Production Of Chitosan Oligosaccharides Research Articles

Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides.

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Structure-based rational design of chitosanase CsnMY002 for high yields of chitobiose

Chitosan oligosaccharides (COS) are attractive active molecules for biomedical applications. Currently, the prohibitively high cost of producing fully defined COS hampers extensive studies on their biological activity and restricts their use in various industries. Thus, cost-effective production of pure COS is of major importance. In this report, chitosanase from Bacillus subtilis MY002 (CsnMY002) was prepared for COS production. The structure of apo CsnMY002 displayed an unexpected tunnel-like substrate-binding site and the structure of the CsnMY002_E19A/(GlcN)6 complex highlighted the “4 + 2″ splitting of hexaglucosamine even though the “3 + 3″ splitting is also observed in the TLC analysis of the enzyme products for hexaglucosamine. Structure based rational design was performed to generate mutants for chitobiose production. The CsnMY002_G21 K mutant produced chitobiose with a relative content > 87 % from chitosan with a low degree of acetylation, and 50.65 mg chitobiose with a purity > 98 % was prepared from 100 mg chitosan. The results provide insight on the catalytic mechanism of chitosanase and underpin future biomedical applications of pure chitobiose.

Biochemical Degradation of Chitosan over Immobilized Cellulase and Supported Fenton Catalysts

This paper describes the application of Fe-MCM-48 (Mobil Composition of Matter No.48) and cellulase-MCM-48 catalysts for the depolymerization of chitosan. The results show that H2O2 is a good oxidant for the depolymerization of chitosan in the presence of Fe-MCM-48. The average polymerization degree of the product decreased to 6.1, and decreased to 29.2 when cellulase-MCM-48 was used as a catalyst, because the effect of the enzyme was affected by the molecular structure of chitosan. When both materials were used for depolymerization, the average degree of polymerization sharply decreased to 3.8. The results show that the two degradation methods can promote each other to obtain oligosaccharides with a lower degree of polymerization. This provides a new method for the controllable degradation of chitosan and lays a good foundation for the industrial production of chitosan oligosaccharides with a low degree of polymerization.

Identification of low molecular weight degradation products from chitin and chitosan by electrospray ionization time-of-flight mass spectrometry

The beneficial effects provided by chitosan oligosaccharides (COS) make them of interest in medical research. The monomers that constitute COS confer distinct properties, so controlling COS composition during their production is significant. In this work, we degraded chitin and chitosan polymers and identified low molecular weight products such as COS that formed, using electrospray ionization time-of-flight mass spectrometry. Our results show that hydrochloric acid, hydrogen peroxide, and nitrous acid generate distinct products from chitin and chitosan. Hydrochloric acid degrades chitin and chitosan to produce glucosamine (GlcN) monomers and oligomers. Hydrogen peroxide degrades chitosan to produce GlcN and N-acetyl-d-glucosamine (GlcNAc) monomers and oligomers, and nitrous acid degrades chitosan to produce 2,5-anhydro- d-mannose. Our studies show that COS composition is dictated by both the degradation protocol and the starting polymer. Additionally, our results enable selection of degradation protocols based on their ability to degrade chitin and chitosan and facilitate the production of COS with desired compositions.

Production of chitosan-oligosaccharides by the chitin-hydrolytic system of Trichoderma harzianum and their antimicrobial and anticancer effects

Chitosan-oligosaccharides (COS) are low-molecular weight chitosan derivatives with interesting clinical applications. The optimization of both COS production and purification is an important step in the design of an efficient production system and for the exploration of new COS applications. Trichoderma harzianum is an innocuous biocontrol agent that represents a novel biotechnological tool due to the production of extracellular enzymes, including those that produce a COS mixture. In this work, we propose different systems for the production of COS using the T. harzianum chitinolitic system. A complete qualitative and quantitative analysis of a partially purified COS mixture were performed. Also, an evaluation of the anticancer and antimicrobial effects of the COS mixture was carried out. Three chitosan variants (colloidal, solid and solution) and two fungus stages (spores and mycelia) were tested for COS production. The best system consisted of the interaction of the solid chitosan and the fungal spores, producing a COS mixture containing species from the monomer to the hexamer, in a concentration range of 7–238 mg/mL, according to chromatographic analysis. The proposed purification method isolated the monomer and the dimer from the COS mixture. Moreover, the COS mixture has an inhibitory effect on the growth of bacteria and changes the morphology of yeasts. As anticancer compounds, COS inhibited the growth of cervical cancer cells at concentration of 4 mg/mL and significantly reduced the survival rate of the cells. In conclusion, T. harzianum proved to be an efficient system for COS mixture production.

Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis.

Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5' untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s-1 in the wild type to 7.56 μM s-1 in the mutant R157T.IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

Enhancement of chitosanase secretion by Bacillus subtilis for production of chitosan oligosaccharides

Chitosan oligosaccharides (COSs) are produced by hydrolysis of chitosan (the deacetylation product of chitin) by chitosanases. COSs are widely used in food, textile, pharmaceutical, and medical applications. The chitosanase (Csn) encoded by the Bacillus subtilis 168 csn gene, which belongs to the glycosyl hydrolase family 46, was recombinantly expressed in B. subtilis PT5. Csn fused with various signal peptides was secreted into culture supernatants and its production evaluated. The highest specific activity achieved was 156 ± 4.68 U/ml using the aprE signal peptide rather than the original csn signal peptide. The optimal temperature and pH for enzyme activity were determined as 47 °C and 5.4, respectively, using central composite design (CCD). Recombinant Csn could efficiently hydrolyze both α and β type chitosan to dimer, trimer, and tetramer COSs. Further, the optimal medium for chitosanase production was predicted and successfully determined using Box–Behnken experimental designs. The maximum Csn protein production level that could be achieved using the optimal fermentation medium (0.76% lactose, 1.63% yeast extract, and 2.31% glutamate) was 208.23 ± 5.19 U/ml in a 5-l fermenter. The present study demonstrates that recombinant B. subtilis Csn has potential for use as a biocatalyst to develop industrial-scale production of COSs.

Selective and stable production of physiologically active chitosan oligosaccharides using an enzymatic membrane bioreactor

We investigated the production of chitosan oligosaccharides by continuous hydrolysis of chitosan in an enzyme membrane bioreactor, with the goal of improving the yield of physiologically active oligosaccharides (pentamers and hexamers) and achieving operational stability. The bioreactor was a continuous-flow stirred-tank reactor equipped with an ultrafiltration membrane with a molecular weight cut-off of 2000 Da, and the hydrolysis was accomplished with chitosanase from Bacillus pumilus. After optimization of the reaction parameters, such as the amount of enzyme, the yield of the target oligosaccharides produced in the membrane bioreactor with free chitosanase reached 52% on the basis of the fed concentration of chitosan. An immobilized chitosanase prepared by the multipoint attachment method was used to improve the operational stability of the membrane bioreactor. Under the optimized conditions, pentameric and hexameric chitosan oligosaccharides were steadily produced at 2.3 g/L (46% yield) for a month. The half-life of the productivity of the reactor was estimated to be 50 d under the conditions examined.

Identification and expression of GH-8 family chitosanases from severalBacillus thuringiensissubspecies

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

Production of Chitosan Oligosaccharides at High Concentration by Immobilized Chitosanase

The production of high concentrations of physiologically active pentameric and hexameric chitosan oligosaccharides was investigated. Chitosanase directly immobilized on an agar gel-coated multidisk impeller was used for the hydrolysis of chitosan. As hydrolysis proceeded, the concentration of chitosan was increased from the saturation concentration (20kg/m3) to 50kg/m3 by stepwise addition of chitosan powder with pH control. The timing of the addition of the chitosan powder was determined by monitoring the change in the torque required to agitate the reaction solution. The target products were produced more efficiently when the chitosan powder was added over a short interval rather than a long interval. When hydrolysis of a 50kg/m3 chitosan solution at 50°C was repeated three times using the same immobilized enzyme, the target products were obtained in high concentrations (20kg/m3) from each of the three reactions with no reduction in the activity of the immobilized enzyme.

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.