知识与财富的链接
本实验通过PCR方法从毕赤酵母GS115-phyA中扩增出不含有信号肽及内含子的黑曲霉NRRL3135植酸酶phyA基因,并将其克隆到表达载体pINA1297中,得到表达载体pINA1297-phyA,利用醋酸锂转化法将线性化载体转化到解脂耶氏酵母po1h中,通过YNBcasa和PPB平板筛选出阳性表达菌株,阳性菌株在YM培养基中28℃培养6d后酶活达到最大为636.23U/mL。表达上清经SDS-PAGE分析得到表达植酸酶分子量约为130kDa,但通过去糖基化处理后其分子量变为51kDa,与理论值相符。经过酶学性质分析表明重组植酸酶最适pH为5.5,最适温度为55℃,该酶在pH2.0~8.0处理1h后仍有较高酶活,并且90℃处理10min后还有86.08%的残留酶活,其抵抗胃蛋白酶和胰蛋白酶能力也较强。
Using the polymmerse chain reaction (PCR), we amplified the phytase gene phyA from Pichia pastoris GS115-phyA in Aspergillus niger NRRL3135 without the signal peptide sequence and intron sequence,. Then, it was cloned into pINA1297 vector to generate a recombinant vector of pINA1297-phyA. pINA1297-phyA was linearized and transformed into Yarrowia lipolytica po1h by the lithium acetate method. The positive transformants were obtained by YNBcasa and PPB plates, after induced in YM medium at 28°C for 6 day. The activity of the expressed phytase phyA reached 636.23 U/mL. The molecular weight of the enzyme was 130 kDa measured with SDS-PAGE analysis, whereas its molecular size reduced to 51 kDa after deglycosylation which is correspond with theoretical value. The enzymatic analysis of the recombinant phytase phyA revealed its optimal pH and temperature was 5.5 and 55°C, which had high activity after incubated in pH ranged from 2.0 to 8.0 for 1 h. Moreover, its activity remained 86.08% after exposure to 90°C for 10 min. It also was resistant to pepsin or trypsin treatment.
