CAJ | 학술논문

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2049bp,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636bp,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（5.8U/mL）和木聚糖酶活性（32.3U/mL）。
이미근매（Rhizopus oryzae）3.866기인조DNA위모판,극륭득도당화매기인（glucoamylase gene,amyA）,기인전장2049bp,편마604개안기산;이류아포간균（Paenibacillus sp.）H10-3기인조DNA위모판,극륭출기인목취당매기인（xylanase A gene,xynA）적성숙태편마서렬,장636bp,편마211개안기산。통과중첩연신PCR（SOE-PCR）득도병접편단amyA-l-xynA,병장기극륭도필적효모표체재체pPIC9중,득도중조질립pPIC9-amyA-l-xynA,중조질립선성화후경전격전화도필적효모（Pichia pastoris）GS115중,득도료표체성공적공정균AX11。재AX11발효상청액중동시검측도당화매활성（5.8U/mL）화목취당매활성（32.3U/mL）。
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.