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     

Production, purification and characterization of chitosanase produced by Gongronella sp. JG

AIMS: To optimize the production condition of chitosanases of Gongronella sp. JG and to characterize the major chitosanase. METHODS AND RESULTS: In the optimized medium and culturing condition, strain JG produced 800 micromol min(-1) l(-1) chitosanase activity at 72 h. The major chitosanase - csn1 was purified through three chromatography steps: CM (carboxymethyl)-Sepharose fast flow (FF), Sephacryl S200, SP (sulfopropyl)-Sepharose FF. The molecular weight and the pI value of csn1 were about 90,000 Da and 5 x 8, respectively. Its specific activity was 82 micromol min(-1) mg(-1). The optimal reaction pH for csn1 was between 4 x 6 and 4 x 8. The optimal reaction temperature was 50 degrees C. The half-life of csn1 at 50 degrees C was estimated to be about 65 min. Mn(2+) was a strong stimulator of csn1 activity, both at 1 and 10 mmol l(-1). csn1 showed its highest activity with chitosan of 85% degree of deacetylation, but did not hydrolyse colloidal chitin and carboxylmethyl cellulose. In 20 mmol l(-1) sodium acetate buffer (pH 4 x 8) and at 50 degrees C, the K(m) of csn1 was calculated to be 4 x 5 mg ml(-1). CONCLUSIONS: The production condition of chitosanases by Gongronella JG was optimized and the major chitosanase, csn1, was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work for the first time reported the production, purification and characterization of chitosanases produced by fungus of Gongronella sp. These results provided us more information on fungal chitosanases.     

Isolation and characterisation of chitosanase from Bacillus sp. 739 strain

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 has been determined. Maximum enzyme activity was observed in a medium containing the biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity using chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme, assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. Temperature and pH optima of the purified chitosanase were in the ranges 45-55 degrees C and 6.0-6.5, respectively. Time to half-maximum inactivation of the enzyme at 50 degrees C was equal to 1 h. With colloidal chitosan as the substrate, the value of K(M) of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

来自拟青霉属真菌的壳聚糖酶的分离纯化、理化性质及降解产物的分析(英文)

从来自拟青霉属真菌Paecilomyces sp.CS-Z的发酵液中获得一种壳聚糖酶,该酶被纯化了9.4倍,产率为48.2%。经SDS-PAGE分析确定为单一条带,分子量为29kDa,其最适pH为6.0–6.5,最适温度为55℃,在80℃处理60min后,能保持较好的热稳定性,Hg2+完全抑制了酶活,对脱乙酰度85%–95%的壳聚糖具有较高的水解活性,而对几丁质和羧甲基纤维素无活性。薄层层析和质谱分析表明该酶是一种内切酶,其水解产物为聚合度大于6的壳寡糖,其理化性质与至今报道的壳聚糖酶有所不同,为壳聚糖酶的开发提供了重要的实验依据。     

烟曲霉菌壳聚糖酶基因的克隆及在大肠杆菌中的表达

根据GenBank中发布的烟曲霉菌壳聚糖酶（Aspergillus fumigatus chitosanase,EC3.2.1.132）基因序列人工合成8条DNA长链及4条引物链。DNA链的设计上在不改变壳聚糖酶氨基酸组成的前提下选择大肠杆菌使用频率高的密码子。PCR拼接法扩增壳聚糖酶基因并克隆入pGEM_T easy载体进行序列分析,进一步亚克隆入表达载体pGEX_3X。重组质粒pGEX_Csn转化E.coli DH5α,IPTG诱导表达,亲和层析及Factor Xa酶解纯化重组Csn。所得重组壳聚糖酶具有降解壳聚糖的生物活性,其活性受温度及pH值的影响。     

Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene

A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized.     

Thermostable chitosanase from Bacillus sp. strain CK4: its purification, characterization, and reaction patterns

A thermostable chitosanase, purified 156-fold to homogeneity in an overall yield of 12.4%, has a molecular weight of about 29,000 +/- 2,000, and is composed of monomer. The enzyme degraded soluble chitosan, colloidal chitosan, and glycol chitosan, but did not degrade chitin or other beta-linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co2+ and 1.4-fold by Mn2+. However, Cu2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable after heat treatment at 80 degrees C for 30 min or 70 degrees C for 60 min and fairly stable in protein denaturants as well. Chitosan was hydrolyzed to (GlcN)4 as a major product, by incubation with the purified enzyme. The effects of ammonium sulfate and organic solvents on the action pattern of the thermostable chitosanase were investigated. The amounts of (GlcN)3-(GlcN)6 were increased about 30% (w/w) in DAC 99 soluble chitosan containing 10% ammonium sulfate, and (GlcN)1 was not produced. The monophasic reaction system consisted of DAC 72 soluble chitosan in 10% EtOH also showed no formation of (GlcN)1, however, the yield of (GlcN)3 approximately (GlcN)6 was lower than DAC 99 soluble chitosan-10% ammonium sulfate. The optimal concentration of ammonium sulfate to be added was 20%. At this concentration, the amount of hexamer was increased by over 12% compared to the water-salt free system.     

Chitosanase activity in<Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The ability to produce extracellular chitosanase (EC 3.2.1.132) was found by plate assays in 18 (23%) out of 77 crystalliferous strains of Bacillus thuringiensis. The best chitosanase producer was selected after the growth chosen in a liquid medium with colloidal chitosan as carbon source. Enzyme production was optimized (a 4-d incubation at 32 degrees C with shaking in a medium of pH 6.5 with 4% colloidal chitosan) and the enzyme was partially characterized. This is the first report on the chitosanase of B. thuringiensis.     

Synthesis of exo-β-glucosaminidase BY FUNGUS <Emphasis Type="Italic">Penicillium</Emphasis> sp. IB-37-2

A new strain Penicillium sp. IB-37-2, which actively hydrolyzes chitosan (SD ～80–85%) but possesses low activity against colloidal chitin, was isolated. The fungus was observed to have a high level chitosanase biosynthesis (1.5–3.0 U/mL) during submerged cultivation at 28°C, with a pH of 3.5–7.0 and 220 rpm in nutrient media containing chitosan or chitin from shells of crabs. Purification of the chitosanase enzyme complex from Penicillium sp. IB-37-2 by ultrafiltration and hydrophobic chromatography, followed by denaturing electrophoresis, revealed two predominant proteins with molecular weights of 89 and 41 kDa. The purified enzyme complex demonstrated maximal activity (maximal rate of hydrolysis of dissolved chitosan) and stability at 50–55°C and a pH of 3.5–4.0. The enzyme preparation also hydrolyzed laminarin, β-(1,3)-(1,4)-glycan, and colloidal chitin. Exohydrolysis of chitosan by the preparation isolated from Penicillium sp. IB-37-2 resulted in the formation of single product, D-glucosamine.     

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.     

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.     

Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001

The extracellular chitosanase (34,000 M(r)) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40 degrees C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag(+). Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.     

Isolation and Characterization of Chitosanase from the Strain Bacillus sp. 739

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 was determined. Maximum enzyme activity was observed in a medium containing biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity by chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. The temperature and pH optima of the purified chitosanase were in the ranges 45–55°C and 6.0–6.5, respectively. Time to half-maximum inactivation of the enzyme at 50°C was equal to 1 h. With colloidal chitosan as the substrate, the value of K of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

Purification and hydrolytic action of a chitosanase from Nocardia orientalis

Chitosanase from the culture filtrate of Nocardia orientalis was purified to apparent homogeneity by precipitation with ammonium sulfate followed by CM-Sephadex chromatography, biospecific affinity chromatography on a Sepharose CL-4B with immobilized chitotriose and by gel filtration on Sephadex G-75. The enzyme specifically acted on chitooligosaccharides and chitosan to yield chitobiose and chitotriose as final products. The mode of action of the chitosanase on chitooligosaccharides and their corresponding alcohols suggests that the enzyme requires substrates with four or more glucosamine residues for the expression of activity and its shows maximum activity on chitohexaose and chitoheptaose. In the hydrolysis of chitosans of varying N-acetyl content, the enzyme cleaved about 30% acetylated chitosan with maximum activity and the enzyme activity decreased with increasing the degree of deacetylation of chitosans tested. The analysis of products formed from 33% acetylated chitosan shows the chitosanase is capable of cleaving between glucosamine and glucosamine or N-acetylglucosamine, but not cleaving between N-acetylglucosamine and glucosamine. On the basis of the results, the whole pathway of enymatic degradation of partially acetylated chitosan by a combination of chitosanase, exo-beta-D-glucosaminidase and beta-N-acetylhexosaminidase is proposed.     

Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls.

A strain of Bacillus sp (Bacillus R-4) produces a protease and a carbohydrolase both of which have the ability to lyse Rhizopus cell walls. Of the enzymes, the carbohydrolase has been purified to an ultracentrifugally and electrophoretically homogeneous state, and identified as a chitosanase. The enzyme was active on glycol chitosan as well as chitosan. Molecular weight of the purified enzyme was estimated as 31 000 and isoelectric point as pH 8.30. The enzyme was most active at pH 5.6 and at 40 degrees C with either Rhizopus cell wall or glycol chitosan as substrate, and was stable over a range of pH 4.5 to 7.5 at 40 degrees C for 3 h. The activity was lost by sulfhydryl reagents and restored by either reduced glutathione of L-cysteine. An abrupt decrease in viscosity of the reaction mixture suggested an endowise cleavage of chitosan by this enzyme.     

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.     

Characterization of a thermostable L-arabinose (D-galactose) isomerase from the hyperthermophilic eubacterium Thermotoga maritima

The araA gene encoding L-arabinose isomerase (AI) from the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli as a fusion protein containing a C-terminal hexahistidine sequence. This gene encodes a 497-amino-acid protein with a calculated molecular weight of 56,658. The recombinant enzyme was purified to homogeneity by heat precipitation followed by Ni(2+) affinity chromatography. The native enzyme was estimated by gel filtration chromatography to be a homotetramer with a molecular mass of 232 kDa. The purified recombinant enzyme had an isoelectric point of 5.7 and exhibited maximal activity at 90 degrees C and pH 7.5 under the assay conditions used. Its apparent K(m) values for L-arabinose and D-galactose were 31 and 60 mM, respectively; the apparent V(max) values (at 90 degrees C) were 41.3 U/mg (L-arabinose) and 8.9 U/mg (D-galactose), and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 74.8 mM(-1).min(-1) (L-arabinose) and 8.5 mM(-1).min(-1) (D-galactose). Although the T. maritima AI exhibited high levels of amino acid sequence similarity (>70%) to other heat-labile mesophilic AIs, it had greater thermostability and higher catalytic efficiency than its mesophilic counterparts at elevated temperatures. In addition, it was more thermostable in the presence of Mn(2+) and/or Co(2+) than in the absence of these ions. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield of 56% for 6 h at 80 degrees C.     

Isolation and purification of Mucor circinelloides intracellular chitosanolytic enzymes

This study aimed at isolation, purification and characterization of a chitosanase from Mucor circinelloides mycelium. The latter contains also a mycelium-bound lipase and lipids. The chitosanase and lipase were extracted from defatted M. circinelloides mycelium with a detergent and purified through a two-step procedure comprising chromatography on bacitracin–CNBr-Sepharose 4B and gel filtration on Sephadex G-100. Purification degree of the chitosanase (endo-type enzyme) and lipase was 23 and 12, respectively. These enzymes were optimally active at pH of 5.5–6.0 (chitosanase) and 7.2 (lipase in olive oil hydrolysis) and at 37 °C. Both purified enzymes were activated by Ca²⁺ and Mg²⁺ ions. The preferred substrates of chitosanase were chitosan preparations with a high degree of deacetylation. This enzyme showed no activity for colloidal chitin, Na-CMC and starch. SDS–PAGE of both purified enzymes showed two bands with molecular masses of 42 and 43 kDa. Our results suggest that M. circinelloides synthesizes an oligomeric (bifunctional) lipase which also efficiently depolymerizes chitosan.     

Gene cloning and biochemical analysis of thermostable chitosanase (TCH-2) from Bacillus coagulans CK108

The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size. The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6%, 48.0%, and 12.6% identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively. C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group. The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C. The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography. The half-life of the enzyme was 40 min at 90 degrees C. The purified protein was also highly stable, retaining above 50% residual activities during treatment with denaturants such as urea (8 M) and guanidine x HCl (4 M) at 37 degrees C for 30 min. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product.