中文摘要：

通过3次PCR程序克隆得到了华根霉（Rhizopus chinensis CCTCCM201021）脂肪酶全基因序列，该基因的开放阅读框长1170bp，不含内含子，编码一个389个氨基酸残基的蛋白质，包括26个氨基酸的信号肽，94个氨基酸的前导序列和269个氨基酸的成熟肽，其推断的氨基酸序列与一些已报道的根霉脂肪酶序列同源性为86％。在巴斯德毕赤酵母（Pichia pastoris）GS115中分泌表达前导肽序列。SDS-PAGE分析表明表达蛋白的分子量约37kD。N-端氨基酸序列分析表明该重组蛋白分泌过程中前导序列N-端的67个氨基酸被切割掉，表达的重组脂肪酶由前导序列C-端的27个氨基酸和成熟肽的269个氨基酸组成。发酵132h后上清中重组脂肪酶的表达量最高，蛋白含量约5．4mg／mL，橄榄油乳化法测水解酶活为161U／mL，其比活比野生型华根霉脂肪酶高约43倍。

英文摘要：

The whole-length sequence of a lipase gene was cloned from the genomic DNA isolated from Rhizopus chinensis CCTCCM201021 by three PCR programs. Its entire open reading frame was 1170bp without intron, encoding a 389 amino acid protein including 26 amino acid signal sequence, 94 amino acid prosequence and 269 amino acid mature lipase sequence. The amino-acid sequence showed 86% similarity to those of the other Rhizopus sp. lipase amino-acid sequences. The RCL gene without its signal sequence was successfully expressed in Pichia pastoris GS115. The result of SDS-PAGE showed that the molecular weight of the aimed protein was nearly 37kD. The N-terminal amino acid sequence analysis showed that the N-terminal 67 amino acids were degraded when the recombinant protein was secreted, the expressed recombinant protein consisted of a 27 amino acid prosequence and a 269 amino acid mature lipase sequence. The highest specific activity of lipase in the supematant was 30U/mg after 132h of culture, which was 43-fold higher than that of the wild-type Rhizopus chinensis CCTCCM201021.