Construction Of Recombinant GdhA And GlnA Pichia Pastoris And Its Ammonia Removal Effect

The emission of odor,especially ammonia,is one of the most important problems that need to be solved urgently in the pollution of aquaculture pollution.In recent years,adding microecological agents to the diet has become one of the main deodorization methods in aquaculture.Previous laboratory studies have confirmed that gdh A and gln A,which regulate glutamate dehydrogenase(GDH)and glutamine synthetase(GS),are key functional genes for ammonia nitrogen removal in animal intestinal ammonia reduction strains.On this basis,we used genetic engineering technology to heterologous expression of gdh A and gln A,the key functional genes of ammonia nitrogen removal,in eukaryotic cells,and verified their deodorization effect by in vitro fermentation test,which provided theoretical basis and data support for further improving the deodorization effect of probiotics in practical production.This study is divided into three parts.In the first experiment,gln A gene from Bacillus coagulans and gdh A gene from Enterococcus faecium were introduced into Pichia pastoris to construct recombinant bacteria by genetic engineering,and the removal of ammonia nitrogen under different initial concentrations of ammonia nitrogen was preliminarily verified.In the second experiment,the activity of the recombinant protein was further improved by optimizing the induced expression conditions.In the third experiment,the effect of recombinant bacteria on ammonia production of laying hens was determined by ammonia production and physical and chemical indexes.The main results are as follows:(1)gln A recombinant bacteria,gdh A recombinant bacteria and double gene recombinant bacteria were successfully constructed.The specific enzyme activity of GS in gln A recombinant bacteria was 17.23 U/mg,the specific enzyme activity of GDH in gdh A recombinant bacteria was 18.11 U/mg,and the specific enzyme activities of GS and GDH in double gene recombinant bacteria were 13.14 U/mg and 15.23 U/mg,respectively.After induced expression,the constructed recombinant bacterias all have a certain effect on the removal of ammonia nitrogen.The ammonia nitrogen removal effect is the best when the initial ammonia nitrogen concentration is 0.1 mg/m L.The removal rates of the double gene recombinant bacteria,gdh A recombinant bacteria and gln A recombinant bacteria are 74.8%,58.2% and 44.6%,respectively,but with the initial ammonia nitrogen as the concentration increases,the removal efficiency of ammonia nitrogen is getting lower and lower.(2)Set different conditions to optimize the induced expression.Under the control of a single variable,the optimal amount of shake flask for inducing expression is 10%,the optimal bacterial inoculum is 2%,and the optimal methanol addition is 2%.(3)Compared with the CK group,the original bacteria group,empty bacteria group,gdh A recombinant bacteria group,gln A recombinant bacteria group and double gene recombinant bacteria group can significantly reduce the ammonia production in the cecum of laying hens(P<0.05),the ammonia production was reduced by 37.58%,38.44%,63.95%,65.68% and 74.04%.The ammonia production of the gdh A recombinant bacteria group,the gln A recombinant bacteria group and the double gene recombinant bacteria group were significantly lower than the original bacteria group and the empty bacteria group(P<0.05).In addition,the recombinant bacteria can also significantly reduce the p H and ammonium nitrogen content of the fermentation broth(P<0.05),and stimulate the conversion of ammonium nitrogen to nitrate nitrogen;the content of uric acid and urea in the fermentation broth and the activity of urease are also significantly reduced(P<0.05).In summary,the constructed recombinant bacterias can significantly reduce the production of ammonia gas in the caecum of laying hens(P<0.05).The lowering of p H in the fermentation broth inhibits the production of ammonia on the one hand,and on the other hand inhibits the growth of harmful bacteria,and at the same time promotes the conversion of ammonium nitrogen to nitrate nitrogen,and reduces ammonia emissions.In addition,the reduction of urease activity also inhibits the conversion of urea,further reducing ammonia production.