Luận văn β d fructofuranosidase production and application to the manufacture of frutooligosaccharides

Preface Oligosaccharides, especially fructooligosaccharides (FOS) are relativelyfood new functional ingredients that have great potential to improve the quality of many foods In addition to providing useful modifications to food favors and physiochemical characteristics, many of these sugar possess properties that ar beneficial to the heath of consumers These include non-cariogenicity, a low calorific value and the ability to stimulate the growth of beneficial bacteria in the colon Both the production and the applications of food-grade oligosaccharides are increasing rapidly Major uses are in beverages, infant milk powders, confectionery, bakery products, yoghurts and dairy desserts Research continues into the development of new oligosaccharides with a range of physiological properties and applications in the food industry FOS has been attracted attention of many researchers with its prebiotical property recently In industrial scale, immobilized fungal β-fructofuranosidase or immobilized cells are used for the manufacture of FOS To improve the yield of FOS, so many studies have been done For this reason, this report will represent some new methods for the production of β-fructofuranosidase and the combination of immobilized fungal βfructofuranosidase with innovated operations to improve the FOS obtained Introduction 1.1 β–D– (15%) β-D-fructofuranosidase (FFase, EC 3.2.1.26) is a glycoenzyme that fructofuranosidase hydrolyses β-such as sucrose, raffinose, stachyose, ( α-DD-fructofuranoside -Fructofuranosides and β D-fructopyranosides are not hydrolysed ), alsoInnamed invertase hydrolysis of sucrose into fructose and glucose addition to releasing FFase catalyses D-glucose and D- the frucose from sucrose, some microbial β-D-fructofuranosidase may catalyse the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked toFFase sucrose different bonds depend 2006) has by been found glycosidic in many different plants on andthe enzyme source (Sangeetha microorganisms FFase from different sources differs in optinum pH of activity (which may be et al., 2005) neutral, acid This or enzyme has been used in food industry to produce inverted sugar and alkaline) (Winter H, 2000), optinum temperature of activity, mostly used for the preparation of jams, candies and soft-centered 1.1.1 Catalytic chocolates C, many researches on the amino acid residues There(Aranda have been mechanism the activethat site present of FFaseat However, amino acid involved at the active site of enzyme from different source may various According to the study of Reddy and Maley (1996), the active site of FFase from Saccharomyces cerevisiae consists of imidazole, carboxylic and thiol groups Reddy and Maley also indicated that carboxylic groups from Asp-23 and Glu-204 play ansucrose important role in catalytic process The catalytic process of the FFase is divided into Nevertheless, amino steeps: -three First, FFase acid links that with sucrose to form enzyme-substrate participate in theatcatalytic complex the Glu- process of FFase from Arabidopsis thaliane’cell wall 04 by hydrogen bond (Arabidopsis thaliane is a small flowering plant native to Europe, Asia, Second, fructosyl residue on sucrose molecule combines and northwestern with Asp-23 of Africa) are Asp-23 Glu-203 FFase by and valent bond(M to Verhaest, break the2006) glycosidic bond between glucosefructose and - Finally, residue combines with free water in media and fructose After separate from that, α-glucose receives proton from Glu-204 and releases Asp-203 from enzyme 1.1.2 Soluble β active – D – site Commercial is often powdered shape and fructofuranosidase Soluble FFase mayFFase be produced from manyinsources but slight it mainly yellow in colour produced from Saccharomyces cerevisiae, As.niger, As.japonicus Soluble enzymes have a high activity but sensity to temperature, pH, During use, the activity of soluble FFase decreases due to the change in pH, temperature, conformational changes as a result of friction, amostic pressure imposed by the environs of their use Furthermore, since it is soluble, its cover from a mixture of subtrate and product for use is not economically practical Thus, the advance of immobilized enzyme technology has led to increasing efforts to replace conventional enzymatic process with the preparation as immobilization 1.1.3 Immobilized β – D – The immobilization of invertase broadens the field of its fructofuranosidase application, since it of sugar in food products and the prevents the crystallization assimilation of alcohol in fortified wines (D N Klimovskii, 1967) and provides the possibility of regulating the composition of the volatile components of wine, brandy, and aqueous liqueurs (S Kh Abdurazakova, 1978) - The effect of pH Characteristics of enzymes important for their practical In use are general, immobilized enzyme are more stable with the their dependences on the temperature substrate of pHAsthan freeeffect enzyme wepH, canthe see from Fig.1 and below that FFase was concentration immobilized to polyamide was more stable than free FFase (D T Mirzarakhmetova, 1998) The activity free FFase the maximum level at pH around 4.5-5.0 of region and reached a narrower symmetrical profile According andD fellT.down to Mirzarakhmetova, the shift of the pH optimum into the neutral quicklyisafter that On the other hand, immobilized FFase had pH region probably optimum due to ain the change in the local concentration of hydrogen ions in the microenviroment of the enzyme through the introduction of amino groups during the modification of the support The observed narrowing of the pH profile of the immobilized FFase may be a consequence of the selective binding of the more neutral forms of the enzyme with the modified support in the immobilization process Fig.1 Dependence of rate of hydrolysis of immobilized (1) and free FFase from yeast on the pH of the medium - The effect of temperature The same as the effect of temperature, immobilized enzyme are more stable with the effect of temperature than free enzyme The optimal pH of FFase from Fig.2 Determination of the pH optima for immobilized (1) and free (2) FFase from yeast The thermostability cureves are shown in Fig.3 As can be seen, the free enzyme was inactivated completely at 60-700C for 0.5-1h In contrary, immobilized enzyme was not inactivated at 700C, event after 3h Fig.3 Thermostabilitis of immobilized (1) and free (2) FFase: 0 A (30 C), B (500 C), C (55 C), D 0(60 C), E (70 C) - The effect of substrate and product concentration It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum After this point, increases in substrate concentration will not increase the velocity (Worthington, Biochemical corporation, 1972) This is represented graphically in Fig.4 Fig.4 Effect of substrate concentration on the reaction velocity of enzyme Acid invertases from plants are also inhibited by their reaction products, with Glc acting as a non-competitive inhibitor and Fru as a competitive inhibitor Figure shows the dependence of the concentration of reaction products on the time for the immobilized and native enzymes The activity of the immobilized enzyme was stable for h, while the native enzyme was inactivated after 15 Fig.5 Kinetics of the formation of the products of the enzymatic hydrolysis of sucrose: 1)immobilized enzyme; 2) native enzyme 1.2 Fructooligosacharides In response to an increasing demand from the customer for (FOS) healthier and controlled foods, a calorienumber of so-called alternative sweeteners such as palatinose and various oligosaccharides including isomaltooligosaccharides, soybean oligosaccharides and especially, fructooligosaccharides have emerged since the 1980s They are important primarily because of their functional properties rather than sweetness All of the new products introduced so far, microbial fructooligosaccharides (FOS) from sucrose have attracted special attention and are attributed to the expansion of the sugar market by several factors First, mass production is not complicated Second, the sweet taste is very similaronly to that sucrose, a traditional name for of fructose oligomers thatsweetener are mainly composed of 1F Various fructans of higher molecular have been produced kestose nystose (GF), and(GF), l -fructofuranosyl nystose (GF) in weight which fructosyl units (F) are by the action of bound at the of β- sucrose (GF), respectively, which should be 2,1 position transfructosylation activity from many plants andand microorganisms distinguished from other (Hidaka kinds of fructose oligomers H Eida, 1986 Hayash, 1989) Depending on the The production yield of FOS using enzymes originated from enzyme sources, plants is lowthey andhave difference linkages; for instance, fructosyltransferase from mass production of enzyme is quite limited by seasonal conditions; fungi such as Aureobasidium pullulans and Aspergillus niger produce only the therefore, industrial F lproduction -type depends chiefly on fungal enzymes from either Aureobasidium sp FOS Claviceps purpurea enzymes and asparagus enzymes produce (Yun,while J W F both - and or 6G A niger (Hidaka H Eida, 1986) In 1984, Meiji Seika Co Jung, l1992) type oligofructosides It is an accepted opinion that in Japan first 1.2.1 fructooligosaccharides is a common succeeded in the commercial production of FOSS (commercial name is Plants Occurrence Neosugar) by A The the fructooligosaccharides are found in the several kinds of plants, transfers terminal fructosyl residue from trisaccharide to niger enzyme as onion, wheat,such asparagus sucrose to reform a root, (Shiomi N, 1976) Allen and Bacon, 1956 found donor molecule was discovered in the Jerusalem artichoke (Edelman, transfructosylation 1966) Onion and activity derived from the leaves of suger beet and F G led toare the lwere fructosylsucrose) with somesources neokestose (6 -β-fructosylsucrose) An asparagus also important of fructosyltransferase (Edelman, conclusion that in the presence of sucrose, the products of transfer are enzymeShiomi whichet 1980) mainly 1-kestose ( the fructosyltransferase extracted from asparagus extensively studied roots They isolated eleven components of FOS Asparagus oligosaccharides are produced by cooperative enzymatic reactions with at least three kinds of fructosyltransferase: sucrose 1fructosyltransferase, 6”- fructosyltransferase, and lFfructosyltransferase They further purified and characterized the individual fructosyltransferases It was found that the general properties resembled those of the Jerusalem artichoke, but its substrate specificity differed Satyanarayana, 1976 described the biosynthesis of oligosaccharides and fructans from agave He isolated various oligosaccharides, (DP 3-15), synthesized them in vitro, and proposed a reaction mechanism Unlike most enzymes, this agave enzyme is capable of synthesizing inulotriose from inulobiose The naturally occurring oligosaccharides in agave consists of l-kestose, neokestose, 6-kestose, and their derivatives These oligosaccharides arise not only by transfructosylation reactions but by the stepwise hydrolysis Table of the 1: Fructooligosaccharide-synthetic higher oligosaccharides and fructans enzymes catalyzed (1) by the inherent from plants hydrolytic activity of the enzyme Table show the fructooligosaccharide-synthetic enzymes from plants that were discover by some workers in the past Microoganisms On the other hand, industrial fructooligosaccharides are mainly produced from sucrose by fungal enzyme During the cultivation of several fungi in the sucrose medium, the synthesis of FOSs was observed the of the concentration 1952 studied transfructosidation of anWhen enzyme A.oryzae He foundof two 1and tetrasaccharides named provisonly 1-inulobiosyl-Dglucose and sucrose supply in the β-2,1 linked triinulotriosyl-Dmedium was inadequate, FOSs were as energy glucose, respectively (they seem to ultilized be 1-kestose and source nystose, (Arcamone, 1970) Pazur, according to Jong Won Yun, 1996) The action of C.purpurea enzyme on sucrose also gives rise to a number of oligofructosides including 1-kestose and neokestose (Dickerson, 1972) Fusarium oxysporum is another important enzyme source functioning transfructosylation activity, which has been studied by many workers Maruyama and Onodera, 1969 isolated two kinds of enzyme showing transfructosylation activity That is everything sciencetists did prior to 1980s Enzymes with the potential for achiving a high yield of FOS production were found in the late 1980s and early 1990s Hidaka et al, 1988 studied A niger enzymes; they fully characterized this enzyme and virtually developed it into the industrial production of FOS syrup By using A niger enzyme, the maximum FOS conversion reached 55 - 60% (w/w) based on total sugars Van Balken et, 1991 reported another fructosyltransferase showing high activity from Aspergillus phoenicis; they produced Table 2: Fructooligosaccharide-producing FOSS microorganisms at a 60% yield Furthermore, Takeda et al, 1994 reported a new fungal strain, Scopulariopsis brevicaulis This strain has the ability of selective production of 1kestose, a major component of FOS Table shows microorganisms that were discovered as the fructooligosaccharide-producing sources Moreover, using imobillized cell and enzyme for the production of FOS has been developing Production of FOS from sucrose catalyzed by β-Dfructofuranosidase was achieved by Chien and Lee (2001) with the use of mycelia of Aspergillus japonicus 400g.L-1 After a reaction period of 5h, the FOS yield was 61% of the total immobilized in gluten Onethe gram of mycelia-immobilized particials sugars When Aspergillus oryzae was used, cultural conditions and reaction having a cellhave content parameters been of 20% (w/w)towas with sucroseRamesh solution with an standardized get incubated FOS yield of 58%100ml (Sangeetha, and Prapulla, initial content of 2002) Besides fungi, bacterial strain have been reported to produce FOS A transfructosylating enzyme, which produces FOS from sucrose, have been isolated from Bacillus macerans FG-6 which, unlike other FTases, produced selectively GF5 and GF6 fructooligosaccharides The final yield of FOS was reported to be 33% when sucrose was50% used as substrate (Park and Oh, 2001) Lactobacillus reutri strainreported 121 has to produce-110g.L (95% kestose and 5% nystose) in the been supernatants when grown on sucrose containing medium (Van Hijum, Van Geel-Schutten, 2002) More recently, high content FOS is produced by removing the liberated glucose and unreacted sucrose from the reaction mixture resulting in up to 98% FOS aspect 1.2.2This Chemical will beFOS represented are easilylater understood as inulin-type oligosaccharides of Dstructure fructose attachedthat carry a D-glucosyl residue at the end of by β-(2 ->1) linkages the chain They constitute a series of homologous oligosaccharides derived from sucrose usually represented by formula GFn as depicted in Figure Fig Chemical structure of fructooligosaccharides A research group of Meiji Seika Co, the first commercial producer of FOS, introduced the chemical structure of FOS produced from A niger fructosyltransferase Until now, FOSs are widely known that oligosaccharides contain 1kestose, nystose and 1f - fructofuranosyl nystose However, this definition is not completely true FOSs are not only contain these sugar but also others sugar with higher polymerization Aspergillus sydowi produce six different FOSs showing a high degree of polymerization (DP 3-13) (Muramasu, 1988) Structure analysis is important in the study of 1.2.3 Enzyme FOSsbecause, as the The reaction mechanism of the fructosyltransferase to form FOSs mechanisms mentioned above, the degree of and polymerization and linkagesa ofseries FOSs on the sourcedepends of the enzyme In plants some microorganisms, vary with the of enzymes act enzyme togethersources whereas a single enzyme works in most other microorganisms For instance, fructosan metabolism in Jerusalem artichoke is established by the combination of two enzyme: sucrose:sucrose l-fructosyltransferase (SST) and β (2->1) fructan:β(2->1) fructan l-fructosyltransferase (FFT) In the first instance, SST converts sucrose into glucose and an oligosaccharide but unable to promote polymerization above the trisaccharide level; further higher polymers are consecutively synthesized FFT Thegroup and n is the number of extrasucrosyl where GF is by a sucrosyl overall mechanism was expressed as follows: fructosereaction residues Agave enzyme catalyzed a stepwise transfructosylation reaction to give rise to higher FOS formation where synthesis of FOSs from sucrose takes place as GF + fructosyltransferase -> Ffollows: fructosyltransferase +G F-fructosyltransferase + GF -> GF2 + frucotsyltransferase Here, it is notable that glucose, not fructose, acts as the acceptor of the fructose molecule from sucrose GF oligosaccharides , GF , and GF , cannot donors of for the synthesis of higher but actact asas acceptors of the fructosyl from moiety fructose sucrose only for the synthesis of higher oligosaccharides This mechanism is identical with that of chicory enzyme reported by Singh and Bhatia, 1971 Gupta and Bhatia, 1980 proposed a model for the fructosyltransferase in F the oqsporum They highest glucofructosan, suggesting that acceptor sitesuggested is perhaps that fructose is to just big enough transferred from donor site to the fructosylated nucleotide bridge accommodate up the to GF seems a similar result with the cases of This and this, in turn, fructosyltransferase transfers the(Hirayama, fructose moiety to the sucrose at the acceptor site to form from A niger 1989 and Hidaka, 1988) and A pullulan (Yun, J W, GF , GF 1992 andwas the Hayashi, 1991) in that GF is the biggest molecule of FOS in both GF4n, + GF GF + n -> n-1 cases The enzyme GFn+1 reaction mechanism (Figure 7) can be expressed as follows: ... -Fructofuranosides and β D- fructopyranosides are not hydrolysed ), alsoInnamed invertase hydrolysis of sucrose into fructose and glucose addition to releasing FFase catalyses D- glucose and D- the frucose... imobillized cell and enzyme for the production of FOS has been developing Production of FOS from sucrose catalyzed by β- Dfructofuranosidase was achieved by Chien and Lee (2001) with the use of mycelia... excessive drying (Mussato and β- D- Fructofuranosidase Mancilha, The 2007) production level of FFase depends to a great extent on the Yeast FFase value have been widely studied fructooligosaccharides