Dissertations / Theses on the topic 'Xylanolytic and chitinolytic enzymes'

It has been envisaged that lytic enzymes may be crucial in determining the morphology and growth of fungi, and may therefore represent a target for antifungal agents. The chitinolytic system of Candida albicans was investigated using a range of 4-methylumbelliferyl glycosides as model substrates, and its potential as a target for antibiotics has been assessed. Maximum hydrolysis was obtained with substrates having monomer and tetramer chain lengths, being attributed to N-acetylglucosaminidase and endochitinase respectively. Activities were investigated in cell fractions, vacuoles, and also in whole cell preparations. The characteristics of both chitinase and N-acetylglucosaminidase were examined, including pH and temperature optima and Km values. Chitinase was semi-purified on Fast Protein Liquid chromatography system and activity could be located after native polyacrylamide gel electrophoresis. Analysis of chitinase activity during the growth of the yeast morphology of C.albicans revealed maximal activities during the logarithmic phase, suggesting a relationship of chitinase levels to active growth of the pathogen. It was found that the antibiotic allosamidin was a potent inhibitor of chitinase activity of C.albicans, but not of N-acetylglucosaminidase. Conversely, an analogue of N-acetylglucosamine was found to be a potent inhibitor of N-acetylglucosaminidase but not of chitinase. Treatment of yeast suspensions with allosamidin resulted in an increased chain length. No cell death, or discernible pattern of change in the radiolabelling was observed in the presence of either allosamidin or N-acetylglucosamine analogue. Similarly no consistent change in the optical density of cultures was observed in the presence of either inhibitor. Even in the presence of the membrane permeabilising agent amphotericin no effects were observed above those achieved with amphotericin alone. Comparative studies were carried out upon the chitinolytic activity of Kluyveromyces lactis toxin and bovine serum. The chitin synthetic system of Benjaminiella poitrasii was compared to that of C.albicans.

There are many diseases and illnesses in the world that require new drug treatments and chitin has been shown to produce chitooligomeric derivatives which exhibit promising antimicrobial and immune-enhancing properties. However, the rate-limiting step is associated with the high recalcitrance of chitinous substrates, and low hydrolytic activities of chitinolytic enzymes, resulting in low product release. To improve and create a more sustainable and economical process, enhancing chitin hydrolysis through various treatment procedures is essential for obtaining high enzyme hydrolysis rates, resulting in a higher yield of chitooligomers (CHOS). In literature, pre-treatment of insoluble biomass is generally associated with an increase in accessibility of the carbohydrate to hydrolytic enzymes, thus generating more products. The first part of this study investigated the effect of alkali- (NaOH) and acid pre-treatments (HCl and phosphoric acid) on chitin biomass, and chemical and morphological modifications were assessed by the employment of scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Energy-Dispersive X-ray spectrometery (EDX) and x-ray diffraction (XRD). Data obtained confirmed that pre-treated substrates were more chemically and morphologically modified. These results confirmed the fact that pre-treatment of chitin disrupts the structure of the biomass, rendering the polymer more accessible for enzymatic hydrolysis. The commercial chitinases from Bacillus cereus and Streptomyces griseus (CHB and CHS) are costly. Bio-prospecting for other chitin-degrading enzymes from alternate sources such as Oidiodendron maius, or the recombinant expression of CHOS, was a more economically feasible avenue. The chit1 gene from Thermomyces lanuginosus, expressed in Pichia pastoris, produced a large range CHOS with a degree of polymerisation (DP) ranging from 1 to above 6. TLC analysis showed that O. maius exhibited chitin-degrading properties by producing CHOS with a DP length of 1 to 3. These two sources were therefore successful in producing chitin-degrading enzymes. The physico-chemical properties of commercial (CHB and CHS) and expressed (Chit1) chitinolytic enzymes were investigated, to determine under which biochemical conditions and on which type of biomass they can function on optimally, for the production of value-added products such as CHOS. Substrate affinity assays were conducted on the un-treated and pre-treated biomass. TLC revealed that chitosan hydrolysis by the commercial chitinases produced the largest range of CHOS with a DP length ranging from 1 to 6. A range of temperatures (35-90oC) were investigated and CHB, CHS and Chit1 displayed optimum activities at 50, 40 and 45 oC, respectively. Thermostability studies that were conducted at 37 and 50oC revealed that CHB and CHS were most stable at 37oC. Chit1 showed great thermostablity at both temperatures, rendering this enzyme suitable for industrial processes at high temperatures. pH optima studies demonstrated that the pH optima for CHB, CHS and Chit1 was at a pH of 5.0, with specific activities of 33.459, 46.2 and 5.776 μmol/h/mg, respectively. The chain cleaving patterns of the commercial enzymes were determined and exo-chitinase activity was exhibited, due to the production of CHOS that were predominantly of a DP length of 2. Enzyme binary synergy studies were conducted with commercial chitinases (CHB and CHS) on colloidal chitin. Studies illustrated that the simultaneous combination of CHB 75%: CHS 25% produced the highest specific activity (3.526 μmol/h/mg), with no synergy. TLC analysis of this enzyme combination over time revealed that predominantly chitobiose was produced. This suggested that the substrate crystallinity and morphology played an important role in the way the enzymes cleaved the carbohydrate. Since CHOS have shown great promise for their antimicrobial properties, the CHOS generated from the chitinous substrates were tested for antimicrobial properties on Bacillus subtilis, Escherichia coli, Klebsiella and Staphlococcus aureus. This study revealed that certain CHOS produced have inhibitory effects on certain bacteria and could potentially be used in the pharamceutical or medical industries. In conclusion, this study revealed that chitinases can be produced and found in alternate sources and be used for the hydrolysis of chitinous biomass in a more sustainabe and economically viable manner. The chitinases investigated (CHB, CHS and Chit1) exhibited different cleaving patterns of the chitinous substrates due to the chemical and morphological properties of the biomass. CHOS produced from chitinous biomass exhibited some inhibitory effects on bacterial growth and show potential for use in the medical industry.

Hydrolytic enzymes such as chitinases and glucanases are implicated in plant defense responses against fungal pathogens. These enzymes are responsible for the breakdown of chitin and glucan, two major components of the fungal cell walls. Genes encoding these enzymes have been used to genetically engineer plants to enhance their protection against fungal pathogens. Western Australia has over 4000 endemic plant species and a largely unknown fungal biota. Given that fungi possessing chitinases and glucanases with novel activities have been isolated in other parts of the world, we propose that fungi from Western Australian soils may possess novel biochemical/enzymatic activities. The aims of this research project were to isolate chitinolytic and glucanolytic fungi from soil and to clone the genes encoding for chitinase and glucanase enzymes. To achieve these aims, fungi with activity against chitin and glucan were isolated, the activity quantified by colorimetric and inhibition assays and gene fragments with homology to known chitinase and glucanase genes were isolated and their sequences determined. Soil fungi were isolated from five locations in and around the Perth Metropolitan area of Western Australia with the use of a medium containing Rose Bengal that eliminates all actinomycetes and most bacteria and reduces the growth of fast growing mold colonies. Forty-one isolates were obtained by this method. Twenty four chitinolytic and glucanolytic fungal isolates were identified by growing them on chitin-containing media to select for those species that utilised chitin/glucan as a carbon source. These were assayed for production of exo- and endochitinolytic and glucanolytic enzymes. Enzyme activity was compared between crude and dialysed supernatants. Exochitinase activity was determined in the supernatants of 4-day old fungal cultures by the release of p-nitrophenol from p-nitrophenyl-N-acetyl-â-D glucosaminide. The supernatants were measured for endochitinase activity determined by the reduction of turbidity of suspensions of colloidal chitin. Glucanase activity was determined by release of reducing sugar (glucose) from laminarin. Supernatants from eleven of the twenty four isolates showed significant levels of enzyme activity. Eleven isolates were assayed for activity against purified cell walls of phytopathogenic fungi. Activity was determined by measuring reducing sugars in the fungal supernatants against cell wall preparations of six economically important plant pathogens. Chitinolytic activity was detected in seven isolates against cell wall preparations of Botrytis cinerea and Rhizoctonia solani, in four isolates against Fusarium solani and Sclerotinia sclerotium; in five isolates against Ascochyta faba and in six isolates against Leptosphaeria maculans. Similarly glucanolytic activity was detected in eight isolates against B. cinerea, in seven against R. solani, in two against F. solani, in three against S. sclerotium and A. faba and in one against L. maculans. The supernatants derived from the isolates were used in a bioassay to determine growth inhibition against live B. cinerea spores by measuring turbidity reduction. Growth inhibition was measured against a control (B. cinerea, grown in medium with no added supernatant). Boiled supernatant did not inhibit the growth of B. cinerea spores but there was 100% inhibition by the crude supernatant from ten of the twenty four isolates. Similarly, supernatants were used to assess growth inhibition against live mycelia cultures of F. solani and S. sclerotium. Growth inhibition of F solani ranged from 9- 59%, boiled and crude supernatants respectively whilst growth inhibition of S. sclerotium ranged from 46-75%, boiled and crude supernatants respectively. Two partial chitinase genes from the soil filamentous ungus Trichoderma asperellum,(ChiA and ChiB) and a novel glucanase gene from the filamentous fungus Aspergillus (Glu1) were cloned. ChiA, was 639 bp long, encoding 191 amino acids with identity to other chitinase genes. Two highly conserved regions, characteristic of glycosyl hydrolases from family 18, were present. ChiB, was 887 bp long and encoded a 293 amino acid sequence that was closely related to an endochitinase gene from the filamentous fungus Trichoderma asperellum. The two highly conserved regions corresponding to the substrate binding and active sites that characterise the glycosyl hydrolases from family 18, also found in ChiA, were found in this gene. Glu1 was 2844 bp long and encoded a 948 amino acid sequence that shared high identity with a â-1, 3-glucanase from the filamentous fungus Aspergillus oryzae. The sequence contained conserved regions found in glycosyl hydrolases from family 17 that encode for substrate binding, N-terminal sequences and putative asparagine linked glycosylation sites. The partial putative sequence ChiA is probably a pseudogene because it has two inframe stop codons. However, once the entire sequence of ChiB is known, both ChiB and the novel glucanase gene Glu1 could be useful contenders for engineering resistance in crop plants.

Thesis (Ph. D.)--University of Georgia, 2005.
Directed by James T. Hollibaugh. Includes an article published in Applied and environmental microbiology, and articles submitted to Aquatic microbial ecology, and Applied and environmental microbiology. Includes bibliographical references.

碩士
國立臺灣大學
農業化學系
81
The partially hydrolyzed products of chitin and chitosan, N- acetylchitooligosaccharides and chitooligosaccharides, are of special interest because these oligosaccharides have been evalu- ated in various applications.This study focused on the production of N-acetylchitooligosaccharides by microbial enzymes. Crab shell chitin prepared by treating crab shell with acid and alkali was used as a substrate for isolating chitinolytic mi- croorganisms. Among the 103 isolated strains, strain No. 16 appeared to be the most potential chitinase-producing strain. It was identified belonging to the genus Aeromonas and named Aero- monas sp. No. 16. When the medium contained 1.5% colloidal chitin , 2.0% tryp- tone and 1.5% yeast extract with initial pH 10, Aeromonas sp. No. 16 could produce 1.4 units/milliliter of chitinase activity after 24 hours cultivation in 500 mL Hinton''''s flask. Using crys- talline chitin as substrate for enzyme productionin a 5L fermen- tor with temperature at 30℃, aeration at 1 vvm, agitation at 400 rpm and pH controlled between 7 and 8, chitinase activity could reach 1.5 units/milliliter after 30 hours cultivation. Chitin and N-acetylglucosamine were both found to be inducers for chitinase synthesis by Aeromonas sp. No. 16, on the other hand, addition of other carbohydrates would repress enzyme production with different degrees. Crude enzyme hydrolyzed chitin and produced N-acetylglucosa- mine as main product ,hydrolyzed chitosan with unknown products formation, which was supposed to be hetero- chitooligosaccharide. In crude enzyme, chitinase activity was most active at pH 6.0 and 50℃, and was stable at pH between 5 and 9 and at temperature be- low 40℃. β-N-acetyl-D- glucosaminidase was less stable at 40℃ and supposed to be an intracellular enzyme. After PAGE and acti-

碩士
國立臺灣大學
生物產業機電工程學研究所
96
Abstract N-acetylchitooligosaccharides (degree of polymerization 4-6) have specific biological activities such as antitumor activity and immuno-enhancing effects. In this study, we aimed to isolate environmental microorganisms which could produce enzymes to hydrolyze chitin into N-acetylchitooligosaccharides. At the initial stage, we hydrolyzed colloidal chitin with crude microbial enzymes and analyzed the products by HPLC. From this screening, we found that the crude enzyme from one bacterial isolate could hydrolyze chitin and produce N-acetylchitooligosaccharides. The bacterial strain was identified by 16S rRNA sequencing and phylogenetic analysis to belong Serratia marcescens and was named S. marcescens NTU-17. We used central composite design (CCD) of response surface methodology (RSM) to obtain the optimal culture condition for chitinase production: 0.4 g/l colloidal chitin, 1.6 g/l casein, 30。C and pH 7.5; the highest chitinase activity was produced at 18 hours after inoculation. The crude enzyme from culture broth of S. marcescens NTU-17 was subjected to successive steps of purification. After ammonium sulfate fractionation (35-70%), gel filtration-Sephacryl 200 chromatography, and DEAE-Sephacel column chromatography, two species of chitinase were purified and the molecular weights were determined by SDS-PAGE to be 53 kDa (chitinase 1) and 39 kDa (chitinase 2). The chitinase activity of chitinase 1 and 2 were also verified by an in-gel chitinase activity assay. After purification, the specific activity of chitinase was increased by 2.5 fold and the yield was 12%. Chitinase 1 exhibited the optimal activity at pH 3 and 50℃, and chitinase 2 showed the optimal activity at 30℃ and similar activities at pH 3-12.

碩士
臺灣大學
微生物與生化學研究所
98
It was reported that N-acetylchitooligosaccharides (degree of polymerization 4-7) (GlcNAc)4-7 have specific biological activities such as antitumor activity and immuno-stimulating effects. In this study, we aimed to screen microorganisms from a chitin-rich environment and isolate the ones that can produce enzymes to hydrolyze chitin into N-acetylchitooligosaccharides. At the initial stage, we used the selection medium with colloidal chitin as the sole carbon source to select for microorganisms which could utilize chitin for growth. The microbial isolates were further screened by incubating the culture broth with colloidal chitin and analyzing the products by HPLC and TLC. Using this screening strategy, we obtained one bacterial isolate that could produce enzymes to hydrolyze chitin into (GlcNAc) 4-5 and hydrolyze chitosan into (GlcN/GlcNAc) 4-6. The bacterial strain was identified by 16S rDNA sequencing and phylogenetic classification to belong to Aeromonas caviae and was named Aeromonas caviae No. 2. To optimize the culture condition for producing higher amounts of chitinase, we first tested various culture pHs and temperatures and found that the highest chitinase activity was produced at pH 6 and 25℃. Using the central composite design (CCD) of response surface methodology (RSM), we further obtained the optimal concentrations of chitin and nitrogen sources in the culture medium to be colloidal chitin: 1.098%, soybean flour: 0.735% and yeast extract: 0.74%. The crude enzyme produced in selection medium went through successive steps of purification including ammonium sulfate precipitation (40-70%), chitin affinity-hydrolysis method and gel filtration chromatography (Sephacryl 200). After purification, four major proteins were found range from 55 to 100 kDa on the SDS-PAGE, and two of them showed chitinase activity range from 55 to 70 kDa on the in-gel chitinase activity assay. When proteins from the optimized culture medium were purified using nine major bands appeared range from 40 to 100 kDa on the SDS-PAGE, and three of them showed chitinase activity on the in-gel chitinase activity assay. Moreover, the purified proteins could hydrolyze chitin primarily into (GlcNAc)2 and hydrolyze chitosan into (GlcN/GlcNAc) 4-6.

碩士
靜宜大學
食品營養研究所
92
Partially purified chitotriosidase and chitosanase were obtained from a commercial crude papain preparation using the sequential steps of buffer (containing 1.66 mM pHMB) extraction, 70% saturation ammonium sulfate precipitation and Sephacryl S-100 gel filtration. For hydrolysis of chitotriose derivative 4-methylumbelliferyl-β-D-N,N’,N” triacetylchitotriose (4-MU-β-GlcNAc3), the partially purified chitotriosidase had an optimal pH of 4.0, an optimal temperature of 40~50℃, a Km of 9.33μM and a Vmax of 0.476 nmol/min/mg. For hydrolysis of p-nitrophenyl-β-D-N-acetyl-glucosamine oligomer (pNP-β-GlcNAc1-5), the enzyme had much higher activity toward pNP-β-GlcNAc than toward other p-nitrophenyl-β-D-N-acetylglucosamine oligomers for release of p-nitrophenol. This suggests that the partially purified enzyme contained exo-type β-N-acetylglucosaminidase activity. In addition to pNP-β-GlcNAc hydrolysis activity, the enzyme also showed activity toward pNP-β-(GlcNAc)3-5 oligomer. The rate of the reaction was observed in the following order, pNP-β-(GlcNAc)4＞pNP-β-(GlcNAc)3＞pNP-β(GlcNAc)5. However, the enzyme had no activity toward pNP-β-(GlcNAc)2. These results indicate that the partially enzyme contained endo-type chitotriosidase and the cleavage site of the chitotriosidase may be the third and fourth linkages from the non-reducing terminus of pNP-β-(GlcNAc)n. Partially purified chitosanase consisted of several isoforms of chitinase and chitosanase, as analyzed by native-PAGE (pH 4.3) and chitinase and chitosanase activity staining. For hydrolysis of ethylene glycol chitin, the enzyme had an optimal pH of 3.0, an optimal temperature of 60℃, a Km of 1.24 mg/mL and a Vmax of 263 nmol GlcNAc/min/mg. Relative rate for hydrolysis of colloidal chitin, CM-chitin and ethylene glycol chitin were 30, 40 and 100, respectively. After incubation at 70℃ for 1 hour, 86% of the chitinase activity still remained. For hydrolysis of chitosan, the enzyme had an optimal pH of 4.6, an optimal temperature of 70℃, a Km of 1.58 mg/mL and a Vmax of 61.35 nmol GlcN/min/mg. Relative rate for hydrolysis of glycol chitosan, fungial chitosan (MW 390 kDa), shrimp chitosan (MW 340 kDa), crab chitosan (MW 300 kDa) and crab chitosan (MW 1,100 kDa) were 5, 24, 53, 73 and 100, respectively. After incubation at 70℃for 1 hour, 75% of the chitosanase activity still remained. The main products of chitosan hydrolysis catalyzed by the enzyme were low molecular weight chitosan (48 kDa~1.7 kDa) and some chitooligosaccharides.

碩士
淡江大學
化學學系碩士班
99
Application of NF and Crude SSC on the purification of enzymes to achieve rapid purification purposes. Adsorbent : SP, AC, pure chitin, Crude SSC, Crude CSC, Crude SPC , Choose a more suitable adsorbent Crude SSC. Studying temperature, weight, time on the adsorption effect of the pineapple enzyme. By experiment that at 4 ℃, 30 min, 0.1 g of the conditions to get specific activity 0.0918 U / mg. Studying temperature, weight, time on the effect of NF adsorption bromelain enzyme. Learned from the experiment at 25 ℃, 30 min, 0.1 g of the conditions to get specific activity 0.1357 U / mg. Use the above conditions, NF, and Crude SSC absorption bacterial enzyme purified enzyme to discuss the results. Purified enzyme affected by the experimental conditions known isoelectric point is not the only factor, but also need to consider the interaction between enzyme and adsorbents as well as physical absorption and other factors. Results show that absorption is a viable way to purify the enzymes, it can improve the purification efficiency.

博士
中山醫學大學
醫學研究所
97
Backgrounds Little is known about the association between human haptoglobin phenotypes and diseases. Also, not much is known about the association between blood chitinolytic enzyme levels and human diseases. Recent studies have suggested a link between haptoglobin and the molecules associated with inflammation. Subsequent discoveries on its roles in T-cell development and the governing signaling pathways have further enlightened both basic and epidermiological studies. The understanding of human chitinases, on the other hand, is relatively limited. To our knowledge, lacking of rapid and convenient analytical methods has limited the progress on studying both molecules in human. Our study aimed to develop rapid and accurate biochemical methods in analyzing (1) the haptoglobin phenotypes in human plasma, and (2) the specific activity of chitinolytic enzymes. These analytical methods were then applied to plasma samples from clinical and general populations in investigating the possible associations between human diseases and these traits. Methods We have developed a new method, termed mCBB-R250 staining, for rapid and sensitive typing of haptoglobin phenotypes from human plasma. The percentage of frequencies and the proportion of the three haptoglobin phenotypes, Hp 1-1, Hp 2-1, and Hp 2-2, in our collection of human plasma samples were analyzed using descriptive statistic, one sample t test and Chi-square tests. Using glycol chitosan as substrate, an analysis method using (K3Fe(CN)6) as indicator were developed to determine the chitioson-degrading enzymes in human plasma. An in-gel activity staining method was also developed for the detection and separation of chitinolytic enzymes. The specific activities of chitinolytic enzymes were analyzed using the normality test, independent-sample parametrical test, or non-parametrical test. Results We have successfully applied the mCBB-R250 method to determine the Hp phenotypes of 1148 samples. Among these, 11.67% are Hp 1-1, 42.60% are Hp 2-1, and 45.73% are Hp 2-2 type. As a comparison, the distribution of the three Hp phenotypes, as determined for 151 plasma samples from patients with cardiovascular diseases, are 11.26%, 41.06%, and 47.68% respectively for Hp 1-1, 2-1, and 2-2 type. These percentages are similar to that of the general population. Among the 151 samples from cardiovascular disease patients, 101 are stable angina (CAD), and the rest 50 are acute coronary syndrome (ACS) patients. While the percentages of Hp 2-2 type are statistically indifferent between these groups, the percentages of the Hp 1-1 and Hp 2-1 types are significantly different among CAD and ACS groups, in which we found that Hp 2-1 type is higher in CAD, but Hp 1-1 is higher in ACS. Using glycol chitosan and (K3Fe(CN)6) solution, the specific activities of chitinoltic enzymes, after analyzing 899 human plasma samples, was determined to be a normal distribution, with an average specific activity of 92.00 U/mg (95% CI 90.584，93.416). We have found that, among 151 patients of cardiovascular diseases, the specific chitinolytic enzyme activities in CAD group (102.724 U/mg) are higher than those in the ACS group (90.513 U/mg). We have also developed an in-gel detection method for rapid separation and detection of chitinolytic enzymes. With this new method, we have identified a new chitinolytic enzyme with a molecular weight of about 30 kDa. Conclusion We have developed rapid, accurate, and inexpensive methods for haptoglobin phenotyping and chitinolytic enzyme activity staining. The methods were applied to the Hp phenotyping and chitinolytic enzyme activity detection of 1178 plasma samples. With their application in clinical studies, these methods may grant us further understandings on the possible association with human diseases.

碩士
淡江大學
生命科學研究所碩士班
97
The purpose of this thesis is to study the production of chitinolytic enzymes and other antioxidants from Bacillus cereus TKU006. The optimum culture conditions for chitosanase and other antioxidants production were composed of 1% squid pen powder, 0.1% K2HPO4, 0.05% MgSO4．7H2O shaken at 25℃ in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) for 3 days and 25 mL of liquid medium for 2 days, respectively. The optimum culture carbon/nitrogen source for chitinase production was 2% shrimp head powder incubated for 2 days. A chitinase and a chitosanase were purified by chromatography procedures of ion exchange and gel filtration. The molecular mass of the chitinase and chitosanase determined by SDS-PAGE were approximately 39 kDa and 44 kDa, respectively. The chitinase was completely inactivated by Fe2+, and the chitosanase was completely inactivated by Fe2+ and Cu2+. The optimum temperature, optimum pH, thermal stability and pH stability of chitinase were 60℃, pH 5, ≦50℃, pH 2-8 . The optimum temperature, optimum pH, thermal stability and pH stability of chitosanase were 60℃, pH 7, ≦50℃, pH 3-10. After squid pen powder fermented, the antioxidant properties by the assays of radical scavenging, metal chelating, reducing power and total phenolic content were reached at 75%, 97%, 441 μg/mL cysteine equivalent and 815 μg/mL gallic acid equivalent, respectively. Correlation between DPPH scavenging ability and total phenolic content were r=0.92.

Submitted in complete fulfillment for the Degree of Master of Technology (Biotechnology), Durban University of Technology, Durban, South Africa, 2014.
Chitin, a highly insoluble 1,4- -linked polymer of N-acetyl- -D-glucosamine, is the second-most abundant bio-polysaccharide in nature after cellulose. Most chitinolytic fungi are known to produce more than one kind of chitinase. The recent sequencing of the Thermomyces lanuginosus SSBP genome by our group has revealed four putative family 18 chitinases. In this study, three novel chitinase genes (chitl, chit2 and chit3) and the previously reported chit4 gene were cloned from Thermomyces lanuginosus SSBP and their gene structures were analysed. chit3, encoding a 36.6 kDa protein, and chit4, encoding a 44.1 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit3 and Chit4 enzymes exhibited optimum activity at pH 4.0 and 5.0 and at 40oC and 50°C, respectively. Chit3 was stable at 40oC and retained 71% of its activity at 50°C after 60 min, while Chit4 was stable at 50°C and retained 56% of its activity at 60°C after 30 min. Both enzymes produced chitobiose as the major product using colloidal chitin, chitooligosaccharides and shrimp shell powder as substrates. Of the fungal strains tested, Chit3 displayed antifungal activity against Penicillium sp. and Aspergillus sp. This is the first report on the multi-chitinolytic system of T. lanuginosus and enzyme characterization has shown the potential of the enzymes to be used in degradation of the under-utilized bio-resource chitin.
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