黑曲霉QU10果糖基转移酶的克隆表达（英文）  

Molecular cloning and over-expression of a fructosyltransferase from Aspergillus niger QU10

作　　者：张国青[1] 杨敬[1] 石家骥[1] 钱世钧[1] 钞亚鹏[1] 

机构地区：[1]中国科学院微生物研究所传感技术联合国家重点实验室,北京100101

出　　处：《生物工程学报》2015年第4期512-522,共11页Chinese Journal of Biotechnology

基　　金：National High Technology Research and Development Program of China(863 Program)(No.2012AA101807)~~

摘　　要：微生物果糖基转移酶能够以蔗糖为底物产生低聚果糖。为获得更多新酶资源,通过PCR法成功地克隆出黑曲霉QU10的果糖基转移酶基因(Gen Bank Accession No.KF699529),基因片段长度为1 941 bp,包含一个54 bp的内含子。进一步利用RT-PCR法克隆了果糖基转移酶的c DNA,其编码628个氨基酸。将所得片段定向克隆到p ET-22b、p GAPZA及p GAPZαA载体,并转化至大肠杆菌或毕赤酵母中,通过筛选获得果糖基转移酶表达活力高的转化子。利用α信号肽的毕赤酵母转化子获得最高果糖基转移酶胞外酶活力为431 U/m L,是原始菌株酶活力的35倍。此毕赤酵母重组酶为同源二聚体,半天然PAGE表观分子量约200 k Da。以蔗糖为底物,果糖基转移酶在p H 5.0、45℃下反应4 h,酶解产物中主要是蔗果三糖和四糖,蔗果寡糖最高可占总质量的58%。结果表明,果糖基转移酶酵母工程菌具有很高的转果糖基的能力,而且表达活力高,具有潜在的工业应用价值。The main commercial production of fructooligosaccharides（FOS） comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated fromAspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to Gen Bank（KF699529）. The c DNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/m L after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 k Da as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.

关 键 词：黑曲霉 果糖基转移酶 克隆 表达 低聚果糖 

分 类 号：Q936[生物学—微生物学]