产低温脂肪酶非极端细菌的筛选、产酶发酵及粗酶性质研究

Isolation,Fermentation and the Crude Enzyme's Characterization of a Cold-active Lipase Producing Mesophilic Bacterium

目的:筛选产低温脂肪酶非极端细菌菌株,扩大脂肪酶的应用范围。方法:利用维多利亚蓝B平板显色法和摇瓶发酵法,从土壤中筛选产脂肪酶菌株,通过菌落形态和菌体特征观察初步对菌种进行鉴定,并对该菌株的产酶发酵培养基进行了优化。结果:得到一株产低温脂肪酶非极端细菌菌株sybc-li-1,该菌株适宜产酶培养基(%)为淀粉1、牛肉浸膏1、NaNO3 0.08、CaCl2 0.04、MgSO4 0.04、橄榄油2和OP1;初始pH8、30℃、200r/min培养72h,脂肪酶活力可高达到30.2U/mL;所产脂肪酶粗酶最适作用温度20℃,最适pH9.5,0℃时仍能保持70%的酶活性,属于低温酶;该酶与目前报道的低温脂肪酶相比,有较好的热稳定性,粗酶在pH8.5、70℃条件下保温60min,酶活力损失30%。结论:该菌株为自然环境中筛选的非极端细菌,所产脂肪酶为低温脂肪酶,在开发应用上有良好的前景。

Objective： A strain of mesophilic bacterium producing cold- active fipase was screened in order to enlarging the application of lipase in industry. Method： A lipase - producing strain was isolated from soil samples by screening with plates containing Victoria Blue B and the flask- shaking fermentation. Then it was identified and demonstrated by the morphological and physiological characteristics. The optimal lipase - produc- ing fermentation conditions were also studied. Result： A strain of mesophilic bacterium producing cold - active lipase named sybc - li - 1 was ob- tained in this paper. The optimal fermentation medium （ % ） was starch 1, beef extract 1, Na-NO3 0.08, CaC12 0.04, MgSO4 0.04, olive oil 2 and OP 1. The yield of lipase was up to 30.2 U/mL at the original fermentation condition of pH 8, temperature 30℃, shaking frequency 200r/ min, and time 72 h. The optimum pH and temperature of the lipase were pH 9.5 and 20℃ respectively. Furthermore, the thermal stability of the cold = adapted lipase was better than lipases those had been reported. Only 30% of the original activity was lost when incubated at 70℃ and pH 8.5 for 60 min. Conclusion： Tiffs strain was a mesophilie bacterium strain screened from nature environment. It produced a novel lipase belong to cold- active lipase, and the characteristic above implied good prospects in the development and application.