Study on characterization of chitinase from streptomyces
Nguyen Thanh Huong STUDY ON CHARACTERIZATION OF CHITINASE FROM STREPTOMYCES MASTER THESIS MAJOR BIOTECHNOLOGY HANOI – 2011 UNIVERSITY OF LIEGE *** VIETNAM NATIONAL UNIVERSITY, HANOI INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY *** Nguyen Thanh Huong STUDY ON CHARACTERIZATION OF CHITINASE FROM STREPTOMYCES Speciality: Biotechnology Code: 60 42 80 MASTER THESIS MAJOR BIOTECHNOLOGY SUPERVISOR: Dr. DUONG VAN HOP HANOI - 2011 LIEGE UNIVERSITY *** VIETNAM NATIONAL UNIVERSITY, HANOI INSTITUTE OF MICROBIOLOGY AND BIOTECHNOLOGY *** TABLE OF CONTENTS ABSTRACT Error! Bookmark not defined. CHAPTER 1. INTRODUCTION Error! Bookmark not defined. 1.1. Chitin and application of chitin and chitinoligosaccharides Error! Bookmark not defined. 1.1.1. Application of chitin in Agriculture and Environment Error! Bookmark not defined. 1.1.2. Application of chitin in Medicine Error! Bookmark not defined. 1.1.3. Application of chitin in cosmetic and industry Error! Bookmark not defined. 1.2. Compositions and methods for producing chitin Error! Bookmark not defined. 1.3. Chitinase Error! Bookmark not defined. 1.3.1. Main chitinase sources Error! Bookmark not defined. 1.3.2. Chitinase from Streptomyces and other sources Error! Bookmark not defined. 1.3.3. Purification of chitinase Error! Bookmark not defined. 1.3.4. Recombinant chitinase Error! Bookmark not defined. 1.3.5. Diversity of chitinase Error! Bookmark not defined. 1.4. Potential of chitin product application in Vietnam Error! Bookmark not defined. 1.5. All domestic related studies Error! Bookmark not defined. CHAPTER 2. MATERIALS AND METHODS Error! Bookmark not defined. 2.1. Analytical instruments Error! Bookmark not defined. 2.2. Microbes Error! Bookmark not defined. 2.3. Media Error! Bookmark not defined. 2.4. Methodology Error! Bookmark not defined. 2.4.1. Screening of chitinase-producing Streptomyces and culture conditions Error! Bookmark not defined. 2.4.2. Selecting good chitinese producers by chitinase activity assayError! Bookmark not defined. 2.4.3. Identification of Streptomyces strain Error! Bookmark not defined. 2.4.4. Effect of culture conditions (temperature, pH, aeration, carbon, nitro sources) for chitinase fermentation from Streptomyces Error! Bookmark not defined. 2.4.5. Purification of chitinase Error! Bookmark not defined. 2.4.6. SDS-PAGE and activity gel (zymogram) Error! Bookmark not defined. 2.4.7. Characterization of the partly purified chitinase Error! Bookmark not defined. CHAPTER 3. RESULTS AND DISCUSSION Error! Bookmark not defined. 3.1. Screening of chitinase-producing Streptomyces Error! Bookmark not defined. 3.1.1. Primary screening good Streptomyces strains for chitinase production Error! Bookmark not defined. 3.1.2. Chitinase activities of 60 Streptomyces strains in liquid mediumError! Bookmark not defined. 3.2. Identification of Streptomyces strain VN08-A0438 Error! Bookmark not defined. 3.2.1. Morphology of strain VN08-A0438 Error! Bookmark not defined. 3.2.2. Studying carbon sources assimilation of the culture Error! Bookmark not defined. 3.2.3. Some physiological criteria of the culture Error! Bookmark not defined. 3.2.4. 16S rDNA sequencing of Streptomyces VN08-A0438 Error! Bookmark not defined. 3.3. Selecting medium and conditions for chitinase production Error! Bookmark not defined. 3.4. Purification of chitinase Error! Bookmark not defined. 3.5. Characterization of the partly purified chitinase Error! Bookmark not defined. CONCLUSION Error! Bookmark not defined. FURTHER STUDIES Error! Bookmark not defined. BIBLIOGRAPHY Error! Bookmark not defined. ABSTRACT Chitin (C 8 H 13 O 5 N) n which is one of the natural polysaccharides is very popular and can be found in a variety of species such as in shells of crustaceans, in cuticles of insects or in the cell wall of fungi and some algae. Chitin and its deacetylated product named chitosan have great benefits in agriculture and environment, for instance, act as fertilizers to help plants develop (chitin) or decompose toxic compounds (chitosan). Besides, chitin also plays important parts in other fields such as medicine (possible pathway in human allergic disease, component of skin dressing), cosmetic and industry (additive to thicken and stabilized foods, substance improving paper’s size and strength)… Streptomyces species are important soil microorganisms. Some studies have been done on chitinase from several streptomyces strains, nevertheless, these studies did not fully concentrate on characteristics of chitinase. This study will be the one that completely solve that problems including identifying streptomyces strains, purifying chitinase and determining chitinase characterization from streptomyces strains in Hoang Lien Son national park, Vietnam. The objectives of this study: The aim of study was screened strains that are capable of producing chitinase. Since then, isolated strains were identified and determined the characterization of their chitinase. Content of study: To discover the streptomyces strain having the highest chitinase activity from 500 strains of isolated Streptomyces, the highest chitinase-producing streptomyces strain will be determined by primary screening on agar Petri dish and P.V. Suresh and M. Chandrasekaran method. To Identify of Streptomyces strain based on the morphology, biological criteria, chemotaxonomic study together with 16S rDNA sequencing. To purified the enzyme received from the target streptomyces strain. To characterize the purified enzyme produced by the target streptomyces strain including pH and temperature stability, TLC test. Practical applicability: In the future, we intend to apply these isolated strains in different field scales. Contribution of the study: Based on the morphology, biological criteria, chemotaxonomic study together with 16S rDNA sequencing, strain VN08-A0438 which showed the highest ability in producing chitinase was identified as Streptomyces chromofuscus. This is the first time the enzyme produced by Streptomyces chromofuscus was partly purified and characterized (including pH and temperature stability, TLC test) in Vietnam. Distribution of thesis: thesis contained 67 pages, 8 tables, 17 figures, and 43 references. Distribution of thesis concluded: Abstract (1 page), Foreword (1 page), Introduction (24 pages), materials and methods (11 pages), results and discussion (13 pages), conclusions and futher study (1 page), and reference (4 pages), appendix (6 pages). CHAPER 1. INTRODUCTION 1.1. Chitin and application of chitin and chitinoligosaccharides Chitin (C 8 H 13 O 5 N) n is one of the natural polysaccharides including a copolymer of N-acetyl-D- glucosamine and D-glucosamine residues. These two components are linked together by β-1,4 glycosidic bonds. According to scientists, chitin is a useful substance that helps plants develop. It has been known to take part in a popular phenomenon named defense mechanisms in plants as an extreme good inducer. Beside, in agriculture and horticulture, chitosan-a substance derived from chitin can be used as a bio- control elictor [26]. Chitin oligosaccharides are also well-known by their abilities in “fast turning on” plant’s defense mechanisms against some invasion by fungi, therefore, enhance the plant disease resistance. Chitosan - a substance derived from chitin can trigger plant defensive mechanisms as a vaccine in human, stimulate plant growth and induce unexpected effects of certain enzymes. Scientists believe that chitin is used for environmental treatment because of its features: natural origin and being biodegraded. Occupations associated with high environmental chitin levels, such as shellfish processors, are prone to high incidences of asthma. Recent studies have suggested that chitin may play a role in a possible pathway in human allergic disease. Specifically, mice treated with chitin develop an allergic response, characterized by a build-up of expressing innate immune cells. In these treated mice, additional treatment with a chitinase enzyme abolishes the response [26]. chitosan’s cicatrizant properties have illustrated its role as a component, notably in all types of dressings (artificial skin, corneal dressings, etc.), surgical sutures, dental implants, and in rebuilding bones and gums. It is said that the role of chitin in industry is of great value. Chitin has been previously used as an additive to thicken and stabilized foods. Besides, chitin acts as a binder in dyes, fabrics and adhesives. Industrial separation membranes and ion-exchange resins can be made from chitin. In paper production, chitin is known to be a substance improving paper’s size and strength. Chitosan also has been widely used in food production, preservation and in diet diagrams. Chitosan is applied in cosmetics in the name of formulating moisturizing agents such as sunscreens and organic acids protector… with these characteristics, chitosan can enhance skin bioactivity and effectiveness. Besides, due to its antibacterial properties, chitosan is widely used in the composition of skin-care creams, shampoos and hair spray. 1.2. Methods for producing chitin Heterologous genes from viral, fungal, insect or other orsanisms can be achieved by many methods to increase the amounts of chitin or to directly produce chitosan without the chemical modified chitin. the compositions include polynucleotides encoding enzymes or polypeptides, a coding sequence for one or more polypeptides, transformed fungi, bacteria, plants, plant cells, tissues and seeds. It is almost easy and quick to produce chitin from shrimp waste by chemical methods. However, people believed that this process may not be considered as a good recovery option because of expensive cost and non - environmental friendly. It can be stated that physical methods are of great value in producing chitin from seafood. Scheme 1: Conventional method Shrimp shells → Demineralization with 2 N HCl for 48 hr → Deproteinization with 1 N NaOH at 1000 o C for 8 hours → Chitin. Scheme 2:Radiation method Shrimp shells → Irradiated to 25 kGy with gamma radiation →Demineralization with 2 N HCl for 48 hr → Deproteinization with 1 N NaOH at 1000 o C for 1 hour → Chitin. Physical method in producing chitosan from chitin is more effective in comparison with chemical method because of its simple and fast features in degradation. In biological method, Proteases can be used for the deproteinization of crustacean shells for the production of chitin or chitosan 1.3. Chitinase Chitinase (EC 3.2.1.14) is a member of the glycoside hydrolyse family, and are characterized by their ability to catalyze the hydrolytic cleavage of chitin. Chitinase have been detected in a wide variety of organisms including organisms that do not contain chitin, such as bacteria, fungi, viruses, plants and insects. The roles of chitinases in different organisms are diverse. Chitinases from all organisms are classified into two phylogenetically distinct families 18 and 19 among the 87 families of glycosyl hydrolases based on amino acid sequence of their catalytic domains. Chitinase is difficult to purify and modify chemically. Plant chitinase is specific example for chitinase purification. Chitinases can be purified from a total homogenate, from the intercellular fruid or from latex. Affinity chromatography, using chitin or colloidal chitin, is a successful method for the purification of the chitinases from barley leaf intercellular fluid, soybean, bean, tobacco and wheat. Chitinases have a very basic or very acid isoelectric points (and they are described as basic or acidic proteins). The development in recombinant DNA technology has helped to reduce chitinase production cost and enhance its production. Bacterial expression systems are the most common and preferred method for the overexpression of recombinant proteins because of fast cell growth. There are four forms of chitinase: endochitinase is defined as an enzyme splitting within the chitin polymer, exochitinase (EC 3.2.1.14) as an enzyme releasing chitobiose, β-N-acetylglucosaminidase releases N-acetylglucosamine monomers from chitin and chitobiase hydrolyses chitobiose. Endochitinases (EC 3.2.1.14) cleave chitin polymers at randominternal sites, whereas exochitinases cleave chitin progressively starting at the non-reducing ends of the chains and release N-acetyl-D- glucosamine (GlcNAc) monomers or diacetylchitobiose by b-(1,4)-N-acetylglucosaminidase activity (EC 3.2.1.30) or 1,4-b-chitobiosidase activity (EC 3.2.1.29), respectively. 1.4. Potential of chitin product application in Vietnam Crustacean is an abundant aquatic product source accounting for one - third of total number of fisheries production in Vietnam. The production of chitosan from shrimp shells can bring high economic benefit. With the chitin and chitosan’s abilities in widen its applications, many countries including Vietnam have studied to produce these products. 1.5. All domestic related studies Scientists from Vietnam Academy of Science and Technology have found out 3 bio-procedures based on the use of enzyme proteolytic to extract chitin from shrimp’s head and shell. (i) Method that used enzyme bromelain in pineapple extracted solution. (ii) Fermentation of the bacteria producing proteinase; (iii) Natural fermentation Doctors from Vietnam National Cancer Hospital have taken a research in 2003 on 60 patients from ages 35 to 76 and found out that chitosan supported effectively in cancer treatments. In medical-pharmaceutical, Vietnamese scientists produced Glusivac – a specific medicine for osteoarthritis treatment. Besides, there are some weight loss pills made from Chitozan. Vietnamese scientists have gained success in constructing chitin and chitosan production techonology from seafood’s shell (shrimp, crab, shell, squid) for health and food sectors. CHAPTER II. MATERIALS AND METHODS 2.1. Analytical instruments The stuffs used in this research are standard ones belonging to the institute of Microbiology and Biotechnology - Vietnam National University, Hanoi. 2.2. Microbes The soil samples used for this experiment were collected from Hoang Lien Son national park. A number of 500 strains of Streptomyces isolated from these samples were kept in VTCC and used for the study on characteristics of chitinase. 2.3. Media * Agar medium (yeast extract - starch YS), Medium 1 (M1) (culture fermentation) and medium ISP4. 2.4. Methodology 2.4.1. Screening of chitinase-producing Streptomyces and culture conditions. - Preparing culture broth of Streptomyces - Primary screening on agar Petri dish 2.4.2. Selecting good chitinese producers by chitinase activity assay 2.4.3. Identification of Streptomyces strain - Morphology and growth characterization - Some biological criteria of Streptomyces target strain - Chemotaxonomic Study of Streptomyces - 16S rDNA sequencing 2.4.4. Effect of culture conditions (temperature, pH, aeration, carbon, nitro sources) for chitinase fermentation from Streptomyces 2.4.5. Purification of chitinase 2.4.6. SDS-PAGE and activity gel (zymogram) 2.4.7. Characterization of the partly purified chitinase CHAPTER III. RESULTS AND DISCUSSION 3.1. Screening of chitinase-producing Streptomyces 3.1.1.Primary screening good streptomyces strains for chitinase production: Five hundred collected Streptomyces strains in Hoang Lien Son national park were isolated and maintained at Vietnam Type Culture Collection - Institute of Microbiology and Biotechnology - Vietnam National University, Hanoi. Agar Petri disc method was used in primary screening step. Chitinase activity was determined according to the clear zones’ diameters (fig 1). The zone diameters on plates varied in size (2 - 30 mm). Fig 1. Clear zones’ diameters illustrated Chitinase activity of Streptomyces strains. A number of 60 strains showing strong chitinase activities, 146 strains showing medium chitinase activity and weak chitinase activity belong to remaining 186 strains (Table 2) Table 1. Summary chitinase activities of 500 strains Streptomyces. Chitinase Activity No observation Weak Medium Strong Total Number of strains 186 108 146 60 500 Ratio (%) 37.2 21.6 29.2 12.0 100 Note: Diameter of growth zones D (mm); No activity: D ≤ 2; Weak: 2 < D ≤ 10; Medium: 10 < D < 20; Strong: D ≥ 20. All 60 strains with high chitinase activity will be used for next secondary screeing for chitinase activity on liquid medium. 3.1.2. Chitinase activities of 60 strains Streptomyces in liquid medium Sixty selected strains of streptomyces were grown on the medium (as described in the methodology). The broths were taken for chitinase assay and measure as number of unit/ml. Almost tested strains possess chitinase activities in the rank from 10 to 20 U/ml. There are 3 strains (VN10A- [...]... Selecting medium and condition for chitinase production We conducted study for selecting conditions for chitinase production from strain VN08-A0438 In this study, we focused on some key factors on chitinase production: optimum temperature, pH, aeration, fermentation time, carbon and nitrogen sources From the experimental results in fig 5 A, B, C, D, E, F we selected medium M1 with carbon source as glucose... Fig 5 Effect of some parameters on chitinase activity of Streptomyces VN08-A0438 (A: Temperature; B: pH; C: Shaking rate; D: Carbon sources; E: Nitrogen sources; F: Time of fermentation) 3.4 Purification of chitinase The cell free supernatant was subjected to fractional ammonium sulfate (NH4)2SO4 precipitation, chitinase enzyme was precipitated in 60-80-100% ammonium sulfate saturation as described... bioassay of the active fraction It was clear that peak of chitinase activity and protein was not matching The high chitinase activity was found in fractions from 45 to 70 Those fractions were pooled for further study We checked activity of fractions from 55 to 62 (fig 6), it was obvious that those fractions having good chitinase activity To confirm the chitinase activity, the sample was concentrated... grow at higher concentration of NaCl 3.2.4 16S rDNA sequencing of Streptomyces VN08-A0438 + Extract total DNA and amplification of 16S rDNA Fig 3 Extraction (A) and 16S rDNA (B) of the total DNA amplification of from strain VN08A-438 The value of optical (260/280) was about 1.95, the extracted DNA is good enough for processing PCR amplification The result of PCR amplification (Figure 3B) showed that... About of 3 ml of the chitinese was applied on a Sephadex G100 for further purification Table 3 Summary of partly purification Steps of purification Total activity (U) Protein Specific activity Purity (fold) Recovery (%) The supernatant 6000 320 mg 18 1 100 Ammonium Sulphate precipitation 2700 68 mg 40 2.1 45 Sephadex G100 1100 8.5 130 7.2 18 (mg) Fig 6 Chromatographygram of chitinase enzyme on Sephadex... methodology section The precipitate was collected and dialyzed for the removal of ammonium sulfate The fractions collected from ammonium sulfate at 3 concentration 60, 80, 100% saturation were determined the specific activity The results showed that fraction of 80 % saturation gave higher specific activity (40U/mg protein) in comparing to the others (22 U/mg and 31 U/mg for 60% and 100 % saturation respectively)... utilization (+): when growth on tested carbon was significantly train better than on basal medium without carbon Negative ultization (-): when growth was similar to or less than growth on basal medium without carbon 3.2.3 Some physiological criteria of the culture The strain grow well at temperature (30-35oC), pH (6-7) It only could grow in medium with 1% - 2% NaCl and could not grow at higher concentration... evaporation at room temperature to 10 folds The sample (40µl) was loaded on SDS-PAGE and activity gel to check zymogram Native gel electrophoresis showed two bands which one band corresponded to chitinase activity as shown by the hydrolysis zone in the zymogram The other band on native gel might be a strange protein which was unattended Fig 7 Zymogram and SDS-PAGE of chitinase 3.5 Characterization of the... products containing ranks of mono-, di-, tri-, tetra- , penta-, hexa- of N-acetyl glucosamine For completely degaradation of chitin, it was reported that there were a number of three enzymes involved: endochitinase with the main product of chitobiose and chitinooligosacharide, exochitinase with the main products of N-acetyl glucosamine and chitinooligosaccharide, chitobiose with the final products of N-acetyl... single enzyme protein in next studies Fig 9 TLC analyzing of final chitinase reaction products CONCLUSION 1 From five hundred screened Streptomyces strains, sixty good chitinase producers (account for 12 percent- 12%) which have chitinase activities from 15 to 39 U/ml were selected Further screening showed that strain VN08-A0438 showed the highest chitinase activity (39.9 U/ml), followed by strains VN10A-0046 . Identification of Streptomyces strain Error! Bookmark not defined. 2.4.4. Effect of culture conditions (temperature, pH, aeration, carbon, nitro sources) for chitinase fermentation from Streptomyces. of Streptomyces chromofuscus. Fig 4. Phylogenetic tree contruction for VN08-A0438 strain. 3.3. Selecting medium and condition for chitinase production We conducted study for selecting conditions. conditions for chitinase production from strain VN08-A0438. In this study, we focused on some key factors on chitinase production: optimum temperature, pH, aeration, fermentation time, carbon and