Expression and enzymatic characterization of a chitosanase with tolerance to a wide range of pH from <i>Bacillus atrophaeus</i>.

Wenjuan DU; Awagul TURSUN; Zhiqin DONG; Huijuan MA; Zhenghai MA

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Expression and enzymatic characterization of a chitosanase with tolerance to a wide range of pH from Bacillus atrophaeus.

Author: Wenjuan DU ¹ ; Awagul TURSUN ¹ ; Zhiqin DONG ¹ ; Huijuan MA ¹ ; Zhenghai MA ¹
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1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Urumqi 830017, Xinjiang, China.
Publication Type:Journal Article
Keywords: Bacillus atrophaeus; chitosanase; enzymatic properties; expression and purification; pH tolerance
MeSH: Bacillus/genetics*; Hydrogen-Ion Concentration; Escherichia coli/metabolism*; Glycoside Hydrolases/biosynthesis*; Substrate Specificity; Enzyme Stability; Chitosan/metabolism*; Temperature; Kinetics; Cloning, Molecular; Bacterial Proteins/biosynthesis*; Recombinant Proteins/genetics*
From: Chinese Journal of Biotechnology 2025;41(1):352-362
CountryChina
Language:Chinese
Abstract: To screen and identify a chitosanase with high stability, we cloned the chitosanase gene from Bacillus atrophaeus with a high protease yield from the barren saline-alkali soil and expressed this gene in Escherichia coli. The expressed chitosanase of B. atrophaeus (BA-CSN) was purified by nickel-affinity column chromatography. The properties including optimal temperature, optimal pH, substrate specificity, and kinetic parameters of BA-CSN were characterized. The results showed that BA-CSN had the molecular weight of 31.13 kDa, the optimal temperature of 55 ℃, the optimal pH 5.5, and good stability at temperatures below 45 ℃ and pH 4.0-9.0. BA-CSN also had good stability within 4 h of pH 3.0 and 10.0, be activated by K⁺, Na⁺, Mn²⁺, Ca²⁺, Mg²⁺, and Co²⁺, (especially by Mn²⁺), and be inhibited by Fe³⁺, Cu²⁺, and Ag⁺. BA-CSN showcased the highest relative activity in the hydrolysis of colloidal chitosan, and it had good hydrolysis ability for colloidal chitin. Under the optimal catalytic conditions, BA-CSN demonstrated the Michaelis constant K_m and maximum reaction rate V_max of 9.94 mg/mL and 26.624 μmoL/(mL·min), respectively, for colloidal chitosan. In short, BA-CSN has strong tolerance to acids and alkali, possessing broad industrial application prospects.