Depolymerization of High-Molecular-Weight Chitosan by the Enzyme Preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55°C and an enzyme to substrate ratio of 1 : 400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol:distilled water) yielded fractions with molecular weights in the range 3.2–26.4 kDa.     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Application of a biosensor for monitoring of ethanol

An alcohol biosensor for the measurement of ethanol has been developed. It comprises an alcohol oxidase/chitosan immobilized eggshell membrane and a commercial oxygen sensor. Ethanol determination is based on the depletion of dissolved oxygen content upon exposure to ethanol solution. The decrease in oxygen level was monitored and related to the ethanol concentration. The biosensor response depends linearly on ethanol concentration between 60 microM and 0.80 mM with a detection limit of 30 microM (S/N=3) and 1 min response time. In the optimization studies of the enzyme biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30% (w/v), respectively. The phosphate buffer (pH 7.4, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. In the characterization studies of the ethanol biosensor some parameters such as interference effects, operational and storage stability were studied in detail. The biosensor was also tested with various wine samples. The results of this newly developed biosensor were comparable to the results obtained by a gas chromatographic method.     

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability

Abstract

In this study, the different mole ratios of glucose oxidase/chitosan/dextran–aldehyde and glucose oxidase/chitosan/dextran–sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by C_chitosan/C_{dextran–aldehyde} = 10/1 ratio and C_chitosan/C_{dextran–sulfate} = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60?°C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1?M NaCl or CaCl₂. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8?±?9.3?nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Effect of bipolar membrane electrobasification on chitosanase activity during chitosan hydrolysis

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.     

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.     

Development of a method for immobilization of non-flocculating cells in loofa (Luffa cylindrica) sponge

Both the matrix structure of loofa sponge and the flocculating property of cells were necessary for efficient immobilization. The addition of chitosan to a reactor containing a bed of loofa sponge and a Candida brassicae cell suspension induced cell flocculation and the cells were efficiently immobilized. During ethanol production by the immobilized cells, the free cell concentration in the broth was controlled at the desired level by intermittent addition of chitosan to the reactor. The immobilized cell concentration increased but their specific ethanol productivity decreased with an increase in the chitosan concentration. The maximum ethanol productivity was obtained at a low chitosan concentration of 0·03 g/litre. With this optimal concentration, the cell concentration, ethanol yield and productivity were, respectively, 2, 1·3 and 3 times higher than those of the suspension culture.     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

Hydrolysis of chitosan sulfate by an enzyme complex from Streptomyces kurssanovii

The possibility of enzymatic hydrolysis of chitosan was shown. The optimum conditions for the process are: sodium acetate buffer pH 6.0, 37 degrees C, 24 h, and the chitosan sulfate-protein volume ratio of 500:1 in the enzyme preparation. During hydrolysis, the intrinsic viscosity of chitosan sulfate solution decreased by a factor of 2.7.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

Dehydration from alcohols by polyion complex cross-linked chitosan composite membranes during evapomeation

This study describes the dehydration of an ethanol/water azeotrope during evapomeation using polyion complex cross-linked chitosan composite (q-Chito-PEO acid polyion complex/PES composite) membranes, constructed from quaternized chitosan (q-Chito) and poly(ethylene oxydiglycolic acid) (PEO acid) on a porous poly(ether sulfone) (PES) support. Both the q-Chito/PES composite and the q-Chito-PEO acid polyion complex/PES composite membranes showed high water permselectivity for an ethanol/water azeotrope. Both the permeation rate and the water permselectivity of the q-Chito/PES composite membranes were enhanced by increasing the degree of quaternization of the chitosan molecule because the affinity of the q-Chito/PES composite membranes for water was increased by introducing a quaternized ammonium group into the chitosan molecule. q-Chito-PEO acid polyion complex/PES composite membranes prepared from an equimolar ratio of carboxylate groups in the PEO acid versus quaternized ammonium groups in the q-Chito showed the maximum separation factor for water permselectivity without lowering the permeation rate. With an increasing molecular weight of PEO acid, the separation factor for water permselectivity increased, but the permeation rate almost did not change. The mechanism responsible for the separation of an ethanol/water azeotrope through the q-Chito-PEO acid polyion complex/PES composite membranes was analyzed by the solution-diffusion model. The permeation rate, separation factor for water permselectivity, and evapomeation index of q-Chito-PEO acid 400 polyion complex/PES composite membrane with an equimolar ratio of carboxylate groups in PEO acid 400 and ammonium groups in q-Chito were 3.5 x 10(-1) kg/(m(2) hr), 6300, and 2205, respectively, and very high membrane performance. The separation factor for water permselectivity for aqueous solutions of n-propyl and isopropyl alcohol was also maximized at an equimolar ratio of carboxylate groups and ammonium groups and was greater than that for an ethanol/water azeotrope. The above results were discussed from the viewpoint of the physical and chemical structure of the q-Chito-PEO acid polyion complex/PES composite membranes and the permeants.     

Effect of the degree of acetylation of chitosan on its enzymatic hydrolysis with the preparation Celloviridin G20x

The degree of acetylation exerted only insignificant effects on the enzymatic hydrolysis of chitosan, while affecting the composition of the resulting hydrolysates and their water solubility. Chitosan with various degrees of acetylation was produced by reacetylation of the original chitosan (the solvents, methanol and 2% acetic acid, were present at a ratio of 54:51 v/v; the amount of acetic anhydride was in the range 0.1-2.0 mmol per 1 g chitosan). Hydrolysis by the enzymatic preparation Celloviridin G20x was performed at the enzyme to substrate ratio of 1:400 in sodium-acetate buffer, pH 5.2 (55 degrees C) for 1 h.     

Matrices (re)loaded: Durability,viability, and fermentative capacity of yeast encapsulated in beads of different composition during long-term fed-batch culture

Encapsulated microbes have been used for decades to produce commodities ranging from methyl ketone to beer. Encapsulated cells undergo limited replication, which enables them to more efficiently convert substrate to product than planktonic cells and which contributes to their stress resistance. To determine how encapsulated yeast supports long-term, repeated fed-batch ethanologenic fermentation, and whether different matrices influence that process, fermentation and indicators of matrix durability and cell viability were monitored in high-dextrose, fed-batch culture over 7 weeks. At most timepoints, ethanol yield (g/g) in encapsulated cultures exceeded that in planktonic cultures. And frequently, ethanol yield differed among the four matrices tested: sodium alginate crosslinked with Ca²⁺ and chitosan, sodium alginate crosslinked with Ca²⁺, Protanal alginate crosslinked with Ca²⁺ and chitosan, Protanal alginate crosslinked with Ca²⁺, with the last of these consistently demonstrating the highest values. Young's modulus and viscosity were higher for matrices crosslinked with chitosan over the first week; thereafter values for both parameters declined and were indistinguishable among treatments. Encapsulated cells exhibited greater heat shock tolerance at 50°C than planktonic cells in either stationary or exponential phase, with similar thermotolerance observed across all four matrix types. Altogether, these data demonstrate the feasibility of re-using encapsulated yeast to convert dextrose to ethanol over at least 7 weeks.     

Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production

Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6×His tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1 g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry.     

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.     

Continuous chitosan hydrolyzate production by immobilized chitosanolytic enzyme from Enterobacter sp. G-1.

Chitosanolytic enzymes from Enterobacter sp. G-1 were immobilized on various carriers to continuously hydrolyze chitosan. Four different carriers were tested: FE-3901 (strong basic anion exchange resin, ionic binding), glutaraldehyde-treated FE-4612 (weak basic anion exchange resin, cross-linking), Chitopearl (chitosan beads), and alginate calcium. Glutaraldehyde-treated FE-4612 and Chitopearl immobilized more protein than the others. The enzyme immobilized on FE-3901 had the greatest activity. The activity of enzyme immobilized on FE-3901 decreased rapidly when exposed to a continuous flow of 1% chitosan. The enzyme immobilized with Chitopearl retained more than 50% of its original activity after 17 days, and the activity was fully restored by re-immobilization.     

Ethanol and leucine oxidation--II. Leucine oxidation by rat tissue in vitro

The effect of ethanol upon leucine oxidation by rat tissues in vitro is reported. The activities of branched chain amino acid aminotransferase and 2-oxo acid dehydrogenase were decreased by chronic administration of ethanol (20% v/v solution as drinking water for 35 d) in muscle and kidney but were increased, although not significantly, in liver. Acute administration of ethanol (8 g kg-1 body-weight 0.73) did not affect enzyme activities. Tissue NAD+:NADH ratios, calculated from lactate:pyruvate ratios, were significantly decreased in the liver and kidney of rats receiving ethanol acutely. These data are consistent with the view that ethanol decreases leucine oxidation by decreasing availability of NAD+ when given acutely and by decreasing enzyme activity when administered chronically.     

Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan

The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5–150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, >70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9–6.2) by the complex containing 0.006–0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase.