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 β-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.
Highlights
Polymers 2021, 13, 290. https://Chitosanases (EC 3.2.1.132) catalyze the hydrolysis of β-1,4 glycosidic linkages in chitosan, a deacetylated derivative of chitin extracted from an abundant source of shellfish exoskeletons
The recombinant chitosanase was composed of 260 amino acids, including one from the start codon of pET20b and 259 from the mature Bacillus
According to our 2008 study [30], recombinant chitosanase is expressed intracellularly and is found in the soluble fraction of the cell lysate when harvested recombinant E. coli cells are suspended in 20 mM phosphate buffer with a pH of 7.0 and disrupted by sonication
Summary
Chitosanases (EC 3.2.1.132) catalyze the hydrolysis of β-1,4 glycosidic linkages in chitosan, a deacetylated derivative of chitin extracted from an abundant source of shellfish exoskeletons. This polysaccharide and its derivatives have diverse applications in science, medicine, and the cosmetic industry [1]. Chito-oligosaccharides have received a great deal of attention owing to their interesting biological properties such as their inhibitory effects on the growth of microorganisms and tumor cells [2,3] They are able to induce the expression of disease-resistance-response genes in higher plants [4,5]. Because of the high cost of commercial chitosanase, studies have been focused on several enzyme immobilization recycling techniques [7,8], increasing the enzymatic concentration through stepwise substrate addition [7], continual production in a dual reactor system [9,10], and the use of cheap, crude, non-specific enzymes [11,12]
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