Purification and some properties of chitinase produced by Vibrio sp.

Chitinase was purified from the culture filtrate of a Vibrio sp. isolated from soil and its enzymatic properties were examined. The molecular weight measured by SDS-gel electrophoresis was approximately 100,000. The chitinase hydrolyzed colloidal chitin, chitin powder, chitosan and chitin oligosaccharides more than chitotriose but did not hydrolyze glycolchitin and chitobiose.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

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 K_m, k_cat and catalytic efficiency (k_cat/K_m) 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.     

N,N-Diacetylchitobiose production from chitin by Vibrio anguillarum strain E-383a

Vibrio anguillarum strain E-383a, isolated from sea water, accumulated a considerable amount of N,N '-diacetylchitobiose when it was cultivated in a medium containing colloidal chitin. The maximum conversion of chitin to chitobiose was found to be 40·3%. The rate of chitobiose accumulation was accelerated after the cessation of bacterial growth. Small amounts of N -acetylglucosamine and N,N',N -triacetylchitotriose were also accumulated but no other saccharides were detected. These results may suggest that strain E-383a produces an exo-type chitinase which successively hydrolyses the glycosidic linkages of chitin into biose units. The exclusive accumulation of chitobiose by the bacterial cells may provide a new, selective method for the production of this substance.     

Chitinolytic activity of the sheep rumen ciliate Diploplastron affine

Supplementation of the rumen ciliate Diploplastron affine growth medium with commercial chitin stimulated growth of ciliates and the density of their population was positively correlated with chitin doses (r = 0.95; p < 0.01). The cell-free extracts prepared from bacteria-free ciliates degraded chitin to N-acetyl-D: -glucosamine and chitobiose. Three exochitinases, two endochitinases and two beta-N-acetylglucosaminidases were identified in the cell-free extract of protozoa. The molar mass of exochitinases was 80, 65 and 30 kDa, and endochitinases 75 and 50 kDa; the molar mass of one of the identified beta-N-acetylglucosaminidases was 45 kDa.     

Reverse hydrolysis reaction of chitin deacetylase and enzymatic synthesis of beta-D-GlcNAc-(1-->4)-GlcN from chitobiose

We found that a chitin deacetylase from Colletotrichum lindemuthianum could acetylate free amino sugar residues into N-acetylated forms in the presence of 3.0 M sodium acetate. The result was analyzed using a beta-N-acetyl-hexosaminidase-coupled assay system with p-nitrophenyl 2-amino-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta- D-glucopyranoside as the substrate, and the liberation of p-nitrophenol was observed as a consequence of enzymatic N-acetylation of the glucosamine residue at the nonreducing end of the substrate. The chitin deacetylase also acetylated chitobiose and chitotetraose as substrates, which was evidenced by the decrease in the amount of free amino sugar residues in the chitooligosaccharides. The reaction product of chitobiose after the acetylation reaction was exclusively 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->4)-2-amino-2-deoxy-D-gluc ose [GlcNAcGlcN], the structure of which was determined by FABMS and NMR analyses. This study offers a novel method for enzymatic N-acetylation of amino sugars, and especially with chitobiose as substrate, a selectively N-acetylated product, GlcNAcGlcN, can be synthesized.     

Identification and characterization of genes underlying chitinolysis in Collimonas fungivorans Ter331

Through a combinatorial approach of plasposon mutagenesis, genome mining, and heterologous expression, we identified genes contributing to the chitinolytic phenotype of bacterium Collimonas fungivorans Ter331. One of five mutants with abolished ability to hydrolyze colloidal chitin carried its plasposon in the chiI gene coding for an extracellular endochitinase. Two mutants were affected in the promoter of chiP-II coding for an outer-membrane transporter of chitooligosaccharides. The remaining two mutations were linked to chitobiose/N-acetylglucosamine uptake. Thus, our model for the Collimonas chitinolytic system assumes a positive feedback regulation of chitinase activity by chitin degradation products. A second chitinase gene, chiII, coded for an exochitinase that preferentially released chitobiose from chitin analogs. Genes hexI and hexII showed coding resemblance to N-acetylglucosaminidases, and the activity of purified HexI protein towards chitin analogs suggested its role in converting chitobiose to N-acetylglucosamine. The hexI gene clustered with chiI, chiII, and chiP-II in one locus, while chitobiose/N-acetylglucosamine uptake genes colocalized in another. Both loci contained genes for conversion of N-acetylglucosamine to fructose-6-phosphate, confirming that C. fungivorans Ter331 features a complete chitin pathway. No link could be established between chitinolysis and antifungal activity of C. fungivorans Ter331, suggesting that the bacterium's reported antagonism towards fungi relies on other mechanisms.     

Properties of Chitosanase from Bacillus cereus S1

Chitosanase from Bacillus cereus S1 was purified, and the enzymatic properties were investigated. The molecular weight was estimated to 45,000 on SDS-PAGE. Optimum pH was about 6, and stable pH in the incubation at 40°C for 60 min was 6–11. This chitosanase was stable in alkaline side. Optimum temperature was around 60°C, and enzyme activity was relatively stable below 60°C. The degradations of colloidal chitosan and carboxymethyl cellulose (CMC) were about 30 and 20% relative to the value of soluble chitosan, respectively, but colloidal chitin and crystalline cellulose were not almost hydrolyzed. On the other hand, S1 chitosanase adsorbed on colloidal chitin completely and by about 50% also on crystalline cellulose, in contrast to colloidal chitosan, which it did not adsorb. S1 chitosanase finally hydrolyzed 100% N-deacetylated chitosan (soluble state) to chitobiose (27.2%), chitotriose (40.6%), and chitotetraose (32.2%). In the hydrolysis of various chitooligosaccharides, chitobiose and chitotriose were not hydrolyzed, and chitotetraose was hydrolyzed to chitobiose. Chitobiose and chitotriose were released from chitopentaose and chitohexaose. From this specificity, it was hypothesized that the active site of S1 chitosanase recognized more than two glucosamine residues posited in both sides against splitting point for glucosamine polymer. Received: 8 June 1999 / Accepted: 20 July 1999     

Purification and properties of chitinase from Streptomyces cinereoruber

A chitinase (EC 3.2.1.14) was purified from the culture filtrate of Streptomyces cinereoruber, selected as a microorganism which produces enzymes lysing Aspergillus niger cell wall, by fractional precipitation with ammonium sulfate and column chromatographies on DEAE-cellulose, Sephadex G-100 and CM-Sephadex C-50. The final preparation was homogenous in polyacrylamide gel disc electrophoresis. The molecular weight of the enzyme was about 19,000 daltons and its isoelectric point was pH 8.6. The optimum pH and temperature for chitinase activity were 4.5 and at 50°C, respectively. The enzyme was stable in the pH range from 4.0 to 10.0. The activity was inhibited by Ag⁺, Hg⁺, Hg²⁺ and p-chloromercuribenzoate. Paper chromatographic analysis demonstrated that the hydrolytic products of colloidal chitin and chitotriose with the enzyme were N-acetylglucosamine and chitobiose. The lysis of A. niger cell wall with the enzyme is discussed.     

弧菌几丁质降解代谢及调控机理的研究进展

对近年来在弧菌中几丁质降解代谢的主要过程及调控机理方面的研究进行了综述,指出弧菌降解几丁质主要分为3个步骤,几丁质的水解、几丁寡糖和N-乙酰基葡萄糖胺的运输、几丁二糖和N-乙酰基葡萄糖胺-6-磷酸的进一步降解等,且此过程的许多环节均受到二元信号传导系统的调控。     

Production of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

The pharmaceutically important compound N-acetylglucosamine (NAG), is used in various therapeutic formulations, skin care products and dietary supplements. Currently, NAG is being produced by an environment-unfriendly chemical process using chitin, a polysaccharide present in abundance in the exoskeleton of crustaceans, as a substrate. In the present study, we report the potential of an eco-friendly biological process for the production of NAG using recombinant bacterial enzymes, chitinase (CHI) and chitobiase (CHB). The treatment of chitin with recombinant CHI alone produced 8% NAG and 72% chitobiose, a homodimer of NAG. However, supplementation of the reaction mixture with another recombinant enzyme, CHB, resulted in approximately six fold increase in NAG production. The product, NAG, was confirmed by HPLC, TLC and ESI-MS studies. Conditions are being optimized for increased production of NAG from chitin.     

Genetics and Regulation of Chitobiose Utilization in Borrelia burgdorferi

Borrelia burgdorferi spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of N-acetylglucosamine (GlcNAc). The B. burgdorferi celA, celB, and celC genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the celB gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes chbA, chbB, and chbC, since they probably encode a chitobiose transporter. We also found that the chbC gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.     

Chitinase in bean leaves: induction by ethylene,purification, properties,and possible function

Ethylene induced an endochitinase in primary leaves of Phaseolus vulgaris L. The enzyme formed chitobiose and higher chitin oligosaccharides from insoluble, colloidal or regenerated chitin. Less than 5% of the total chitinolytic activity was detected in an exochitinase assay proposed by Abeles et al. (1970, Plant Physiol. 47, 129–134) for ethylene-induced chitinase. In ethylene-treated plants, chitinase activity started to increase after a lag of 6 h and was induced 30 fold within 24 h. Exogenously supplied ethylene at 1 nl ml^?1 was sufficient for half-maximal induction, and enhancement of the endogenous ethylene formation also enhanced chitinase activity. Cycloheximide prevented the induction. Among various hydrolases tested, only chitinase and, to a lesser extent, β-1,3-glucanase were induced by ethylene. Induction of chitinase by ethylene occurred in many different plant species. Ethylene-induced chitinase was purified by affinity chromatography on a column of regenerated chitin. Its apparent molecular weight obtained by sodium dodecyl sulfate-gel electrophoresis was 30,000; the molecular weight determined from filtration through Sephadex G-75 was 22,000. The purified enzyme attacked chitin in isolated cell walls of Fusarium solani. It also acted as a lysozyme when incubated with Micrococcus lysodeikticus. It is concluded that ethylene-induced chitinase functions as a defense enzyme against fungal and bacterial invaders.     

Fractionation of oligosaccharides containing N-acetyl amino sugars by reverse-phase high-pressure liquid chromatography

High-pressure liquid chromatography (hplc) of N-acetylglucosamine (GlcNAc) and galactosamine (GalNAc) containing carbohydrates was performed on several reverse-phase silica columns. Nanomolar level detection was accomplished using far uv-absorbance monitoring. Baseline separations of the α and β anomers of GlcNAc, chitobiose, chitotriose, and chitotetraose were observed with water elution of the reverse-phase column. With the addition of up to 3% acetonitrile to the eluting solvent, similar resolution of chitin oligomers up to a chain length of seven was observed. Anomerization of the residue could be followed by isolation of either anomeric peak with subsequent rechromatography. Reduction of chitotriose with borohydride yielded a single sharp peak with a retention volume similar to that of the reducing trisaccharide. Semipreparative reverse-phase hplc allowed for the separation and identification by ¹³C NMR of the GlcNAc-α-1→6 GlcNAc disaccharide from the β-1→6 isomer. Methyl glycosides of GalNAc and GlcNAc were shown to have retention times much longer than the free sugar.     

The chitobiose transporter, <Emphasis Type="Italic">chbC</Emphasis>, is required for chitin utilization in <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background  

The bacterium Borrelia burgdorferi, the causative agent of Lyme disease, is a limited-genome organism that must obtain many of its biochemical building blocks, including N-acetylglucosamine (GlcNAc), from its tick or vertebrate host. GlcNAc can be imported into the cell as a monomer or dimer (chitobiose), and the annotation for several B. burgdorferi genes suggests that this organism may be able to degrade and utilize chitin, a polymer of GlcNAc. We investigated the ability of B. burgdorferi to utilize chitin in the absence of free GlcNAc, and we attempted to identify genes involved in the process. We also examined the role of RpoS, one of two alternative sigma factors present in B. burgdorferi, in the regulation of chitin utilization.     

Chitinase is an inducible enzyme in Beauveria bassiana

Sterilization of chitin by autoclaving or boiling causes release of d-glucosamine and N-acetylglucosamine from the macromolecule and these solubilized components actually function as the inducers for synthesis of chitinase. The insoluble macromolecule is not an inducer of chitinase since sterilization by dry heat or chloroform will not bring about release of the amino sugars or induction of the enzyme. Free glucosamine, N-acetylglucosamine, and chitobiose are all good inducers of chitinase. Most sustained synthesis of the enzyme occurs in an autoclaved chitin-salts medium.     

Enhanced enzymatic hydrolysis of langostino shell chitin with mixtures of enzymes from bacterial and fungal sources

A combination of enzyme preparations from Trichoderma atroviride and Serratia marcescens was able to completely degrade high concentrations (100 g/L) of chitin from langostino crab shells to N-acetylglucosamine (78%), glucosamine (2%), and chitobiose (10%). The result was achieved at 32 degrees C in 12 days with no pre-treatment (size reduction or swelling) of the substrate and without removal of the inhibitory end-products from the mixture. Enzymatic degradation of three forms of chitin by Serratia/Trichoderma and Streptomyces/Trichoderma blends was carried out according to a simplex-lattice mixture design. Fitted polynomial models indicated that there was synergy between prokaryotic and fungal enzymes for both hydrolysis of crab chitin and reduction of turbidity of colloidal chitin (primarily endo-type activity). Prokaryotic/fungal enzymes were not synergistic in degrading chitosan. Enzymes from prokaryotic sources had much lower activity against chitosan than enzymes from T. atroviride.     

β-N-Acetylglucosaminidase from Aspergillus nidulans which degrades chitin oligomers during autolysis

A hexosaminidase from autolyzed cultures of Aspergillus nidulans was purified 196 fold and characterized as a beta-N-acetylglucosaminidase (EC 3.2.1.30). The enzyme has a MW of 190000, a pI of 4.3, and optimum pH of 5.0 and is unstable at temperatures above 50 degrees C. The enzyme is a glycoprotein with 19.5% sugars, mannose being the principal component. It binds strongly to chitin. The enzyme hydrolyzes different substrates. The Ki with the competitive inhibitor 2-acetamido-2-deoxy-D-gluconolactone was independent of the substrate used. The enzyme was inhibited by Hg2+, Ag+, acetate and other organic anions. The kinetics of hydrolysis of chitin oligosaccharides from 2 to 6 units was studied by HPLC. This enzyme is an exoenzyme which degraded chitin oligomers gradually with the production of N-acetylglucosamine. The hydrolysis of N-N'-diacetylchitobiose was inhibited non-competitively by glucosamine and N-acetylglucosamine. In mixtures of chitin oligosaccharides, the hydrolysis of chitobiose was competitively inhibited by each of the other oligomers.     

Induction and repression of a Streptomyces lividans chitinase gene promoter in response to various carbon sources

Induction and repression of a gene for chitinase (chiA) in Streptomyces lividans was investigated using a catechol 2,3-dioxygenase gene (xylE) as the reporter gene. Of various substrates examined, expression of the promoter (PchiA) was observed after a delay when colloidal chitin or small chitin-oligosaccharides were added to the medium. N-acetylglucosamine completely repressed the chiA promoter. The duration of the delay in expression of PchiA differed with the inducer used, with chitobiose inducing the activity most rapidly. The minimum concentration of chitobiose needed for induction was 1 microM. It appears, therefore, that an efficient inducer of the gene for chitinase in S. lividans is chitobiose.     

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the ¹⁴C-labeled crystalline polymeric substrates ¹⁴C chitin nanowhiskers and ¹⁴C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed K_i(obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the K_i(obs) of Cel7A was an order of magnitude higher than the true K_i value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.     

Detection of bacterial chitinase activity in transformed plant tumour cells using a specific exochitinase substrate

Methods for the detection of bacterial chitinase activity were compared. The soluble substrate p-nitrophenyl-ß-D-N,N diacetyl chitobiose (NDC) was more sensitive in detecting purified chitinase of Serratia marcescens than assays measuring degradation of a solid chitin substrate by either radiochemical or colorimetric means. A chimaeric gene containing a S. marcescens chitinase gene under control of a Cauliflower Mosaic Virus 35S promoter and nopaline synthase terminator sequences was constructed and transferred to tobacco tumour cells using Agrobacterium tumefaciens as a vector. The rate of hydrolysis of the NDC substrate was three fold greater with cell extracts of both pooled and individual tumours carrying the chimaeric chitinase gene than in control tumours. It was calculated from the enzyme activity data that the foreign bacterial chitinase contributed 0.1% of the total soluble protein in transformed plant cells. This level of expression of this gene was not detectable using the less sensitive assays employing solid chitin substrate. These results indicate that NDC is a preferable substrate for assaying bacterial chitinase in transformed plant cells.