高效产壳聚糖酶海洋菌株Mitsuaria sp.SH-50的筛选、鉴定及产酶条件优化

Screening,Identification and Optimization of Enzyme Production Conditions of the Chitosanase-Producing Marine Strain Mitsuaria sp.SH-50 with High Efficiency

旨在从滨海虾蟹养殖区土壤中筛选出高效产壳聚糖酶的菌株,并获得其最优产酶条件。利用以壳聚糖为唯一碳源的培养基,对海南三亚滨海虾蟹养殖区土壤中的微生物进行富集、分离和筛选,获得高效产壳聚糖酶的菌株。通过生理生化特性分析及16S rDNA测序对其菌属进行鉴定,然后采用单因素试验、爬坡试验(Plackett-Burman)和响应曲面法(Box-Behnken Design)优化其产酶条件。筛选得到一株高效产壳聚糖酶的菌株,经鉴定为松江菌属(Mitsuaria sp.),并将其命名为Mitsuaria sp.SH-50。单因素试验及爬坡试验结果表明,壳聚糖浓度、初始pH、发酵温度对菌株产酶影响最为显著。通过响应面回归分析得出其最佳产酶培养基组分为:粉末壳聚糖7.52 g/L,酵母粉1.0 g/L,K_(2)HPO_(4) 4.0 g/L,NaCl 4.0 g/L,MgSO_(4) 1.0 g/L;最佳培养条件为:初始pH 6.75,温度30.5℃,接种量1%,180 r/min摇瓶培养12 h。优化后SH-50菌株产壳聚糖酶活性为26.6 U/mL,较优化前提高了5.3倍。SH-50菌株的产酶活性高于目前报道的大多数菌株,且产酶周期仅为12 h。该研究为壳聚糖酶的大规模生产和工业化制备低聚壳聚糖提供了重要的理论依据。

The aim of this study is to screen chitosanase-producing strains with high efficiency from the soil of coastal shrimps and crabs culture areas,and to obtain the optimal conditions for the production of enzyme.The microorganisms in the soil of coastal shrimps and crabs culture areas in Sanya,Hainan are enriched,separated and screened with chitosan as the only carbon source,and a chitosanase-producing strain with high efficiency is obtained.Physiological and biochemical characteristic analysis and 16S rDNA sequencing are used to identify the bacterial genus,and then single factor test,climbing test(Plackett-Burman)and response surface methodology(Box-Behnken Design)are used to optimize its enzyme production conditions.A chitosanase-producing strain with high efficiency is obtained by screening,and it is identified as Mitsuaria sp..The strain is named Mitsuaria sp.SH-50.The results of single factor test and climbing test show that the concentration of chitosan,initial pH and fermentation temperature have the most obvious effect on the enzyme production of Mitsuaria sp.SH-50.The optimal components of enzyme-producing medium obtained through response surface regression analysis are as follows:powdered chitosan 7.52 g/L,yeast powder 1.0 g/L,K_(2)HPO_(4) 4.0 g/L,NaCl 4.0 g/L,MgSO_(4) 1.0 g/L;the optimal culture conditions are as follows:initial pH 6.75,temperature 30.5℃,inoculation amount 1%,shake flask culture at 180 r/min for 12 hours,and the chitosanase production activity of SH-50 strain is 26.6 U/mL after optimization,which is 5.3 times higher than that before optimization.SH-50 strain has higher enzyme production activity than most strains that have been reported so far,and the enzyme production period is only 12 hours,which provides an important theoretical basis for the large-scale production of chitosanase and the industrial preparation of oligo chitosan.