脂肪酶产生菌的筛选及基因的克隆表达

目的筛选出脂肪酶活性较高菌株,通过脂肪酶基因克隆技术获得高表达的脂肪酶,根据酶活曲线确定该酶的最适温度和最适pH.方法采用透明圈法,以三丁酸甘油酯为底物筛选脂肪酶活性较高菌株;通过PCR扩增获得其脂肪酶基因Pseudomonas peli(PP)序列,构建pET-28 a表达载体,并在大肠杆菌Escherichia coli BL21(DE3)中进行异源表达;通过Ni柱将脂肪酶纯化,并根据酶活曲线确定该酶的最适温度和最适pH.结果筛选到一株脂肪酶活性较高的菌株,16S rRNA基因序列比对结果初步鉴定为Pseudomonas peli.PCR扩增得到的基因片段长度为801 bp,其序列与Pseudomonas peli中的脂肪酶基因序列相似性达到100%,PP基因在大肠杆菌中异源表达蛋白的分子量约29 KD,该蛋白在55℃、pH 7.0时活性最高.结论通过基因克隆表达后得到的脂肪酶Ni柱纯化后纯度达95%以上,且耐高温,为脂肪酶工业化应用奠定了基础.

Isolation of Lipase-producing Strain and the Cloning Expression of Pseudomonas Peli

Objective To isolate lipase-producing strains from mud,to clone and characterize the lipase gene, and to determine the optimum temperature and pH.Method High activity lipase-producing strains were screened using tributyrin as substrate by transparent ring method.Lipase gene sequence of Pseudomonas peli (PP)was obtained by PCR amplification using the genomic DNA as template.The expression plasmid pET-28a-PP(rePP)was constructed and expressed in Escherichia coli BL21(DE3).Lipase enzyme was purified by Ni column,and the optimum temperature and pH were determined according to the activity curve.Results A high lipase activity strain was isolated, and was preliminarily identified as Pseudomonas peli.The fragment obtained from PCR amplification was 801 bp,which shared 100% sequence identity with that from PP.The protein of PP was expressed in E.coli BL21(DE3)with the molecular of 29 KD.The purified recombinant enzyme showed high lipolytic activity at temperature 55 ℃ and pH 7.0.Conclusion The PP gene was obtained, and the over expression protein with high lipolytic activity was obtained in this study,which maybe widely used in industry.