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pH、pH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/mL,适宜pH为6.5,适宜温度为50℃;在pH 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(Fe2+)、钠离子(Na+)、锰离子(Mn2+)、钙离子(Ca2+)对重组酶L-4相对活性具有激发作用,钡离子(Ba2+)、锌离子(Zn2+)、铜离子(Cu2+)、镍离子(Ni2+)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 was cloned and heterologous expressed in this research. The target gene of recombinant enzyme L-4, which was 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 opti-mum pH, pH stability, temperature, temperature stability and effects of organic solvents and metal ions on rel-ative activity of the recombinant enzyme L-4 were determined, and its application on keratin K hydrolyzed nat-ural feather keratin was also investigated. The results showed that the length of lipid hydrolase gene was 747 bp, encoded 248 amino acids, the recombinant enzyme L-4 was successfully expressed in Escherichia co-li, and the molecular weight was about 28.3 ku, the esterase activity was 0.41 U/mL, the optimum pH was 6.5, the optimum temperature was 50℃. The relative activity of recombinant enzyme L-4 kept above 80%treated with 30 min under pH 6. 5 to 9. 5 condition, and the relative activity kept above 70% treated with 30 min under below 50℃ condition. The ferrous iron ( Fe2+) , sodion ( Na+) ,manganese ion ( Mn2+) and cal-cium ion ( Ca2+) had an stimulative effect on the relative activity of recombinant enzyme L-4, while the barium ion ( Ba2+) , zinc ion ( Zn2+) , copper ion ( Cu2+) and nickel ion ( Ni2+) had an disincentive effect on the rela-tive activity of recombinant enzyme L-4. When the concentration of organic solvent was 30%, the relative ac-tivity 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, while the relative activity was complete loss in acetonitrile solution. The pretreatment of natural feath-er substrates with 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.
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