地衣芽孢杆菌CP-16脂类水解酶的研究

Research on Lipolytic Enzymes from Bacillus licheniformis CP-16

本试验旨在通过异源表达获得地衣芽孢杆菌(Bacillus licheniformis)CP-16的脂类水解酶,并探究其在羽毛降解过程中的作用。试验以地衣芽孢杆菌CP-16基因组DNA为模板,扩增脂类水解酶基因,转化入大肠杆菌中表达,获得重组酶L-4。研究重组酶L-4的适宜p H、p H稳定性、适宜温度、温度稳定性以及有机溶剂和金属离子对其相对活性的影响,同时探究其对角蛋白酶K水解天然羽毛角蛋白的作用。结果显示,获得的脂类水解酶基因大小为747 bp,编码248个氨基酸,在大肠杆菌中成功表达出重组酶L-4,其分子质量约为28.3 ku,酯酶活性为0.42 U/m L,适宜p H为6.5,适宜温度为50℃;在p H 6.5~9.5条件下处理30 min相对活性保持80%以上,在低于50℃温度条件下处理30 min相对活性保持70%以上。二价铁离子(Fe^(2+))、钠离子(Na^+)、锰离子(Mn^(2+))、钙离子(Ca^(2+))对重组酶L-4相对活性具有激发作用,钡离子(Ba^(2+))、锌离子(Zn^(2+))、铜离子(Cu^(2+))、镍离子(Ni^(2+))对重组酶L-4相对活性具有抑制作用。当有机溶剂浓度为30%时,重组酶L-4在二甲基亚砜(DMSO)和甲醇中保存97%和85%的相对活性,在丙酮、乙醇中保存45%以上的相对活性,在异丙醇中保存不到20%的相对活性,而在乙腈中相对活性基本完全丧失。用重组酶L-4预处理天然羽毛底物,可提高角蛋白酶K对底物的水解效率,促进率为4.32%。由此可见,脂类水解酶可降解羽毛表层脂质,可在促进角蛋白酶水解羽毛角蛋白中发挥作用。

In order to explore the influence of lipolytic enzyme on feather degradation,the lipolytic enzyme gene of Bacillus licheniformis CP-16 w as cloned and heterologous expressed in this research. The target gene of recombinant enzyme L-4,w hich w as used Bacillus licheniformis CP-16 genome DNA as a template,amplified lipid hydrolase gene,and then transferred into Escherichia coli for expression of the targeted gene. The optimum p H,p H stability,temperature,temperature stability and effects of organic solvents and metal ions on relative activity of the recombinant enzyme L-4 w ere determined,and its application on keratin K hydrolyzed natural feather keratin w as also investigated. The results show ed that the length of lipid hydrolase gene w as747 bp,encoded 248 amino acids,the recombinant enzyme L-4 w as successfully expressed in Escherichia coli,and the molecular w eight w as about 28. 3 ku,the esterase activity w as 0. 41 U/m L,the optimum p H w as6.5,the optimum temperature w as 50℃. The relative activity of recombinant enzyme L-4 kept above 80%treated w ith 30 min under p H 6. 5 to 9. 5 condition,and the relative activity kept above 70% treated w ith30 min under below 50 ℃ condition. The ferrous iron（ Fe^（2＋））,sodion（ Na~＋）,manganese ion（ Mn^（2＋）） and calcium ion（ Ca^（2＋）） had an stimulative effect on the relative activity of recombinant enzyme L-4,while the barium ion（ Ba^（2＋））,zinc ion（ Zn^（2＋））,copper ion（ Cu^（2＋）） and nickel ion（ Ni^（2＋）） had an disincentive effect on the relative activity of recombinant enzyme L-4. When the concentration of organic solvent w as 30%,the relative activity of recombinant enzyme L-4 kept 85% and 97% in methanol and dimethyl sulfoxide solutions,the relative activity kept above 45% in acetone and ethanol solutions,the relative activity kept less than 20% in isopropanol solution,w hile the relative activity w as complete loss in acetonitrile solution. The pretreatment of natural feather substrates w ith recombinant enzyme L-4 promoted the hydrolysis of keratinase K to the substrate,and the promotion rate is 4.32%. In conclusion,lipolytic enzymes can degrade feather surface lipids and it may play a role in promoting keratin hydrolysis of feather keratin.