzymes in organic media Enzyme immobilization is one of the methods of increasing the activity of enzymes in organic media, which is the main objective of this work; hence, it is discussed in details At first, immobilization of enzymes onto polymers for use in organic media is given followed by the enzyme immobilization in controlled pore glass,... functionalization of SBA-15 with organic moieties in the framework was also developed, attracting enzymologist to use this material as a host for the immobilization of enzymes 1.3 Research Objectives The objective of this work is to use unfunctionalized and thiol functionalized SBA-15 as a host for the immobilization of enzymes and to find out the activity of the immobilized enzymes in aqueous and organic... in the case of aqueous media, enzyme is immobilized in order to recycle the enzyme, but in the case of organic media enzyme is immobilized in order to suppress the diffusional limitation which leads to increased activity 1.2 Immobilization of Enzymes Immobilization is the process of arresting the mobility of enzyme Numerous methods are available in the literature for immobilization of enzymes and each... adsorption of enzymes on the surface and porous materials are some of the examples of non-covalent method of immobilization Selection of a particular method depends on the requirements to be met and the efficiency of the selected method depends on the nature of the enzyme and supports In this study, enzyme is adsorbed onto porous support which is segregated under non-covalent method of immobilization. .. changes and the role of salt matrix as the immobilization support suggested as the reason for the increased activity of enzyme upon addition of salt during lyophilization (Griebenow and Klibanov, 1997) However, Laszlo and Compton, (2001) showed that α-chymotrypsin lyophilized in the presence of potassium chloride possessed negligible activity in acetonitrile and they concluded that the salt activation was... the importance of mesoporous silicate a host for the immobilization of enzymes for catalysis in organic media 2.3 Enzyme Immobilization in Mesoporous Silicate for Use in Aqueous Media Application of mesoporous silicate as a host for the immobilization of enzymes commence in 1996 Initial attempt on the immobilization of enzymes onto mesoporous silicate showed that the loading efficiency of the enzyme... peroxidase as received from company The adsorption of horseradish peroxidase were found to be high when the material pore size was larger then the molecular dimension of horseradish peroxidase, FSM-16 and MCM-41 were found to possess highest enzyme loading of 183 and 147 mg g-1 respectively The activities of horseradish peroxidase immobilized in FSM-16 and MCM-41 were higher when pore size of the support... pore sizes in the range of 2- 10 Ǻ However, they are not suitable for the immobilization of enzymes due to their smaller pore sizes as the molecular dimension of enzymes are several angstroms larger than the pore size of zeolites Hence, zeolites receive less attention as supports for the immobilization of enzymes The discovery of highly ordered mesoporous silica materials (M41S) has opened the way to... protocol, 2.3 g of pluronic P123 and 2.2 g of potassium chloride was dissolved in 59.8 g of water containing 4.4 g of hydrochloric acid at 38±1○C Then 4.2 g of TEOS was added and the resulting solution was stirred vigorously for about 8 min and kept at the same temperature under static condition for 24 hours The slurry was then maintained at 100○C for 24 hours The solid was collected by filtration and dried... materials are classified by International Union of Pure and Applied Chemistry (IUPAC) into three classes such as microporous (pore size < 20 Å), mesoporous (pore size 20 - 500 Å), and macroporous materials (pore size >500 Å) (Taguchi and Schuth, 2005) Zeolite is a well known member of the microporous materials but its application as a host for immobilization of enzymes is limited due to its smaller pore ... objective of this work is to use unfunctionalized and thiol functionalized SBA-15 as a host for the immobilization of enzymes and to find out the activity of the immobilized enzymes in aqueous and. .. various reasons for the low activity of enzyme in organic media as well as the ways to increase the activity of enzymes in organic media Enzyme immobilization is one of the methods of increasing... g of pluronic P123 and 2.2 g of potassium chloride was dissolved in 59.8 g of water containing 4.4 g of hydrochloric acid at 38±1○C Then 4.2 g of TEOS was added and the resulting solution was