【摘要】 【目的】克隆伯克霍尔德菌ZYB002菌株中的新型脂肪酶lip C24基因,测定其基本酶学性质,为后续深入研究该基因在菌株中的生理功能奠定基础。【方法】根据洋葱伯克霍尔德菌JK321菌株的全基因组DNA信息,直接设计引物从伯克霍尔德菌ZYB002菌株基因组中扩增出lip C24基因,并对之进行原核表达、重组蛋白的纯化及酶学性质分析。【结果】lip C24基因全长1317 bp,编码438个氨基酸残基;多肽链中具有保守五肽-G-X1-S-X2-G-序列;重组蛋白Lip C24的分子量为45 k Da;能有效水解各种对硝基苯酯,对中链脂肪酸的对硝基苯酯表现出偏爱性;其催化水解反应的最适温度为40℃,最适p H7.5;40℃下的半衰期为15.72 min,在p H 7.0-8.0的条件下,具有较好的稳定性。【结论】lip C24的编码产物为一个45 k Da蛋白,具有明显的脂肪酶活性,为中温中性脂肪酶。更多还原

【Abstract】 [Objective] We cloned a lipase gene, lip C24, from Burkholderia sp. ZYB002 and characterized the recombinant lipase Lip C24. [Method]Based on the known genomic DNA sequence from Burkholderia cecapia JK321,we designed a pair of specific primers for the lip C24 gene and then obtained the full length of lip C24 gene. The lip C24 gene fragment enconding the mature peptide Lip C24 was then subcloned into expression plasmid,p ACYC-Duet-lip B,and expressed in E. coli. The recombinant protein,Lip C24,was purified to homogeneity by His Trap HP chromatography column and Hi Trap DEAE FF chromatography column. [Results] We expressed the lip C24 gene from Burkholderia sp.ZYB002 in E. coli Origami 2( DE3). Nucleotide sequencing revealed that the lip C24 gene had an open reading frame of1317 bp,and the deduced amino acid sequence of Lip C24 corresponded to 438 amino acid residues,including a conserved-G-X1-S-X2-G- motif. The relative molecular weight of the purified Lip C24 was about 45 k Da. The purified Lip C24 displayed hydrolysis activity to various 4-nitrophenyl esters and substrate preference for the medium chain length 4-nitrophenyl-esters. The optimal temperature was 40℃ and the optimal p H was 7. 5. The lipase was stable between p H 7. 0and 8. 0 for 24 hours. However,the half-life was only 16 min at 40℃. [Conclusion]The Lip C24 was a 45 k Da protein,a mesotherm and neutral lipase.更多还原