Heterologous Expression Of Urease-encoded Gene From Bacillus Amyloliquefaciens In Recombinant Bacillus Subtilis And Its Application In Urea Degradation

Ethyl carbamate(EC)is a potential carcinogenic compound in rice wine.Urease degradation of urea,the precursor of EC,has attracted much attention.To guarantee the food safety of rice wine,the gene encoding B.amyloliquefaciens IT-45 urease(Ba-urease)was obtained by gene mining and expressed in recombinant B.subtilis.In this study,the recombinant strain was successfully optimized for fermentation,and its function of effectively degrading urea was verified.This provides a practical solution for the degradation of urea in rice wine,and has broad application prospects in traditional fermented food industry.(1)Through gene mining and structural analysis,the gene encoding Ba-urease,which contains only three structural subunits(Ure A,Ure B and Ure C),was obtained.The urease gene cluster was recombined and expressed in B.subtilis WB168,B.subtilis WB600 and B.subtilis WB800.By comparing the growth trend and enzyme activity,it was found that the recombinant strain B.subtilis WB600/p P43NMK-Ure obtained high urease activity,which was 6.85 U·m L^-1.Therefore,B.subtilis WB600/p P43NMK-Ure was selected for follow-up research.(2)The expression of all subunits in the urease gene is essential for the production of enzyme activity.After the optimization of codon usage and ribosome binding site,the urease gene cluster was reconstructed and enzyme activity was increased from 6.85 U·m L^-1 to 9.01U·m L^-1;by adjusting the position of the gene ure C,the enzyme activity was further increased to 10.15 U·m L^-1,which was increased by 48%compared with the original urease activity.The urease expression strain was reconstructed by replacing the high-strength constitutive promoter,but the urease activity was not successfully increased due to the instability.Thus,the P₄₃ promoter was still selected as the expression promoter.The activity of recombinant urease further reached 12.5 U·m L^-1 in 500 m L shake-flask after culture conditions optimization,which was increased by 82%compared with the original urease activity.By the way,TB medium,3%inoculum volume fraction,28?expression temperature,4 mmol·L^-1Ni²⁺addition amount are the best fermentation conditions.(3)The recombinant urease exhibited the highest activity at the optimal temperature37?and in the optimal p H range 6.5-7.0,which was neutral urease.The recombinant urease has good stability in the temperature range of 30?-50?,relatively good stability in the p H range of 6.0-7.0,and can also tolerate a certain low concentration(0%-15%)of ethanol.It was detected by high performance liquid chromatography that the recombinant urease can degrade urea in the urea solution directly and effectively,with a degradation rate of about 36%-68%.When applied to a simulated wine sample,the degradation rate of urea is 20%-35%;while in the rice wine system,the degradation rate is reduced to 7%.At this time,the degradation rate of the recombinant urease strain can only reach 11%.The immobilization method of embedding method and cross-linking method was used to treat the recombinant urease strain,the urea degradation rate was about 30%,20%.The recombinant urease is safe,effective and easy to operate.The p H of the reaction and its stability still need to be further improved.