Efficient Expression Of PNP In Bacillus Subtilis And Its Preliminary Application Study

Purine nucleoside phosphorylase(PNPase,PNP)is a key enzyme in the purine remedy synthesis pathway.A purine degradation pathway involving the cleavage of the oxidized purine ring,which forms the end product uric acid in the body and increases the risk of developing gout.Gout is the most common joint inflammation and is characterized by crystalline deposits of uric acid at the joints.Studies have shown that in addition to several non-modifiable risk factors such as genetics,gender and age,some modifiable risk factors such as alcohol consumption and lifestyle are also associated with gout.Alternative approaches such as consuming foods or beverages with low purine and sugar content,and lifestyle changes can help control uric acid levels in the body while reducing the associated medical financial burden.During the saccharification of the raw wort for beer production,purines in the barley malt are released,and during later fermentation,yeast can only use the free purine bases,while the bound purine nucleosides remain in the finished brew,which in turn triggers the risk of gout.Purine nucleoside phosphorylase is able to use purine nucleoside as a substrate and break it down into phosphoric acid and free purine bases,which greatly improves the utilization of purine substances in the wort by yeast,thus reducing the purine content of the finished wine.To improve the fermentation level of purine nucleoside phosphorylase,the purine nucleoside phosphorylase genes from Kuyveromyces lactis(K.lactis or Klac)and human(Homo sapiens or Hs)were codon optimized,and the target genes before and after optimization were ligated into the secretory expression vector the optimal fermentation conditions were determined by single-factor optimization: fermentation for 60 h,fermentation temperature of 37 ℃,and initial p H of the medium at 7.The results showed that the enzyme activity of Klac PNP before codon optimization was 333.69 U/m L;after optimization,the enzyme activity of Klac PNP reached 351.61 U/m L;the enzyme activity of Hs PNP before codon optimization was 315.06 U/m L;after optimization,the enzyme activity of Hs PNP strain PNP-YO-p BSA43 reached 327.67 U/m L.The enzyme activity of PNP-YO-p BSA43-WB600 was the best,and it was suitable for the expression and purification of the recombinant protein in the later stage.In order to further improve the recombinant protein expression,a 3-factor,3-level response surface analysis test was conducted by Box-Behnken response surface design,and the optimal fermentation conditions for PNPase production by the optimized recombinant strain were: initial p H=6.67,fermentation time 55 h,and fermentation temperature 28.7 ℃.Under these conditions,the recombinant purine nucleoside phosphorylase activity was 372.79 U/m L,which was 1.12 times higher than that before optimization.The optimized PNP crude enzyme solution was added to the wort,and it was found that PNP could break down the bound purine nucleosides in the wort into free purine bases,increase the free purine base content in the wort,promote the yeast to absorb and utilize more free purine bases in the fermentation stage,and finally reduce the total purine content in the fermentation broth.The free guanine in the original wort was increased from 3.64 μmol/L to 4.77 μmol/L;free hypoxanthine from 1.48 μmol/L to 3.94 μmol/L;free xanthine from 15.70 μmol/L to 21.07 μmol/L.The free guanine in the wort was increased from 8.69 % to 11.14 %;free hypoxanthine from17.41 % to 11.14 %.The free rate of hypoxanthine increased from 17.41 % to 45.45 % and the free rate of xanthine increased from 58.82 % to 75.76 %.Total guanine in the fermentation broth decreased from 36.82 μmol/L to 31.13 μmol/L;total hypoxanthine from 4.85 μmol/L to 3.59μmol/L;total xanthine from 24.24 μmol/L to 17.97 μmol/L.In the later stages of fermentation,purine uptake increased from 60.84 % to 64.27 %;hypoxanthine The uptake rate increased from58.76 % to 66.88 % and xanthine uptake rate increased from 16.81 % to 19.40 %.The codon optimized recombinant expression of purine nucleoside phosphorylase gene from Saccharomyces cerevisiae and human was significantly improved,which laid the foundation for the efficient expression of purine nucleoside phosphorylase in Bacillus subtilis.Its addition to wort successfully hydrolyzed the purine nucleosides in the bound state in wort to purine bases in the free state,increased the free rate of purine in wort,improved the utilization of free purine by yeast in fermentation and reduced the total purine content in fermentation broth.