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