Alkaline Serine Protease AprE Plays an Essential Role in Poly-γ-glutamate Production during Natto Fermentation

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.     

Production of Pseudomonas Cells Having a High Fumarate Hydratase Activity

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.     

Effect of chitin sources on production of chitinase and chitosanase byStreptomyces griseus HUT 6037

The advantage of usingStreptomyces griseus HUT 6037 in the production of chitinase or chitosanase is that the organism is capable of hydrolyzing amorphous or crystal-line chitin and chitosan according to the type of the substrate used. We investigated the effects of the enzyme induction time and chitin sources, CM-chitosan and deacetylated chitosan (degree of deacetylation 75–99%), on production of chitosanase. We found that this strain accumulated chitosanase when cells were grown in the culture medium containing chitosanaceous substrates instead of chitinaceous substrates. The highest chitosanase activity was obtained at 4 days of cultivation with 99% deacetylated chitosan. Soluble chitosan (53% deacetylated chitosan) was found to induce chitinase as well as chitosanase. The specific activities of chitinase and chitosanase were 0.91 and 1.33 U/mg protein at 3 and 5 days, respectively. From the study of the enzymatic digestibility of various degrees of deacetylated chitosan, it was found that (GlcN)₃, (GlcN)₄ and (GlcN)₅ were produced during the enzymatic hydrolysis reaction. The results of this study suggested that the sugar composition of (GlcN)₃ was homogeneous and those of (GlcN)₄ and (GlcN)₅ were heterogeneous.     

Purification and Characterization of Two Types of Chitosanase from a Microbacterium sp.

Two extracellular chitosanases (ChiX and ChiN) were extracted from Microbacterium sp. OU01 with Mr values of 81 kDa (ChiX) and 30 kDa (ChiN). ChiN was optimally active at pH 6.2 and 50°C and ChiX at pH 6.6 and 60°C (assayed over 15 min). Both the activities increased with the degree of deacetylation (DDA) of chitosan. ChiN hydrolyzed oligomers of glucosamine (GlcN) larger than chitopentaose, and chitosan with 62–100% DDA; but ChiX acted on chitosan and released GlcN. Hydrolysis of chitosan with 99% DDA by ChiN released chitobiose, chitotriose and chitotetraose as the major products.     

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.     

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.     

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     

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 an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.     

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.     

Effect of dietary chitosans on trace iron, copper and zinc in mice

Dietary chitosans with different molecular weight M_w and the degree of deacetylation DDA (high molecular weight chitosan HCS with M_w 7.60 × 10⁵ and DDA 85.5%, middle molecular weight chitosan MCS with M_w 3.27 × 10⁴ and DDA 85.2%, chito-oligomer COS with M_w 0.99 × 10³ and DDA 85.7% and water-soluble chitosan WSC with M_w 3.91 × 10⁴ and DDA 52.6%) were used at the 1.05% level to feed mice for 90 days. Afterwards no pathological symptoms, clinical signs or deaths were observed. The body weight of mice in chitosan group and control group showed no significant difference. Although HCS, COS and WSC had no significant effect on the level of Fe, Zn and Cu in the tested mice’s liver, spleen, heart and kidney, MCS significantly increased the level of Fe, Zn and Cu in liver. Therefore dietary ingestion of chitosan did not depress the level of Fe, Zn and Cu in mice.     

Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase from a Koji Mold,Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

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.     

Characterization and kinetics of 45 kDa chitosanase from Bacillus sp. P16

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 degrees C, and was stable between pH 4.5-10.0 and under 50 degrees C. The Km and Vmax were measured with a chitosan of a D.A. of 20.2% as 0.52 mg/ml and 7.71 x 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, k1 values, of 4.98 x 10(-4), 2.3 x 10(-4), and 9.3 x 10(-6) sec(-1), respectively. The enzyme hardly catalyzed degradation of chitooligomers smaller than the pentamer.     

Chitosan-Soyprotein Interaction as Determined by Thermal Unfolding Experiments

Chitosan interaction with soybean β-conglycinin β₃ was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T _m) was 63.4 °C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T _m value was elevated by 7.7 °C, whereas the T _m elevation upon addition of chitosan hexamer (GlcN)₆ was 2.2 °C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T _m elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T _m elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with β-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.     

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.     

Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101

A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 °C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca²⁺. However, Hg²⁺ and Ni⁺ ions strongly inhibited the enzyme. The enzyme produced, GlcN_2–4, were the major products from a soluble chitosan.     

A novel nanobiosensor for the detection of paraoxon using chitosan-embedded organophosphorus hydrolase immobilized on Au nanoparticles

Organophosphorus (OP) compounds are one of the most hazardous chemicals used as insecticides/pesticide in agricultural practices. A large variety of OP compounds are hydrolyzed by organophosphorus hydrolases (OPH; EC 3.1.8.1). Therefore, OPHs are among the most suitable candidates that could be used in designing enzyme-based sensors for detecting OP compounds. In this work, a novel nanobiosensor for the detection of paraoxon was designed and fabricated. More specifically, OPH was covalently embedded onto chitosan and the enzyme–chitosan bioconjugate was then immobilized on negatively charged gold nanoparticles (AuNPs) electrostatically. The enzyme was immobilized on AuNPs without chitosan as well, to compare the two systems in terms of detection limit and enzyme stability under different pH and temperature conditions. Coumarin 1, a competitive inhibitor of the enzyme, was used as a fluorogenic probe. The emission of coumarin 1 was effectively quenched by the immobilized Au-NPs when bound to the developed nanobioconjugates. However, in the presence of paraoxon, coumarin 1 left the nanobioconjugate, leading to enhanced fluorescence intensity. Moreover, compared to the immobilized enzyme without chitosan, the chitosan-immobilized enzyme was found to possess decreased K_m value by more than 50%, and increased V_max and K_cat values by around 15% and 74%, respectively. Higher stability within a wider range of pH (2–12) and temperature (25–90°C) was also achieved. The method worked in the 0 to 1050?nM concentration ranges, and had a detection limit as low as 5?×?10^?11 M.     

Action pattern of Bacillus sp. no. 7-M chitosanase on partially N-acetylated chitosan.

The hydrolyzate of partially N-acetylated chitosan by Bacillus sp. No. 7-M chitosanase was separated by gel filtration on Bio-Gel P-2. Sugar compositions and sequences of the oligosaccharides were identified by exo-splitting with beta-GlcNase, fast atom bombardment mass spectroscopy, and proton NMR spectroscopy. In addition to chitooligosaccharides, (GlcN)2, (GlcN)3, and (GlcN)4, hetero-chitooligosaccharides such as (GlcN)2.GlcNAc.(GlcN)2, GlcN.GlcNAc.(GlcN)3, (GlcN)2.GlcNAc.(GlcN)3, and GlcN.GlcNAc.(GlcN)4 were detected. These results indicate that Bacillus sp. No. 7-M chitosanase is absolutely specific toward the GlcN.GlcN bonds in partially N-acetylated chitosan and at least three GlcN residues were necessary to the hydrolysis of chitosan by chitosanase.     

Detection of Polygalacturonase in Kiwifruit during Ripening

Oligosaccharides produced during the course of the hydrolysis of 25% N-acetylated chitosan by Streptomyces griseus chitinase were fractionated by CM-Sephadex C-25 and Toyopearl HW-40F column chromatographies. Sugar compositions and sequences of main oligosaccharides were identified by N-acetylation, exo-splitting with β-GlcNAcase and β-GlcNase, and nitrous acid degradation. In addition to N-acetylated saccharides, GlcNAc, (GlcNAc)₂, and (GlcNAc)₃, hetero-chitooligosaccharides such as GlcN · GlcNAc, GlcN · GlcNAc · GlcNAc, GlcN · GlcN · GlcNAc, GlcN · GlcNAc · GlcNAc · GlcNAc, GlcNAc · GlcN · GlcNAc · GlcNAc, GlcN · GlcNAc · GlcN · GlcNAc, and GlcN · GlcN · GlcNAc · GlcNAc were identified. These results indicate that Streptomyces griseus chitinase specifically cleaves the N-acetyl-β-d-glucosaminidic linkages in partially N-acetylated chitosan.