耐有机溶剂蛋白酶产生菌MSP05的筛选、鉴定及酶学性质研究

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

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,Cu^(2+)、Mn^(2+)对酶有激活作用,Mg^(2+)、Fe^(3+)和Ba^(2+)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。

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 amyloliquefacieus 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.