共查询到20条相似文献,搜索用时 15 毫秒
1.
Properties of Chitosanase from Bacillus cereus S1 总被引:3,自引:0,他引:3
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 相似文献
2.
Production, purification, and characterization of an extracellular chitosanase from Streptomyces. 总被引:5,自引:1,他引:4
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The synthesis by Streptomyces sp. no. 6 of an extracellular chitosanase was induced by glucosamine. The enzyme was purified to homogeneity by Sephadex G-100, carboxymethyl-cellulose, and diethylaminoethyl-cellulose chromatography. The purified enzyme hydrolyzed chitosan (the beta-1,4-linked polymer of glucosamine) but not chitin nor carboxymethyl-cellulose. The only products of the hydrolysis detectable by paper chromatography were di- and triglucosamine. Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular weight of the enzyme was between 29,000 and 26,000. Acid hydrolysates of the enzyme contained no cysteic acid or glucosamine or other carbohydrate. At 25 C, maximum activity was obtained between pH 4.5 and 6.5. The enzymatic hydrolysis of chitosan occurred over a wide range of temperatures and was maximal at 60 C. The rate of the reaction was inhibited by concentrations of soluble chitosan higher than 0.5 g/liter. The apparent Km calculated from a Lineweaver-Burke plot was 0.688 g/liter at pH 5.5. The enzyme prevented spore germination and caused a significant decrease in the turbidity of germinated spore suspensions of the Mucor strains tested. Such a decrease was the result of a partial lysis of the cell wall. 相似文献
3.
Isabelle Boucher Agnés Dupuy Pierre Vidal Witold A. Neugebauer Ryszard Brzezinski 《Applied microbiology and biotechnology》1992,38(2):188-193
A highly efficient chitosanase producer, the actinomycete N174, identified by chemotaxonomic methods as belonging to the genus Streptomyces was isolated from soil. Chitosanase production by N174 was inducible by chitosan or d-glucosamine. In culture filtrates the chitosanase accounted for 50–60% of total extracellular proteins. The chitosanase was purified by polyacrylic acid precipitation, CM-Sepharose and gel permeation chromatography. The maximum velocity of chitosan degradation was obtained at 65° C when the pH was maintained at 5.5. The enzyme degraded chitosans with a range of acetylation degrees from 1 to 60% but not chitin or CM-cellulose. The enzyme showed an endo-splitting type of activity and the end-product of chitosan degradation contained a mixture of dimers and trimers of d-glucosamine.Correspondence to: R. Brzezinski 相似文献
4.
Chitosanase is an enzyme that hydrolyzes chitosan, a beta-(1-4) glucosamine polymer, into size-specific oligomers that have pharmaceutical and biological properties. The aim of the present work was to use the bipolar membrane technology, in particular the OH(-) stream produced by water splitting, for inactivation of chitosanase at alkaline pH in order to terminate the enzymatic reaction producing chitosan oligomers. The objectives consisted of studying the effect of pH: (a) on the stability of chitosanase, and (b) on the catalytic activity of chitosanase during chitosan hydrolysis. The enzyme was found to be stable in the pH range of 3-8 during at least 7h, and partially lost its activity after 1h at pH 8. The catalytic activity of chitosanase during chitosan hydrolysis decreased after pH adjustment by electrobasification. The reaction rate decreased by 50% from pH 5.5 to 6, whereas the reaction was completely inhibited at pH>7. The decrease of reaction rate was due to chitosan substrate insolubilization and chitosanase denaturation at alkaline pH values. 相似文献
5.
G. E. Aktuganov N. F. Galimzyanova G. A. Teregulova A. I. Melentjev 《Applied Biochemistry and Microbiology》2016,52(5):531-536
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. 相似文献
6.
Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation 总被引:1,自引:0,他引:1
Abstract From autolysed cultures of Mucor rouxii , two chitosanases, A and B, were purified to electrophoretic homogeneity. Apparent M r values of 76 000 and 58 000 and p I values of 4.9 and 4.7 were determined for A and B, respectively. Both chitosanases showed a high specificity for chitosan and chitosan derivatives. They had optimum activities at pH 5.0 and at temperatures of 55°C and 50°C for A and B, respectively. Enzyme A was inhibited by acetate ions and enzyme B by high substrate concentration. Both enzymes showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of dimer, trimer and higher molecular mass oligomers of glucosamine. Glucosamine oligosaccharides were poorly hydrolysed by these enzymes. Both enzymes extensively degraded the chitosan extracted from M. rouxii cell walls. 相似文献
7.
Katarzyna Struszczyk Mirosława Szczęsna-Antczak Marta Walczak Emilia Pomianowska Tadeusz Antczak 《Carbohydrate polymers》2009,78(1):16-24
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. 相似文献
8.
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. 相似文献
9.
Takuo Sakai Minora Okushima Masahiko Sawada 《Bioscience, biotechnology, and biochemistry》2013,77(9):2223-2231
Some properties of the endo-polygalacturonase from Trichosporon penicillatum were investigated. The enzyme showed the highest activity around pH 5.0 and was stable at this pH up to 50°C. The enzyme catalyzed the hydrolysis of galacturonic acid oligomers as well as its polymer. The pentamer was degraded to a trimer and a dimer, the tetramer to a trimer and a monomer, and the trimer to a dimer and a monomer, respectively, whereas the dimer was not degraded. The kinetic constant Vmax and Km values changed with the substrate chain-length; the Km values tended to decrease, whereas the Vmax values tended to increase with increasing chain-length of the substrate. The amino acid residue participating in the active site of the enzyme was studied and it was found to be histidine. 相似文献
10.
《Bioscience, biotechnology, and biochemistry》2013,77(4):802-809
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 β-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°C and 6.5, respectively. The enzyme was stable after heat treatment at 80°C for 30 min or 70°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 ~ (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. 相似文献
11.
Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan,
0.25% casiton, 1% MgSO4, 1.4% K2HPO4, 0.02% CaCl2·2H2O, 0.002% FeSO4·7H2O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system
(butyl Sepharose 4 FF) resulted in two major active fractions; the F2 fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis
with apparent molecular mass of 75 kDa. The enzyme worked best at 70°C and pH between 6.0 and 7.0. When incubated at 70, 80,
and 90°C, the t1/2 values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90°C, the enzyme retained its activity up to 8 h in the presence
of 1mM MnCl2. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan
up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this
enzyme, followed by 90% and 100% DDA chitosan. The K
m app
values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the Vmax values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F2 chitosanase at 24 h incubation (70°C) were pentasaccharide (GlcN)5 and hexasaccharide (GlcN)6. The prelimiaary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium. Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus. 相似文献
12.
Purification and Characterization of Three Chitosanase Activities from Bacillus megaterium P1 总被引:1,自引:0,他引:1
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. 相似文献
13.
Extracellular Enzyme From Myxobacter AL-1 That Exhibits Both β-1,4-Glucanase and Chitosanase Activities 总被引:5,自引:5,他引:0
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
An enzyme that has both beta-1,4-glucanase and chitosanase activities is characterized. Evidence for homogeneity was obtained from electrophoresis and sedimentation velocity studies; only one N-terminal amino acid, valine, was found. Results of denaturation studies showed that beta-1,4-glucanase and chitosanase activities decreased at equal rates. With carboxymethylcellulose as the substrate, a K(m) of 1.68 g of carboxymethylcellulose per liter of solution and a V(max) of 2.20 x 10(-9) mol/min were found. With chitosan (the beta-1,4-polymer of glucosamine) as the substrate, a K(m) of 0.30 g of chitosan per liter of solution and a V(max) of 0.75 x 10(-9) mol/min were found. A pH optimum of 5.0 was found for beta-1,4-glucanase activity, and pH optima of 5.0 and 6.8 were found for chitosanase activity. beta-1,4-Glucanase activity had a temperature optimum of 38 C, and chitosanase activity had a temperature optimum of 70 C. Chitosan stabilized both enzyme activities at 70 C. Cellotriose was the smallest polymer capable of hydrolysis. Glucosamine was released by action of the enzyme upon cell wall preparations of several fungi. 相似文献
14.
球孢白僵菌Beauveria bassiana 1316-V1的培养上清液经硫酸铵分级沉淀,Sephadex G-75凝胶过滤,Chitosan-bead亲和层析,第二次Sephadex G-75凝胶过滤, 得到电泳纯的一种胞外壳聚糖酶,比活力达到45u/mg 。此酶的分子量为36 kD; 最适酶反应温度为60℃;最适pH为4.0;最适离子强度为 0.25mol/L NaCl; 37℃以下,pH 2.0~5.0之间稳定性好; Cu2+、Hg2+、Pb2+、Ni2+ 对该酶有强烈抑制作用;Ag+、Mn2+也有较强抑制作用;Fe2+有轻微激活作用。该壳聚糖酶是一种糖蛋白,含糖约为12.6%。酶的最适底物为脱乙酰度为90%的胶体壳聚糖;也能轻微水解CMC、DEAE-Cellulose和胶体几丁质;但不能水解片状的壳聚糖和几丁质。 相似文献
15.
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. 相似文献
16.
K Hirano S Arayaveerasid K Seki DJ Adams M Mitsutomi 《Bioscience, biotechnology, and biochemistry》2012,76(8):1523-1528
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. 相似文献
17.
P. Zaffaroni N. Oddo R. Olivieri L. Formiconi 《Bioscience, biotechnology, and biochemistry》2013,77(9):1875-1877
An extracellular 45 kDa endochitosanase was purified and characterized from the culture supernatant of Bacillus sp. P16. The purified enzyme showed an optimum pH of 5.5 and optimum temperature of 60°C, and was stable between pH 4.5-10.0 and under 50°C. The K m and V max were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71×10?6 mol/sec/mg protein, respectively. The enzyme did not degrade chitin, cellulose, or starch. The chitosanase digested partially N-acetylated chitosans, with maximum activity for 15-30% and lesser activity for 0-15% acetylated chitosan. The chitosanase rapidly reduced the viscosity of chitosan solutions at a very early stage of reaction, suggesting the endotype of cleavage in polymeric chitosan chains. The chitosanase hydrolyzed (GlcN)7 in an endo-splitting manner producing a mixture of (GlcN)2-5. Time course studies showed a decrease in the rate of substrate degradation from (GlcN)7 to (GlcN)6 to (GlcN)5, as indicated by the apparent first order rate constants, k 1 values, of 4.98×10?4, 2.3×10?4, and 9.3×10?6 sec?1, respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer. 相似文献
18.
Arvind Kumar Bhatt Tek Chand Bhalla Hari Om Agrawal N. Sharma 《Letters in applied microbiology》1992,15(1):1-4
An extracellular chitosanase produced by Rhodotorula gracilis CFR-1 that catalyses a limited degradation of chitosan with no detectable generation of glucosamine or reducing groups was identified. Ultracentrifugation, polyacrylamide gel electrophoresis and gel permeation studies suggest that chitosan of average molecular mass 36000 Da was reduced by the enzymic catalysis to nearly one-fourth this size without further hydrolysis of the products. The enzyme, produced constitutively by this yeast, was partially purified and some of its properties were studied. 相似文献
19.
San-Lang Wang Chiann-Wen Yang Tzu-Wen Liang Juo-Hua Peng Chuan-Lu Wang 《Carbohydrate polymers》2009,78(2):205-212
Serratia marcescens TKU011, a protease- and chitosanase-producing bacterium, the optimized condition for protease and chitosanase production was found after the media were heated at 121 °C for 120 min and the culture was shaken at 25 °C for 5 days in 100 mL of medium containing 1% squid pen powder (SPP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4. An extracellular metalloprotease with novel properties of solvent stable, and alkaline was purified from the culture supernatant of S. marcescens TKU011 with squid pen wastes as the sole carbon/nitrogen source. The enzyme was a monomeric protease with a molecular mass of 48–50 kDa by SDS–PAGE and gel filtration chromatography. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU011 protease were 8, 50 °C, pH 5–11, and <40 °C, respectively. Besides protease and chitosanase, with this method, deproteinization of squid pen for β-chitin, the production of peptide and reducing sugar may be useful for biological applications. 相似文献
20.
A new spectrophotometric method for the assay of chitosanase based on complex formation of the substrate chitosan with Calcofluor white dye is described. The absorption maximum for the chitosan-Calcofluor complex is determined to be 406 nm. The apparent minimum size of chitosan for complex formation is 5–7 kDa. Therefore, those enzymes that do not generate glucosamine or reducing groups as products of hydrolysis at levels not measurable by the available methods of assay can be assayed by the present method. In the standardized procedure 200 μg of chitosan in acetate buffer pH 4.5 with the enzyme in a reaction volume of 1.5ml is incubated at 45°C for 1 h, after which 1.5 ml of Calcofluor white (0.05%) is added, kept for 1h and absorbance at 406 nm measured by a spectrophotometer. The chitosanase unit is arbitrarily defined as the reduction in absorbance by 0.01/min. 相似文献