长梗木霉内切葡聚糖酶I基因的克隆及其在毕赤酵母中的表达  

Cloning of Endoglucanase I Gene from Trichoderma longi-brachiatum and Its Expression in Pichia pastoris

作　　者：刘海英[1] 王娟[1] 舒正玉[1] 吴松刚[1] 黄建忠[1] 

机构地区：[1]福建师范大学工业微生物教育部工程研究中心生命科学学院,福建福州350108

出　　处：《微生物学通报》2009年第3期355-359,共5页Microbiology China

基　　金：福建省发改委重大项目[No.(2004)477];福建省科技厅重大项目(No.2005Q007)

摘　　要：一株纤维素酶高产菌株经ITS序列鉴定并命名为长梗木霉SSL(Trichoderma longi-brachiatum,SSL)。利用RT-PCR的方法从该菌株中克隆出内切-1-4-β-D-葡聚糖酶I的基因(eg1),该基因全长1386bp,编码461个氨基酸。序列分析表明:该基因序列与T.longibrachiatum egl1基因具有90%以上的同源性。将该基因的成熟肽编码序列插入到Pichia pastoris表达载体pPIC9k中,构建重组表达质粒pPIC9k-eg1,转化P.pastoris GS115。重组P.pastoris菌株,经甲醇诱导后,胞外重组内切葡聚糖酶I的活力达73U/mL。SDS-PAGE中出现一条明显加强的蛋白质条带,其分子量大约为58kD。A cellulase high-yield strain was identified and named as Trichoderma longibrachiatum SSL by ITS sequence identification. The endoglucanasel gene （egl） encoding endo-1,4-β-D-glucanase Ⅰ was ampli- fied by RT-PCR method, which including 1386 bp and encoding 461 amino acid. Sequence analysis showed that： This gene has a more 90% homology with the T. longibrachiatum egl gene. The egl gene encoding the mature peptide was inserted into the Pichia pastoris expression vector pPIC9K, which resulted in construc- tion of the recombinant expression plasmid, pPIC9k-eg1. The pPIC9k-eg1 was then introduced into the host Pichia pastoris GS 115. After the induction of methanol, extracellular recombinant endoglucanase I from the supernatant of the recombinant Pichia pastoris strain reached 73 U/mL. A clear strengthening of the protein bands, whose molecular weight is about 58 kD, appeared in the SDS-PAGE.

关 键 词：分子标记 内切葡聚糖酶Ⅰ 毕赤酵母 表达 

分 类 号：Q93[生物学—微生物学] Q78