溶剂稳定性蛋白酶产生菌的筛选和鉴定

Screening and identification of an organic solvent-stable protease producer

自油污土样等样品中分离获得一株具有产胞外溶剂稳定性蛋白酶的耐有机溶剂极端微生物,经BIOLOG系统鉴定及16S rDNA序列(GenBank,EF105377)分析,该菌株为Bacillus licheniformis YP1。该菌株能耐受中浓度盐、强碱性环境(pH12)及多种不同浓度的有机溶剂,但对多种抗生素敏感。在YP1产蛋白酶发酵过程中添加各种有机溶剂结果表明,丙酮虽抑制了菌体生物量的生长却促进了单位菌体的蛋白酶分泌,而长链烷醇如辛醇、十二醇等能强烈抑制该蛋白酶的分泌。该菌株所产蛋白酶经11种50%(V/V)有机溶剂处理后均能保留高活力。该溶剂稳定性蛋白酶在有机相生物催化等领域具有良好的应用前景。

An organic-solvent-tolerant bacterium strain YP1 producing organic-solvent-stable protease was isolated from crude oil contaminant soil. Strain YP1 was strictly aerobic, motile, gram positive, spore-forming, and rod shaped. The YPlstrain was identified as Bacillus licheniformis using culture system BIOLOG analysis （SIM = 0.62,16 - 24h）. The 16S rDNA sequence analysis （GenBank accession number EF105377 ） suggested that strain YP1 was clustered together with B. licheniformis in phylogenetic tree. Based on all the taxonomy, strain YP1 was identified as B. licheniformis. YP1 strain could tolerant organic solvents at different levels, especially it can grow well in the presence of water-miscible solvents dimethylformamide （DMF, log P =-1.0） and dimethylsulphoxide （ DMSO, logP = - 1.35 ） at a concentration of 10% [V/V]. Strain YP1 can also tolerant middle concentrations of NaCl and extra alkaline conditions（pHI2）. More than 80% of the biomass remained at pH range 10.5 - 12. However strain YP1 was sensitive to antibiotics such as ampicillin, tetracycline, kanamycin and chloromycetin. The protease production could be enhanced by acetone and repressed by alkanols such as dodecylalcohol and octanol during the fermentation. Compared to trypsin, the YP1 protease had a wider tolerance for organic solvents. YP1 protease tolerated up to at least 11 organic solvents with logP ranging from - 1.35 to 5.6 including benzene, toluene, DMSO and DMF etc at 50% （V/V） concentration. Moreover, when solvents such as decane and dodecyl alcohol with log P values above 4.0 were added to the crude protease, the enzyme activity levels were 1.08 and 1.21 times higher than the control respectively. Its high tolerance for water-miscible solvents DMF and DMSO makes it an ideal catalyst for kinetic- and equilibrium-controlled synthesis. This organic solvent stable protease could be used as a biocatalyst for enzymatic synthesis in the presence of organic solvents.