萎缩芽孢杆菌酸碱耐受性壳聚糖酶的表达及酶学性质分析

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

摘要:

为筛选鉴定具有良好稳定性的壳聚糖酶，从源于贫瘠盐碱土壤的高产蛋白酶萎缩芽孢杆菌中克隆壳聚糖酶基因并在大肠杆菌中表达，利用镍柱纯化等方法获得萎缩芽孢杆菌壳聚糖酶(Bacillus atrophic chitosanase,BA-CSN)，并对BA-CSN的最适温度、最适pH、底物特异性、动力学参数等性质进行表征。结果表明，BA-CSN的分子量为31.13 kDa，最适反应温度为55℃，最适pH为5.5，在温度低于45℃、pH 4–9时具有良好的稳定性，且在pH为3.0和10.0时处理4 h以内也具有较好的稳定性；K⁺、Na⁺、Mn²⁺、Ca²⁺、Mg²⁺、Co²⁺对BA-CSN具有激活作用，其中Mn²⁺的激活作用最为显著，而Fe³⁺、Cu²⁺、Ag⁺对BA-CSN有抑制作用；BA-CSN水解胶体壳聚糖的相对酶活最高，对胶体几丁质也有较好的酶解能力；在最适催化条件下，BA-CSN对胶体壳聚糖催化反应的米氏常数K_m、最大反应速率V_max分别为9.94 mg/mL、26.624 μmol/(mL·min)。本研究证明BA-CSN对酸碱有较强的耐受性，具有广阔的工业应用前景。

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.