嗜热毛壳菌热稳定脂肪酶基因(lm)的克隆及其在毕赤酵母中的表达(英文)  

Cloning of a novel thermostable lipase gene(lm) from Chaetomium thermophilum and its expression in Pichia pastoris

作　　者：李萌[1] 郭晓红[1] 李多川[1] 

机构地区：[1]山东农业大学环境生物系,山东泰安271018

出　　处：《菌物学报》2015年第3期434-442,共9页Mycosystema

基　　金：Supported by the National Programs for High Technology Research and Development(2012AA10180402);the National Marine Renewable Energy Research Foundation(SDME2011SW01)

摘　　要：研究从嗜热毛壳菌Chaetomium thermophilum中克隆了一个新的脂肪酶基因(lm)。其中DNA序列包含一个由870个碱基构成的开放阅读框,编码289个氨基酸,含有4个内含子,没有信号肽序列。序列提交Gen Bank,登录号为GU338248。将该基因在毕赤酵母中表达。在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,蛋白达到0.428mg/m L,菌物学报酶活力为19.77U/mg。SDS-PAGE检测该蛋白的分子量为35k Da。该脂肪酶的最适反应温度为60℃,具有热稳定性,在40–80℃热稳定,80℃处理60min仍有65%的相对酶活。该酶最适反应p H值为10.0,在p H 9.0–12.0酶活相对稳定。该酶具有较好的热稳定性和耐碱性,具有良好的工业应用价值。A novel lipase gene, lm, was cloned from the thermophilic fungus Choetomium thermophilum. DNA sequencing revealed that the genomic DNA of lm had an open read ntrons without any potential signal sequences. The ing frame of 870bp, encoded 289 amino acid residues, and contained four obtained nucleotide sequence of lm was deposited in GenBank under Accession No. GU338248. A recombinant C. thermophilum lipase was expressed in the methylotrophic yeast Pichia pastoris. The highest lipase activity （19.77U/mg） and protein expression level （0.428mg/mL） were detected in the yeast culture after methano nduction for 6 days. SDS-PAGE analysis demonstrated that the recombinant lipase had a molecular mass of 35kDa. Optima temperature for activity of the recombinant lipase was 60℃. The recombinant lipase was thermostable at 40-80℃ and retained 65% relative activity after incubation at 80℃ for 60min. The optimum pH for activity of the recombinant lipase was 10.0 and the pase was stable from pH 9.0 to 12.0. The results indicated that the novel C. thermophilum lipase had high thermostability and alkaline tolerance, thus was of great value for industrial applications

关 键 词：嗜热真菌 分子量 酶活 最适温度 最适PH 热稳定性 

分 类 号：Q78[生物学—分子生物学]