重组Pyrococcus furiosus葡聚糖磷酸化酶催化淀粉合成葡萄糖-1-磷酸

Synthesis of Glucose-1-Phosphate from Starch by Pyrococcus Furiosus Glucan Phosphorylase

将Pyrococcus furiosus来源的编码葡聚糖磷酸化酶的glg P基因进行稀有密码子优化,在E.coli中重组表达并催化淀粉合成G-1-P。在反应条件优化基础上,以不同的淀粉为底物,GPase均表现出较强的催化能力。在原料预处理、高浓度底物催化及补料催化研究的基础上,以7.5%玉米淀粉为初始底物,反应16 h后补入2.5%玉米淀粉,继续反应32 h,G-1-P产量达250.34 mmol·L^(-1),底物转化率达65.1%,与相同浓度的可溶性淀粉相比,G-1-P产量及转化率(252.9mmol·L^(-1),65.9%)均相近,表明廉价易得的玉米淀粉可以代替可溶性淀粉为原料生产G-1-P,大幅降低G-1-P生产的原料成本。

glg P gene encoding glucan phosphorylase（GPase） from thermophile Pyrococcus furiosus was codon-optimized and overexpressed in E. coli, and the recombinant GPase was used to synthesize glucose-1-phosphate（G-1-P） from starch. The results show that the GPase exhibits high activity for all kinds of starch studied under the optimal reaction conditions. Corn starch was selected as the raw material for further investigation. When the initial corn starch concentration is 7.5%, and another 2.5% corn starch is added after 16 h, the final production of G-1-P is 250.34 mmol·L^（-1） and the conversion ratio is 65.1% after 48 h reaction, which is similar to that obtained with 10% soluble starch as the substrate（252.9 mmol·L^（-1）,65.9%）. The results indicate that cheap and widely available corn starch can be used as an alternative of soluble starch for G-1-P production.