海洋菌株Mitsuaria sp. SH-50产嗜热性壳聚糖酶CsnSH50的酶学性质表征及其应用

Enzymatic Characterization and Application of Thermophilic Chitosanase CsnSH50 from Marine Strain Mitsuaria sp. SH-50

摘要:

实验室从中国海南滨海虾蟹养殖区泥土样品中筛选到一株高产壳聚糖酶的海洋菌株Mitsuaria sp. SH-50，优化后该菌株产酶时间仅为12 h，产酶活性可达26.6 U/mL。该研究对其胞外壳聚糖酶进行分离纯化和酶学性质表征并进行壳寡糖制备的小试放大试验，以期进一步明确该菌株在壳寡糖生产上的实际意义。结果表明：壳聚糖酶CsnSH50分子量约为41 ku，纯化后CsnSH50比酶活力为7 462.50 U/mg，最适反应pH值为4.5，温度为75 ℃，在pH值2.5~8.5及温度低于50 ℃条件下稳定性较好。最适条件下，稀释后CsnSH50的最大反应速率Vmax=29.41 U/mL，米氏常数Km=1.71 mg/mL，1 mmol/L的K+、Ca2+、Cu2+、Fe3+、Mn2+对CsnSH50的酶活力有明显正激活效应，10 mmol/L的Mn2+对其正激活效应可达412%。TLC和ESI-MS结果表明：CsnSH50水解壳聚糖的最终产物主要为壳二糖、三糖、四糖。小试放大试验结果显示：制备5%（m/V）壳寡糖溶液时，壳聚糖溶液的初始pH值3.5，温度75 ℃，水解120 min时壳寡糖产率达到92.1%，产物平均聚合度为10.2。综上，CsnSH50具有酶活性高、热稳定性好、耐酸等特性，同时是一种鲜有报道的嗜热性壳聚糖酶，具有良好的工业应用潜力。

Abstract:

This study screened a marine strain of Mitsuaria sp. SH-50 with high chitosanase activity levels from soil samples of the coastal shrimp and crab culture area in Hainan province, China. After optimization, the enzyme production cycle of strain SH-50 was only 12 h, with the enzyme activity reaching 26.6 U/mL. To further clarify the practical significance of SH-50 in the production of chitosan oligosaccharide, its chitosanase was isolated and purified for the characterization of enzymatic properties and a small-scale test of chitosan oligosaccharide preparation. The results show that CsnSH50 has a molecular weight of approximately 41 ku, an enzymatic activity level of 7 462.50 U/mg, an optimum reaction pH of 4.5, an optimum reaction temperature of 75 ℃, and was stable at pHs 2.5~8.5 and temperatures < 50 ℃. Under optimal conditions, the Vmax and Km of diluted CsnSH50 were 29.41 U/mL and 1.71 mg/mL, respectively, and 1 mmol/L K+, Ca2+, Cu2+, and Mn2+significantly activated CsnSH-50 enzyme activity. The positive activation effect of Mn2+ at 10 mmol/L was up to 412%. TLC and ESI-MS demonstrated that the final products of chitosan hydrolysis by CsnSH50 were mainly chitobiose, chitotriose, and chitotetraose. When preparing 5% chitosan oligosaccharide solution in small-scale experiments, the initial pH was 3.5, the temperature was 75 ℃, and the chitosan oligosaccharide yield rate reached 92.1% after 120 min of hydrolysis with an average degree of polymerization of 10.2. In conclusion, CsnSH50 has high enzyme activity levels, good thermal stability, and acid resistance. CsnSH50 is also a rarely reported thermophilic chitosanase and has the potential for industrial application.