Dataset Information

Pilot-Scale Production of Chito-Oligosaccharides Using an Innovative Recombinant Chitosanase Preparation Approach.

ABSTRACT: For pilot-scale production of chito-oligosaccharides, it must be cost-effective to prepare designable recombinant chitosanase. Herein, an efficient method for preparing recombinant Bacillus chitosanase from Escherichia coli by elimination of undesirable substances as a precipitate is proposed. After an optimized culture with IPTG (Isopropyl ?-d-1-thiogalactopyranoside) induction, the harvested cells were resuspended, disrupted by sonication, divided by selective precipitation, and stored using the same solution conditions. Several factors involved in these procedures, including ion types, ionic concentration, pH, and bacterial cell density, were examined. The optimal conditions were inferred to be pH = 4.5, 300 mM sodium dihydrogen phosphate, and cell density below 10¹¹ cells/mL. Finally, recombinant chitosanase was purified to >70% homogeneity with an activity recovery and enzyme yield of 90% and 106 mg/L, respectively. When 10 L of 5% chitosan was hydrolyzed with 2500 units of chitosanase at ambient temperature for 72 h, hydrolyzed products having molar masses of 833 ± 222 g/mol with multiple degrees of polymerization (chito-dimer to tetramer) were obtained. This work provided an economical and eco-friendly preparation of recombinant chitosanase to scale up the hydrolysis of chitosan towards tailored oligosaccharides in the near future.

SUBMITTER: Cheng CY

PROVIDER: S-EPMC7831125 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Pilot-Scale Production of Chito-Oligosaccharides Using an Innovative Recombinant Chitosanase Preparation Approach.

Cheng Chih-Yu CY Tsai Chia-Huang CH Liou Pei-Jyun PJ Wang Chi-Hang CH

Polymers 20210118 2

For pilot-scale production of chito-oligosaccharides, it must be cost-effective to prepare designable recombinant chitosanase. Herein, an efficient method for preparing recombinant <i>Bacillus</i> chitosanase from <i>Escherichia coli</i> by elimination of undesirable substances as a precipitate is proposed. After an optimized culture with IPTG (Isopropyl β-d-1-thiogalactopyranoside) induction, the harvested cells were resuspended, disrupted by sonication, divided by selective precipitation, and ...[more]

PMID: 33477553

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Project description: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.

Dataset Information

Pilot-Scale Production of Chito-Oligosaccharides Using an Innovative Recombinant Chitosanase Preparation Approach.

Publications

Pilot-Scale Production of Chito-Oligosaccharides Using an Innovative Recombinant Chitosanase Preparation Approach.

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