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1.
Chitinase [EC 3.2.1.14] is an enzyme that can hydrolyze the beta-1,4 linkage between N-acetyl-D-glucosamine in chitin. In the genome database of the hyperthermophilic archaeon Pyrococcus furiosus, we found two adjacent genes (PF1233 and PF1234) homologous to those of the chitinase of Thermococcus kodakaraensis. In the cultured medium of P. furiosus, however, no chitinase activity was detected. On analysis of the structural gene of P. furiosus, it appears that one nucleotide insertion in PF1234 caused a frame shift and separated a gene. By deletion of one nucleotide in PF1234, the best match was achieved between chitinases of T. kodakaraenesis and P. furiosus. We succeeded in constructing an artificial recombinant chitinase exhibiting hydrolytic activity toward not only colloidal but also crystalline chitins at high temperature. Furthermore, by analyzing the characteristics of the domains, a recombinant enzyme comprising two domains exhibiting high activity toward crystalline chitin was prepared.  相似文献   

2.
A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.  相似文献   

3.
The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.  相似文献   

4.
Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiABD), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiBBD [11 kDa]), followed by a catalytic domain (ChiBcat [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.  相似文献   

5.
A hyperthermophilic β-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).  相似文献   

6.
A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg−1. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the Km, kcat and catalytic efficiency (kcat/Km) values of recombinant chitinase were found to be 1.27 mg ml−1, 0.69 s−1 and 0.54 s−1M−1 respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.  相似文献   

7.
A gene encoding a putative arylsulfatase from the hyperthermophilic archaeon Pyrococcus furiosus was identified, cloned, and expressed as a fusion protein with a Sce VMA intein and chitin binding domain (CBD) residue. The gene (PF1345) from P. furiosus encoding a 35 kDa protein showed some similarity (17 ~ 19%) with other arylsulfatases from the bacteria. The recombinant fusion arylsulfatase was overexpressed in E. coli and partially purified. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The optimal temperature and pH for arylsulfatase activity were found to be 45°C and 9.5, respectively. Various divalent cations (Ca2+, Mg2+, Co2+, Cu2+, Zn2+, and Mn2+) slightly activated the arylsulfatase activity in a narrow range of concentrations (below 0.5 mM), whereas Zn2+ concentrations above 2.0 mM significantly inhibited the activity. After the reaction of agar with recombinant fusion arylsulfatase for 12 h at 50°C, 75% of the sulfate in the agar was removed, and the DNA migration was greatly enhanced. Therefore, the arylsulfatase in this study could be applicable for the production of electrophoretic grade agarose by removing sulfate groups in agar.  相似文献   

8.
Summary A DNA fragment encoding for 598 amino acids of chitinase protein from Bacillus circulans No. 4.1 was subcloned into pQE-30 expression vector and transformed into Escherichia coli M15 (pREP4). The molecular weight of the expressed protein was approximately 66 kDa. Enzymatic activity of the recombinant protein was assayed after purification using affinity chromatography on a nickel chelating resin. The enzyme hydrolyzed N-acetylchitooligosaccharides mainly to N-acetylchitobiose, and was active toward chitin, carboxymethyl-chitin, colloidal chitin, glycol chitin and 4-methylumbelliferyl-β-d-N, N′-diacetylchitobiose. The pH and temperature optima of the chitinase enzyme were 7.0 and 45 °C, respectively. This enzyme was stable in the pH range of 5.0–9.0 and at temperatures up to 50 °C. In addition, when cleaved by a proteolytic enzyme, the 20-kDa product could retain high chitinolytic activity.  相似文献   

9.
A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeromonas caviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of the truncated version of the gene in Escherichia coli. The chitinase gene product showed amino-acid sequence similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotometrically, using colloidal chitin azure as substrate for extracellular and intracellular fractions. The ability of the chitinase cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host.  相似文献   

10.
Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi.  相似文献   

11.
A new alkalophilic low-molecular-mass chitinase of 14 kD from the potent biocontrol agent Bacillus subtilis JN032305 was partially purified and enzymology of the chitinase was studied. The enzyme showed optimal pH of 9.0 and temperature of 50°C. The enzyme was found stable during the 60-min incubation at 50°C. The chitinase was inhibited by group specific agents like IAA, DAN, TLCK, and SDS and metal ions Mg2+, Ca2+, Fe2+, Mn2+, Ba2+, and Hg2+, whereas Zn2+ did not show significant inhibitory effect against the chitinase. PMSF partially inhibited the enzyme. Substrates specificity tests indicated that the enzyme showed 75% of relative activity on glycol chitin, 58% on carboxymethylcellulose (CMC), 33% on chitin flakes, and 166% laminarin compared to that on colloidal chitin. The enzyme also hydrolyzed 4-methylumbelliferyl-N-acetyl-D-glucosaminide, indicating its chitobiase activity. The chitinase of this study has broad specificity, which could hydrolyze not only the glycosidic bond in GlcNAc–GlcNAc but also that of related carbohydrates with glycosidic linkages. The partially purified chitinase not only showed antifungal activity against Rhizoctonia solani and Colletotrichum gloeosporioides, two potent phytopathogens of chilli, but also increased the germination of chilli seeds when infected with the two potent phytopathogenic fungi.  相似文献   

12.
A gene encoding a novel chitinase chi58 was cloned from the fungus Chaetomium cupreum by using inverse PCR. The DNA sequence of chi58 contains a 1,602 bp open reading frame and two introns that are 52 and 201 bp in length. Regarding our in silico analysis, chi58 is a modular enzyme composed of a family-18 catalytic domain, which is responsible for chitinase activity, and a chitin-binding domain containing several cysteines. Apparently, the function of these domains is to anchor the enzyme tightly onto the large insoluble polymeric substrate. Chi58 has a pI of 4.47 and a deduced molecular mass of 58 kDa. The optimal pH and temperature conditions were determined to be 5.8 and 45°C, respectively, when colloidal chitin was used as the substrate. SDS-PAGE and zymogram analyses indicated the presence of a single active chitinase. Cells with pPIC9K-chi58 produced an extracellular chitinase that had an activity of 39 U/ml protein. Metal ions such as Ba2+, Mg2+, K+, Cu2+, Fe3+, Zn2+, and Co2+ also influenced the activity of the recombinant enzyme.  相似文献   

13.
Chitinases are digestive enzymes that break down glycosidic bonds in chitin. In the current study, an endochitinase gene Lbchi31 was cloned from Limonium bicolor. The cDNA sequence of Lbchi31 was 1,107 bp in length, encoding 322 amino acid residues with a calculated molecular mass of 31.7 kDa. Clustal analysis showed that there was a highly conserved chitin-binding domains in Lbchi31 protein, containing four sulfide bridges. The Lbchi31 gene was inserted into the pPIC9 vector and transferred into yeast Pichia pastoris GS115 and KM71 for heterologous expression. The transformant harboring the Lbchi31 gene showed a clearly visible protein band with a molecular mass of more than 31 kDa in the SDS-PAGE gel, indicating that it had been translated in P. pastoris. Enzyme characterization showed that the optimal reaction condition for chitinase LbCHI31 activity was: 40°C, pH of 5.0 and 5 mmol l−1 of Mn2+. The maximum enzyme activity was 0.88 U ml−1 following exposure to the cell wall chitin of Valsa sordida. The LbCHI31 enzyme can efficiently degrade cell wall chitin of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, V. sordida, Septoria tritici and Phytophthora sojae, suggesting that it has the biocontrol function to fungal phytopathogen.  相似文献   

14.
Streptomyces venezuelae P10 could produce extracellular chitinase in a medium containing 0.6% colloidal chitin that was fermented for 96 hours at 30°C. The enzyme was purified to apparent homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography and DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 66 kDa. The chitinase was characterized, and antifungal activity was observed against phytopathogens. Also, the first 15 N-terminal amino-acid residues of the chitinase were determined. The chitin hydrolysed products were N-acetylglucosamine and N, N-diacetylchitobiose.  相似文献   

15.
The HschiA1 gene of the archaeon Halobacterium salinarum CECT 395 was cloned and overexpressed as an active protein of 66.5 kDa in Escherichia coli. The protein called HsChiA1p has a modular structure consisting of a glycosyl hydrolase family 18 catalytic region, as well as a N-terminal family 5 carbohydrate-binding module and a polycystic kidney domain. The purified recombinant chitinase displayed optimum catalytic activity at pH 7.3 and 40 °C and showed high stability over broad pH (6–8.5) and temperature (25–45 °C) ranges. Protein activity was stimulated by the metal ions Mg+2, K+, and Ca+2 and strongly inhibited by Mn+2. HsChiA1p is salt-dependent with its highest activity in the presence of 1.5 M of NaCl, but retains 20 % of its activity in the absence of salt. The recombinant enzyme hydrolysed p-NP-(GlcNAc)3, p-NP-(GlcNAc), crystalline chitin, and colloidal chitin. From its sequence features and biochemical properties, it can be identified as an exo-acting enzyme with potential interest regarding the biodegradation of chitin waste or its bioconversion into biologically active products.  相似文献   

16.
A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.  相似文献   

17.
A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus (chi19) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N-acetylated chitooligosaccharides, (GlcNAc) n , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc) n at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)2 from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.  相似文献   

18.
Aims: To reveal the cause of the difference in activity of chitinase A from Vibrio proteolyticus and chitinase A from a strain of Vibrio carchariae (a junior synonym of Vibrio harveyi), we investigated the pH‐dependent activity of full‐length V. proteolyticus chitinase A and a truncated recombinant corresponding to the V. harveyi form of chitinase A. Methods and Results: After overexpression in Escherichia coli strain DH5α, the full‐length and truncated recombinant chitinases were purified by ammonium sulphate precipitation and anion exchange column chromatography. Chitinase activity was measured at various pH values using α‐crystal and colloidal chitins as the substrate. The pH‐dependent patterns of the relative specific activities for α‐crystal chitin differed between the full‐length and truncated recombinant chitinases, whereas those for colloidal chitin were similar to each other. Conclusion: The difference in the activity of V. proteolyticus chitinase A and V. harveyi chitinase A might be partly due to a change in the pH dependence of the chitinase activities against α‐crystal chitin, resulting from C‐terminal processing. Significance and Impact of Study: The present results are important findings for not only ecological studies on the genus Vibrio in association with survival strategies, but also phylogenetic studies.  相似文献   

19.
The chitinase (EC 3.2.1.14) of the human malaria parasite Plasmodium falciparum, PfCHT1, has been validated as a malaria transmission-blocking vaccine (TBV). The present study aimed to delineate functional characteristics of the P. vivax chitinase PvCHT1, whose primary structure differs from that of PfCHT1 by having proenzyme and chitin-binding domains. The recombinant protein rPvCHT1 expressed with a wheat germ cell-free system hydrolyzed 4-methylumbelliferone (4MU) derivatives of chitin oligosaccharides (β-1,4-poly-N-acetyl glucosamine (GlcNAc)). An anti-rPvCHT1 polyclonal antiserum reacted with in vitro-obtained P. vivax ookinetes in anterior cytoplasm, showing uneven patchy distribution. Enzymatic activity of rPvCHT1 shared the exclusive endochitinase property with parallelly expressed rPfCHT1 as demonstrated by a marked substrate preference for 4MU-GlcNAc3 compared to shorter GlcNAc substrates. While rPvCHT1 was found to be sensitive to the general family-18 chitinase inhibitor, allosamidin, its pH (maximal in neutral environment) and temperature (max. at ~ 25 °C) activity profiles and sensitivity to allosamidin (IC50 = 6 µM) were different from rPfCHT1. The results in this first report of functional rPvCHT1 synthesis indicate that the P. vivax chitinase is enzymatically close to long form Plasmodium chitinases represented by P. gallinaceum PgCHT1.  相似文献   

20.
Activity was found for chitinase and chitobiase in the crystalline styles of American oysters (Crassostrea virginica Gmelin) collected from the Chesapeake Bay (Maryland, USA). The oysters were maintained in tanks on natural food from a constant flow of unfiltered estuarine water. Chitinase and chitobiase specific activities were compared with total, viable, and chitinoclastic bacterial counts andCristispira counts. Regression analyses revealed that one correlation, chitobiase vsCristispira, was significant (P < 0.05). Several oysters were fed chitin in the presence or absence of chloramphenicol. Although no chitinoclasts were present in the antibiotic-treated oysters, the treatment means did not differ significantly (P > 0.05) for either chitinase or chitobiase activity. In several cases with both chitin-fed and naturally fed oysters, enzyme activity was found when noCristispira were present. The results of the investigations suggest that the oyster produces chitinase and chitobiase endogenously.  相似文献   

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