Expression Of Purine Nucleoside Phosphorylase In Escherichia Coli And Its Application

Purine nucleoside phosphorylase is a reversible phosphorylation enzyme which catalyzes purine nucleoside into ribose-1-phosphate and the corresponding purine bases. It has been proved that excessive drinking of beer that is rich in purine compounds will cause healthy problems, especially goat. It’s of great need to reduce purine contents in beer product. The present study has constructed a PNPase recombinant vector based on p ET-28a（+）: By transforming and expressing E. coli BL21（DE3）, the recombinant PNPase was purified by the affinity column His Trap^TM excel. With purified PNPase supplementing in the mashing process of beer brewing, the purine nucleoside was hydrolyzed into free purine, which could be used by yeast during fermentation process, resulting in a significant decrease of purine content in final beer. The study layed a foundation for the further reduction of purine content in beer industry. The main research results were as follows:To get the highest expression level of PNPase, the culture and inducing parameters were optimized as: The transformants was cultured at 37℃ in LB to OD600 0.6, then cultured at 30℃ for 7 hours with existence of 0.05 mmol·L^-1 of IPTG, to induce the expression of PNPase.The enzyme activities of PNPase was 184.46 U·m L^-1 by HPLC.A His Trap^TM excel column was used for purification of recombinant PNPase. The purity of obtained protein sample was identified by SDS-PAGE and HPLC. The enzymatic characterization of PNPase was then investigated. The optimal p H of recombinant PNPase was 7.0 and it was relatively stable from p H 6.0 to p H 10.0; the optimum reaction temperature was 60℃, and its activity was not decreased even after incubation at 30℃-50℃ for 2 h; PNPase showed variable hydrolysis activities to adenosine, guanosine, inosine, deoxyadenosine, deoxyguanosine and deoxyinosine, among which it was most active to inosine; EDTA, K+ could enhance the enzyme activity by 1.1 times, but Na+, Ca²⁺, Mg²⁺, and urea did not affect the activity of PNPase, moreover Zn²⁺, Fe²⁺, Mn²⁺ could slightly inhibit its activity, and 1 mmol×L^-1 Ag+ caused obvious inhibition to PNPase. PNPase followed Michaelis-Menten equation, and the Vm ax and Km of the enzyme was 58.82 mol·L^-1·min-1 and 2.71 mmol·L^-1, respectively.With PNPase supplemented in beer brewing process, the purine nucleoside could be decomposed into free purine, leading to an increase of free purine content in wort. Therefore, yeast could use more free purine, resulted in a decrease of purine content in beer. It was found that the purine content in the beer had been reduced from 86.23 mg×L^-1 to 64.89 mg×L^-1 by supplementing PNPase in the mashing process; For brewing that use 60% barley wort and 40% corn syrup, the purine content was reduced from 53.12 mg×L^-1 to 37.77 mg×L^-1; For brewing with 30% barley wort, 30% wheat wort and 40% corn syrup, the purine content was reduced from 39.59 mg×L^-1 to 31.00 mg×L^-1; the purine content in the beer had been reduced about 25%.