疏棉状嗜热丝孢菌脂肪酶基因的克隆及其在毕赤酵母中的高效表达  

Cloning of a gene encoding lipase from Thermomyces lanuginosus and effective expression of the gene in Pichia pastoris

作　　者：郑艳[1] 周波[1] 宋宁宁[1] 李多川[1] 

机构地区：[1]山东农业大学微生物系,泰安271018

出　　处：《菌物学报》2009年第2期268-274,共7页Mycosystema

基　　金：国家"863"计划(No.2006AA10Z304)

摘　　要：疏棉状嗜热丝孢菌Thermomyces lanuginosus可产生具有重要工业生产价值的脂肪酶。根据已报道的相应序列设计特异引物,综合运用PCR、RT-PCR技术克隆到脂肪酶基因的全长DNA和cDNA序列。其中DNA序列长1071bp,包含876bp的开放阅读框以及3段内含子;cDNA序列长885bp。结构基因编码蛋白包含292个氨基酸,前17个氨基酸构成信号肽。序列提交GenBank,登录号分别为EU022703和EU370914。将脂肪酶基因cDNA序列的开放阅读框克隆到酵母分泌型表达载体pPIC9K中,转化毕赤酵母GS115得到重组子且实现了分泌表达。将重组子诱导产酶,在培养温度30℃、甲醇添加量1%的情况下,小规模发酵量达0.93mg/mL,其分泌表达的最高酶活为7.2U/mL。重组酶最适反应温度和pH分别是60℃和8.0。表达蛋白在60℃保温1h后仍有完全酶活,具有较高的热稳定性。Thermomyces lanuginosus produces an extracellular lipase that is important for industrial applications. Based on published DNA sequence, we cloned the full length genomic DNA and cDNA of the lipase using PCR and RT-PCR methods. DNA sequencing revealed that the genomic DNA of the T. lanuginosus lipase gene has an open reading frame of 876bp encoding a protein lipase precursor of 292 amino acid residues, and three introns. And the eDNA of lipase gene we cloned is identical to the registered lipase gene of Thermomyces lanuginosus in GenBank （AF054513）. The DNA and eDNA sequence have been registered in GenBank with accession number EU022703 and EU370914, respectively. PCR product consisting of the eDNA of the T. lanuginosus lipase gene coding region without its signal sequences, first 17 amino acid residues, was cloned into the yeast secretive plasmid pPIC9K, which was then transformed into Pichia pastoris GS 115 strain. The T. lanuginosus lipase gene was expressed and recombinant lipase was secreted into the culture medium in an active form. Production of recombinant protein reached 0.93g/L when it was induced by 1% （V/V） methanol at 30℃ in small-scale cultures. The recombinant enzyme exhibited optimum activity 7.2U/mL at pH 8.0 and 60℃. It is a thermostable enzyme since it retained complete activity in lh at 60℃. The effective expression of the recombinant lipase in P pastoris makes it become an attractive system to produce enzyme for industrial applications.

关 键 词：内含子 成熟蛋白 诱导 分泌表达 重组酶 最适反应温度 最适反应pH 热稳定 

分 类 号：Q78[生物学—分子生物学]