基因工程手段提高磷脂酰丝氨酸合成酶活性的研究

为了提高目的蛋白磷脂酰丝氨酸合成酶(PSS)的表达,使用rTaq酶从E coli K12总DNA中PCR扩增获得磷脂酰丝氨酸合成酶基因片段,将其重组于高效表达载体pET28b质粒,转入相应表达菌株进行表达,收集菌体超声破碎获得含PSS的粗酶液,使用两相反应体系进行生物转化,HPLC-ELSD手段进行酶活检测。结果显示,重组菌株中PSS的酶活力比对照有所提高,同时获得酶活力提高的突变基因pssE210G。将突变基因重组于更高效的表达载体pBAD-MCS,转入相应宿主菌株表达。结果显示E.coli TOP10(pBAD-MCS-pss)表达的酶活性明显优于E.coli BL21(pET28b-pss)中表达的酶活性。以上实验结果表明,将目的基因重组于高效表达质粒有助于提高酶活力;组合到不同的表达质粒,酶活提高程度不同;磷脂酰丝氨酸合成酶基因第73位氨基酸发生突变,对其酶结构和酶活力有直接的影响。

Improvement of Phosphatidyl Serine Synthetase Activity by Means of Gene Engineering

In order to improve the expression of phosphatidyl serine synthetase （PSS） of target protein,fragment of gene pss was PCR amplified with rTaq from E.coli K12 total DNA,followed recombining into a high effective expression plasmid pET28b and transferred into corresponding expression strain to express.Crude PSS enzyme solution of E.coli BL21 （pET28b-pss） cells was collected through sonication,then liquid supernatant was conducted bioconversion PC to PS in a two-phase reaction system,and the enzyme activity was determined by HPLC-ELSD.The results showed that activity of PSS in recombined E.coli BL21 （pET28b-pss） was improved than that of the control one,and at the same time obtained an improved enzyme activity mutated gene pssE210G.The mutated gene was recombined into even more effective expression vector plasmid pBAD-MCS,and transferred into a corresponding host to expression.The results showed that the expressed enzyme activity of E.coli TOP10 （pBAD-MCS-pss） significantly better than that of E.coli BL21 （pET28b-pss）.All the results above suggested that recombine the target gene into high-effective expression plasmids was conducive to promote enzyme activity,and recombine into different expression plasmids the improved degree were different; and the mutation of the 73rd amino acid of PSS directly affected the enzyme structure and its activity.