DCE-01菌株果胶酯酶基因克隆与表达

Cloning and expression of the pectin methyl esterase gene from DCE-01 strain

从麻类脱胶高效菌株DCE-01中克隆果胶酯酶基因并进行原核表达。根据全基因组测序注释结果设计引物,PCR扩增果胶酯酶基因连接到pEASY-E1载体上,导入大肠杆菌进行诱导表达,采用水解圈法进行选择和定量分析。结果表明:克隆出全长1107bp的果胶酯酶基因(GenBank登录号:KC422449),编码368个氨基酸;该基因表达蛋白质序列的前26个氨基酸为信号肽,前导蛋白的分子量约为39.6ku,成熟蛋白为36.9ku,pI为9.1;基因工程菌株以高度酯化橘子果胶为底物的发酵液粗酶活为1.5IU/mL,是原始菌株DCE-01的22.4倍。本研究成功发掘出果胶酯酶基因,其表达产物果胶酯酶能降解高甲氧基果胶,在低甲氧基果胶制备方面具有重要应用前景。

Primers were designed by the potential pectin methyl esterase gene annotated from the whole genome sequence of DCE-01 strain, which is an efficient strain for bast fiber bio- extracting. The pectin methyl esterase gene was cloned,kinked to pEASY-E1, and expressed in Escherichia coil BL21 （DE3）.The positive colonies were selected by the hydrolysis circles, and then their pectin methyl esterase activities were analyzed.It was resulted that the pectin methyl esterase gene（GenBank. KC422449）was 1107bp and encoded 368 amino acids. By the bioinformatics software analysis, the 26 amino acids in front of the protein sequence were signal peptide. The molecular weight of pre-PME was approximately 39.6ku,the molecular weight of mature-PME was 36.9ku, and pl was 9.1.With high methoxyl citrus pectin as substrate,the pectin methyl esterase activity secreted by the genetic engineering strain was 1.51U/mL,22.4 times higher than that from the original DCE-01 strain.An efficient pectin methyl esterase gene had been excavated from the DCE-01 strain,and its expression product could degrade high methoxyl pectin,so it might be available for low methoxyl pectin preparation.