Cloning and expression of the pectin methyl esterase gene from DCE-01 strain
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摘要: 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。Abstract: 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, linked to pEASY- E1, and expressed in Escherichia coli 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 pI was 9.1.With high methoxyl citrus pectin as substrate, the pectin methyl esterase activity secreted by the genetic engineering strain was 1.5IU /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.
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