黑曲霉QU10果糖基转移酶的克隆表达

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

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（GenBank 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cDNA，其编码628个氨基酸。将所得片段定向克隆到pET-22b、pGAPZA及pGAPZαA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/mL，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 kDa。以蔗糖为底物，果糖基转移酶在pH 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。

Abstract:

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 from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA 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/ml 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 kDa 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.