黑曲霉α-L-阿拉伯呋喃糖苷酶基因（ABF2）在酿酒酵母中的克隆与表达。

Cloning and expression of the alpha-L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae.

作者信息

Crous J M, Pretorius I S, van Zyl W H

机构信息

Department of Microbiology, University of Stellenbosch, South Africa.

出版信息

Appl Microbiol Biotechnol. 1996 Oct;46(3):256-60. doi: 10.1007/s002530050813.

DOI:10.1007/s002530050813

PMID:8933843

Abstract

First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the alpha-L-arabinofuranosidase gene (abfB) was amplified with the polymerase chain reaction technique. The abfB DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1P) and terminator (PGK1T) sequences on a multicopy episomal plasmid. The resulting construct PGK1P-abfB-PGK1T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional alpha-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf2) that is 94% identical to the alpha-L-arabinofuranosidase B of A. niger N400. Maximum alpha-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.

摘要

以燕麦spelts木聚糖诱导的黑曲霉MRC11624的mRNA为模板制备第一链cDNA。以该cDNA为模板，采用聚合酶链反应技术扩增α-L-阿拉伯呋喃糖苷酶基因（abfB）。将abfB DNA片段插入多拷贝附加体质粒上的酵母磷酸甘油酸激酶I基因启动子（PGK1P）和终止子（PGK1T）序列之间。得到的构建体PGK1P-abfB-PGK1T命名为ABF2。ABF2基因在酿酒酵母中成功表达，酵母细胞分泌出有功能的α-L-阿拉伯呋喃糖苷酶。测定了ABF2核苷酸序列，验证其编码一个449个氨基酸的蛋白质（Abf2），该蛋白质与黑曲霉N400的α-L-阿拉伯呋喃糖苷酶B有94%的同一性。当自选择重组酿酒酵母菌株分别在合成培养基和复合培养基中培养48小时时，α-L-阿拉伯呋喃糖苷酶的最大活性分别为0.020 U/ml和1.40 U/ml。