米根霉糖化酶、类芽孢杆菌木聚糖酶融合蛋白的真核分泌表达

以米根霉(Rhizopus oryzae)3.866基因组DNA为模板,克隆得到糖化酶基因(glucoamylase gene, amyA),基因全长2 049 bp,编码604个氨基酸;以类芽孢杆菌(Paenibacillus sp.)H10-3基因组DNA为模板,克隆出基因木聚糖酶基因(xylanase A gene, xynA)的成熟肽编码序列,长636 bp,编码211个氨基酸。通过重叠延伸PCR(SOE-PCR)得到拼接片段amyA-l-xynA,并将其克隆到毕赤酵母表达载体pPIC9中,得到重组质粒pPIC9-amyA-l-xynA,重组质粒线性化后经电击转化到毕赤酵母(Pichia pastoris)GS115中,得到了表达成功的工程菌AX11。在AX11发酵上清液中同时检测到糖化酶活性(5.8 U/mL)和木聚糖酶活性(32.3 U/mL)。

Eukaryotic Secrete Fusion Expression of Rhizopus oryzae Glucoamylase and Paenibacillus sp. Xylanase

The glucoamylase gene (amyA) was amplified from Rhizopus oryzae 3.866 total DNA extracts. The sequence of amyA gene was consisted of 2 049 bp, and encoded 604 amino acids. The xylanase gene (xynA) was amplified from Paenibacillus sp. H10-3 total DNA extracts. The mature peptide encoding sequence of xynA was 636 bp, and encoded 211 amino acids. The mature peptide sequence of amyA gene, sequence of linker and sequence of xynA gene were spliced by overlap extension PCR (SOE-PCR), then amyA-l-xynA was obtained. The fusion gene was then inserted into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA. By using electroporation, we successfully transformed the recombinant pPIC9-amyA-l-xynA into Pichia pastoris GS115, AX11 was obtained. The maximum yield of the recombinant glucoamylase and xylanase in AX11 culture medium were 5.8 U/mL and 323 U/mL, respectively.