耐有机溶剂蛋白酶产生菌MSP05的筛选、鉴定及酶学性质研究

目的:从燕尾港受污染海域筛选耐有机溶剂蛋白酶产生菌,确定该菌的分类地位,并对其所产溶剂稳定性蛋白酶的酶学性质进行研究。方法:通过在培养基中添加20%(V/V)甲苯作为筛选压力,筛选耐有机溶剂极端微生物,通过产蛋白酶筛选培养基筛选产蛋白酶的菌株,并利用摇瓶发酵进行复筛,最后对产酶菌株进行鉴定并对粗酶的性质进行测定。结果:筛选获得耐有机溶剂蛋白酶产生菌MSP05,结合16S rDNA序列分析、形态学观察和生理生化性质将其鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。该酶的最适反应温度和pH分别为50℃和10.0,Cu2+、Mn2+对酶有激活作用,Mg2+、Fe3+和Ba2+对酶具有抑制作用。在50%乙醇中,25℃、140 r/min振荡72 h后仍能保持50%以上酶活力。结论:分离获得耐有机溶剂蛋白酶产生菌解淀粉芽孢杆菌菌株MSP05,对有机溶剂具有较好的耐受性,具有潜在的工业应用价值。

Screening,Identification and Characterization of an Organic-Solvent Tolerant Protease Producing Strain MSP05

Objective:Organic-solvent tolerant protease-producing strains were isolated from polluted area in Yanwei Port,the taxonomic status were clarified,and the enzymatic characteristics of the protease were studied.Methods:Organic-solvent-tolerant strains were isolated from organic-pollution water by adding 20%(V/V)methylbenzene into medium as selective pressure. Protease-producing strains was obtained through the first screening with selection medium plate. The high-producing strains were re-screened by shake flask fermentation. Then the strains were identified and the enzymatic characterization was measured. Results:MSP05 was obtained and identified as Bacillus amyloliquefaciens by its morphological,physiological,biochemical characteristics and 16S rDNA sequence homology. The optimum pH and temperature values of this protease were pH10 and 50℃,respectively. The enzyme was activated by Cu2+,Mn2+ and inhibited by Mg2+,Fe3+ and Ba2+. This protease has good organic-solvent tolerant. It remained more than 50% activity after 72 h incnbated with 50% enthol at 25℃ and 140 r/min.Conclusion:One high-producing organic-solvent tolerant protease producer Bacillus amyloliquefaciens MSP05 was obtained,which would have potential industrial value.