Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity

Streptomyces griseus HUT 6037 inducibly produced two chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60 degrees C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanases were obtained for identification of the anomeric form of the reaction products. Both chitosanases produced the beta-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding chitosanase II. Sequencing of the choII gene showed an open reading frame encoding a protein with 359 amino acid residues. The deduced primary structure was similar to endoglucanase E-5 of Thermomonospora fusca, which enzyme belongs to family 5 of the glycosyl hydrolases. This is the first report of a family 5 chitosanase with transglycosylation activity.     

Quantitative fluorometric analysis of the protective effect of chitosan on thermal unfolding of catalytically active native and genetically-engineered chitosanases

We have taken advantage of the intrinsic fluorescence properties of chitosanases to rapidly and quantitatively evaluate the protective effect of chitosan against thermal denaturation of chitosanases. The studies were done using wild type chitosanases N174 produced by Streptomyces sp. N174 and SCO produced by Streptomyces coelicolor A3(2). In addition, two mutants of N174 genetically engineered by single amino acid substitutions (A104L and K164R) and one "consensus" (N174-CONS) chitosanase designed by multiple amino acid substitutions of N174 were analyzed. Chitosan used had a weight average molecular weight (Mw) of 220 kDa and was 85% deacetylated. Results showed a pH and concentration-dependent protective effect of chitosan in all the cases. However, the extent of thermal protection varied depending on chitosanases, suggesting that key amino acid residues contributed to resistance to heat denaturation. The transition temperatures (T(m)) of N174 were 54 degrees C and 69.5 degrees C in the absence and presence (6 g/l) of chitosan, respectively. T(m) were increased by 11.6 degrees C (N174-CONS), 13.8 degrees C (CSN-A104L), 15.6 degrees C (N174-K164R) and 25.2 degrees C (SCO) in the presence of chitosan (6 g/l). The thermal protective effect was attributed to an enzyme-ligand thermostabilization mechanism since it was not mimicked by the presence of anionic (carboxymethyl cellulose, heparin) or cationic (polyethylene imine) polymers, polyhydroxylated (glycerol, sorbitol) compounds or inorganic salts. Furthermore, the data from fluorometry experiments were in agreement with those obtained by analysis of reaction time-courses performed at 61 degrees C in which case CSN-A104L was rapidly inactivated whereas N174, N174-CONS and N174-K164R remained active over a reaction time of 90 min. This study presents evidence that (1) the fluorometric determination of T(m) in the presence of chitosan is a reliable technique for a rapid assessment of the thermal behavior of chitosanases, (2) it is applicable to structure-function studies of mutant chitosanases and, (3) it can be useful to provide an insight into the mechanism by which mutations can influence chitosanase stability.     

A common molecular signature unifies the chitosanases belonging to families 46 and 80 of glycoside hydrolases

The 3D structure-oriented alignment of the primary sequences of fourteen chitosanases, mainly of bacterial origin and belonging to families 46 and 80 of glycoside hydrolases, resulted in the identification of the following pattern common to all these enzymes: E-[DNQ]-x(8,17)-Y-x(7)-D-x-[RD]-[GP]-x-[TS]-x(3)-[AIVFLY]-G- x(5,11)-D. This pattern is proposed as the molecular signature of the chitosanases from families 46 and 80. It includes several amino acids essential for enzyme activity and (or) stability as shown by site-directed mutagenesis studies on the chitosanase from Streptomyces sp. N174. In particular, it includes two carboxylic residues directly involved in catalysis. We suggest that there is a continuum of sequence similarity between all the analyzed chitosanases, and that all these enzymes should probably be classified in one family.     

Uncoupling chitosanase production from chitosan

There is a growing interest in chitosanases as enzymatic tools to hydrolyze chitosan into bioactive forms: low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS). However chitosanases are still expensive and methods of large-scale production of these enzymes are not yet established. The article reviews the approaches used for chitosanase production in various bacterial hosts, pointing out the difficulties resulting from the necessity to include chitosan into the medium composition. A mutated Streptomyces host allows for the efficient production of several chitosanases originating from actinobacteria in the absence of chitosan as inducer.     

Isolation, purification, and enzymatic characterization of extracellular chitosanase from marine bacterium Bacillus subtilis CH2

A Bacillus subtilis strain was isolated from the intestine of Sebastiscus marmoratus (scorpion fish) that was identified as Bacillus subtilis CH2 by morphological, biochemical, and genetic analyses. The chitosanase of Bacillus subtilis CH2 was best induced by fructose and not induced with chitosan, unlike other chitosanases. The strain was incubated in LB broth, and the chitosanase secreted into the medium was concentrated with ammonium sulfate precipitation and purified by gel permeation chromatography. The molecular mass of the purified chitosanase was detected as 29 kDa. The optimum pH and temperature of the purified chitosanase were 5.5 and 60°C, respectively. The purified chitosanase was continuously thermostable at 40°C. The specific acitivity of the purified chitosanase was 161 units/mg. The N-terminal amino acid sequence was analyzed for future study.     

Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.     

Modification of genetic regulation of a heterologous chitosanase gene in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> TK24 leads to chitosanase production in the absence of chitosan

Background  

Chitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous chitosanase gene (csnN106) in Streptomyces lividans.     

A Streptomyces chitosanase is active in transgenic tobacco

Summary Growth inhibition towards Rhizopus nigricans, Fusarium oxysporum f. sp. radicis-lycopersici, Verticillium albo-atrum and Pythium ultimum was observed in vitro using a purified chitosanase from an actinomycete, Streptomyces sp, strain N174. The corresponding gene, with its own signal peptide, was inserted into pBI121.7 shuttle vector to transform tobacco. Transgenic plants were analysed for chitosanase activity by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay. Two major and one minor active electrophoretic forms were detected in transgenic tobacco. Some chitosanases were recovered not only in leaf homogenates but also in leaf intercellular fluid extracts. One chitosanase electrophoretic form migrated very closely to the purified Streptomyces mature protein while the others corresponded to molecules of higher molecular mass. The N-terminus sequence was determined for one of the three chitosanase forms. It exhibited a different signal peptide cleavage site when compared to the mature chitosanase from Streptomyces. This is the first report on the expression of an active chitosanase gene with antimicrobial potential in plants.Abbreviations aa amino acid - CIP calf intestinal phosphatase - CM carboxymethyl - GUS ß-glucuronidase - IF intercellular fluid - MS Murashige and Skoog - PAGE polyacrylamide gel electrophoresis - PR pathogenesisrelated - PVDF polyvinylidene difluoride - SP signal peptide     

Identification and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies

The Gram-positive spore-forming bacterium, Bacillus thuringiensis, a member of the Bacillus cereus group, produces chitosanases that catalyze the hydrolysis of chitosan to chitosan-oligosaccharides (COS). Although fungal and bacterial chitosanases belonging to other glycoside hydrolase (GH) families have been characterized in a variety of microorganisms, knowledge on the genetics and phylogeny of the GH-8 chitosanases remains limited. Nine genes encoding chitosanases were cloned from 29 different serovar strains of B. thuringiensis and they were expressed in Escherichia coli. The ORFs of the chitosanases contained 1,359 nucleotides and the protein products had high levels of sequence identity (>96%) to other Bacillus species GH-8 chitosanases. Thin-layer chromatography and HPLC analyses demonstrated that these enzymes hydrolyzed chitosan to a chitosan-trimer and a chitosan-tetramer as major products, and this could be useful in the production of COS. In addition, a simple plate assay was developed, involving a soluble chitosan, for high-throughput screening of chitosanases. This system allowed screening for mutant enzymes with higher enzyme activity generated by error-prone PCR, indicating that it can be used for directed chitosanase evolution.     

Crystal structure of chitosanase from Bacillus circulans MH-K1 at 1.6-A resolution and its substrate recognition mechanism.

Chitosanase from Bacillus circulans MH-K1 is a 29-kDa extracellular protein composed of 259 amino acids. The crystal structure of chitosanase from B. circulans MH-K1 has been determined by multiwavelength anomalous diffraction method and refined to crystallographic R = 19.2% (R(free) = 23.5%) for the diffraction data at 1.6-A resolution collected by synchrotron radiation. The enzyme has two globular upper and lower domains, which generate the active site cleft for the substrate binding. The overall molecular folding is similar to chitosanase from Streptomyces sp. N174, although there is only 20% identity at the amino acid sequence level between both chitosanases. However, there are three regions in which the topology is remarkably different. In addition, the disulfide bridge between Cys(50) and Cys(124) joins the beta1 strand and the alpha7 helix, which is not conserved among other chitosanases. The orientation of two backbone helices, which connect the two domains, is also different and is responsible for the differences in size and shape of the active site cleft in these two chitosanases. This structural difference in the active site cleft is the reason why the enzymes specifically recognize different substrates and catalyze different types of chitosan degradation.     

Characterization of a Chitosanase from Aspergillus fumigatus ATCC13073

Chitosanase II was purified from the culture filtrate of Aspergillus fumigatus ATCC13073. The purified enzyme had a molecular mass of 23.5 kDa. The N-terminal amino acid sequence of chitosanase II was identical to those of other Aspergillus chitosanases belonging to glycoside hydrolase family 75. The optimum pH and temperature were pH 6.0 and 40 °C. Chitosanase II hydrolyzed 70% deacetylated chitosan faster than fully deacetylated chitosan. Analysis of the degradation products generated from partially N-acetylated chitosan showed that chitosanase II split GlcN-GlcN and GlcNAc-GlcN bonds but not GlcNAc-GlcNAc or GlcN-GlcNAc, suggesting that it is a subclass I chitosanase. It degraded (GlcN)(6) to produce (GlcN)(3) as main product and small amounts of (GlcN)(2) and (GlcN)(4). Reaction rate analyses of mono-N-acetylated chitohexaose suggested that the (+3) site of chitosanase II recognizes the GlcNAc residue rather than the GlcN residue of its substrate.     

Production of Two Chitosanases from a Chitosan-Assimilating Bacterium, Acinetobacter sp. Strain CHB101

A bacterial strain capable of utilizing chitosan as a sole carbon source was isolated from soil and was identified as a member of the genus Acinetobacter. This strain, designated CHB101, produced extracellular chitosan-degrading enzymes in the absence of chitosan. The chitosan-degrading activity in the culture fluid increased when cultures reached the early stationary phase, although the level of activity was low in the exponential growth phase. Two chitosanases, chitosanases I and II, which had molecular weights of 37,000 and 30,000, respectively, were purified from the culture fluid. Chitosanase I exhibited substrate specificity for chitosan that had a low degree of acetylation (10 to 30%), while chitosanase II degraded colloidal chitin and glycol chitin, as well as chitosan that had a degree of acetylation of 30%. Rapid decreases in the viscosities of chitosan solutions suggested that both chitosanases catalyzed an endo type of cleavage reaction; however, chitosan oligomers (molecules smaller than pentamers) were not produced after a prolonged reaction.     

Purification and Characterization of Three Chitosanase Activities from Bacillus megaterium P1

Bacillus megaterium P1, a bacterial strain capable of hydrolyzing chitosan, was isolated from soil samples. Chitosan-degrading activity was induced by chitosan but not by its constituent d-glucosamine. Extracellular secretion of chitosanase reached levels corresponding to 1 U/ml under optimal conditions. Three chitosan-degrading proteins (chitosanases A, B, and C) were purified to homogeneity. Chitosanase A (43 kilodaltons) was highly specific for chitosan and represented the major chitosan-hydrolyzing species. Chitosanases B (39.5 kilodaltons) and C (22 kilodaltons) corresponded to minor activities and possessed comparable specific activities toward chitosan, chitin, and cellulose. Chitosanase A was active from pH 4.5 to 6.5 and was stable on the basis of activity up to 45 degrees C. The optimum temperature for enzymatic chitosan hydrolysis was 50 degrees C. Kinetic studies on chitosanase A suggest that the enzyme is substrate inhibited. The apparent K(m) and V(max) determined at 22 degrees C and pH 5.6 were 0.8 mg/ml and 280 U/mg, respectively. End products of chitosan hydrolysis by each of the three chitosanases were identified as glucosamine oligomers, similar to those obtained for previously reported chitosanase digestions.     

Automated screen of a preliminary lead-drug for chitosanase drug design

Chitosan is a naturally occurring component of certain bacterial and fungal cell walls. If some groups of medically and agriculturally significant fungi contain chitosan, chitosan metabolism represents attractive drug targets specific to those fungal systems. Recently, structure-based drug design emerges as a powerful technique in drug screening. The process initially requires three dimensional structure of a target molecule. Because the bacterialStreptomyces lividans N174 chitosanase is only one chitosanase whose X-ray structure has been solved, we begin the process of structure-based drug design with the bacterial enzyme but it should be extended to a fungal one. In order to initiate the process, a preliminary lead-drug was screened by automated computer search from chemical databases. The 5-nitro-isatin showed an inhibitory effect by 50% at 1.5 mM on theStreptomyces lividans N174 chitosanase.     

Molecular Characterization of a Novel Family-46 Chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5–8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.     

New chitosan-degrading strains that produce chitosanases similar to ChoA of Mitsuaria chitosanitabida

The betaproteobacterium Mitsuaria chitosanitabida (formerly Matsuebacter chitosanotabidus) 3001 produces a chitosanase (ChoA) that is classified in glycosyl hydrolase family 80. While many chitosanase genes have been isolated from various bacteria to date, they show limited homology to the M. chitosanitabida 3001 chitosanase gene (choA). To investigate the phylogenetic distribution of chitosanases analogous to ChoA in nature, we identified 67 chitosan-degrading strains by screening and investigated their physiological and biological characteristics. We then searched for similarities to ChoA by Western blotting and Southern hybridization and selected 11 strains whose chitosanases showed the most similarity to ChoA. PCR amplification and sequencing of the chitosanase genes from these strains revealed high deduced amino acid sequence similarities to ChoA ranging from 77% to 99%. Analysis of the 16S rRNA gene sequences of the 11 selected strains indicated that they are widely distributed in the beta and gamma subclasses of Proteobacteria and the Flavobacterium group. These observations suggest that the ChoA-like chitosanases that belong to family 80 occur widely in a broad variety of bacteria.     

Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.

We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.     

壳聚糖酶结构与功能的研究进展

壳聚糖酶是一类对壳聚糖具有较高催化活性而几乎不水解几丁质的糖苷水解酶,其可将高分子量的壳聚糖转化为低分子量的功能性壳寡糖。近年来,对壳聚糖酶的相关研究取得了显著进展,因此,本文对其生化性质、晶体结构、催化机制和蛋白质工程改造进行总结和探讨,并对酶法制备壳寡糖纯品进行展望,这将加深研究者对壳聚糖酶作用机制的认识,推动壳聚糖酶的工业应用。     

Purification and characterization of chitosanase from Bacillus sp. strain KCTC 0377BP and its application for the production of chitosan oligosaccharides

For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.     

Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent K _m and the catalytic constant k_cat were 0.042 mg/ml and 7,588 min^?1, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way (“3?+?3”) but hydrolysis rate was much faster for (GlcN)₆ than (Glc)₆. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.