Expression And Molecular Engineering Of Bacillus Paralichenifromis Acid Urease For Reducing Urethane In A Model Rice Wine

Ethyl carbamate（EC,or urethane）,a carcinogenic compound that naturally present in various kinds of alcoholic beverages,especially in Chinese rice wine,has been attracted much attention.Microbial enzymatic degradation of EC and its precursor urea by urethanase or acid urease are the efficient and attractive methods.However,the unsatisfactory enzymatic properties for urethanase（such as instability under acid or high concentration of ethanol conditions,low EC affinity）and the low production and nickel concerns for acid urease restrict their industrial application.In this study,we found that urease from Bacillus paralicheniformis ATCC 9945A（BpUrease）was an iron-containing acid urease,which shows high ethanol tolerance and can degrade EC.Thus,BpUrease was further heterologous expressed and engineered for desired properties to degrade EC from rice wine.Major results were outlined as follows:（1）Identification of BpUrease as the urethanase in B.paralicheniformis.Urethanase produced by B.paralicheniformis can retain 64%of enzyme activity after treated by 20%（v/v）ethanol at 37°C for 2 h.Then the urethanase was purified by following procedures:ammonium sulfate fractionation,anion exchange chromatography,hydrophobic chromatography,high resolution anionexchange chromatography and gel filtration chromatography.After purification,its purity was increased by 105 times,the recovery rate was 5.5%and the specific activity was 10.5 U·mg^-1.Eventually,the purified urethanase was identified as BpUrease by MALDI-TOF/MS.（2）Identification of BpUrease as an nickle and iron-containing urease.The enzyme activity of recombinant E.coli,in which BpUrease was expressed,confirmed BpUrease has EC-hydrolyzing activity.After analyzing the effects of medium supplementation with NiSO₄,FeCl₃,MnCl₂,CuSO₄ and CoSO₄ on urease activity in recombinant E.coli,we found that only addition of NiSO₄ and FeCl₃ resulted in obvious accumulation of enzyme activity.After fusion of Strep-tag on N-terminal of BpUreC,the recombinant BpUrease was purified.Native PAGE analysis showed that molecular mass of BpUrease was approximately 220 kDa,suggesting it was a（UreABC）₃ structure.BpUrease（Fe）and BpUrease(Ni²⁺)contained 0.3equivalents of Fe or 1.9 equivalents of Ni per heterotrimer,respectively.The results demonstrate that the BpUrease is nickle and iron-containing urease.（3）BpUreH was identified as a transmembrane protein for enrichment of Ni²⁺and Fe³⁺,and the no obvious reducing of EC was ascribed to the low EC affinity of BpUrease.After deletion of the BpureH,urease acitivity decreased about 75%.After overexpressing BpUreH in E.coli and investigating intracellular concentrations of Ni and Fe,BpUreH was identified as a transmembrane protein for enrichment of Ni²⁺and Fe³⁺.Then,the biochemical propertieswere examined.Both of optimal temperature and pH for BpUrease（Fe）and BpUrease(Ni²⁺)toward urea and EC were 50 and 45°C,5.0 and 7.0 respectively.Both two forms of BpUrease were stable at temperatures lower than 50°C or in the pH of 6.0-8.0.Both of two enzymes can retain 64%of their maximal activity after treatment with 15%ethanol for2 h.The K_m and k_cat/K_m value for BpUrease(Ni²⁺)toward EC and urea were 1018 and 23.7mM,425 and 1.2E+05 s^-1·M^-1,respectively.The K_m and k_cat/K_m value for BpUrease（Fe）toward EC and urea were 958 and 28.2 mM,58 and 9557 s^-1·M^-1,respectively.These results indicated that Ni²⁺was preferable compared with Fe³⁺.Eventually,after digested with 6,000U·L^-1 puried enzyme at 37°C for 50 h,approximately 92.1 and 94.3%of the urea was removed from rice wine by BpUrease（Fe）and BpUrease(Ni²⁺),respectively.In contrast,no obvious reducing of EC was observed which could be ascribed to the low EC affinity.（4）EC affinity and catalytic efficiency were significantly enhanced by molecular engineering method.Substrate channel and catalytic pocket were considered as main targets for engineering.After screening by the site-saturation mutagenesis（SSM）libraries,Leu253and Leu287（C subunit）were found to be key residues that alternative EC affinity and catalytic efficiency.Compared with wild-type BpUrease,k_cat/K_mvalue of variants L253P,L287I and L287N increased by 418,333 and 721%,respectively.K_m value of L287N were decreased by 37.5%.K_m and k_cat/K_m values for variant L253P/L287N were 521 mM and 4788s^-1·M^-1,respectively,which was a 51%decrease in K_m and 1080%increase in k_cat/K_m.Homology modeling analysis suggests that Leu253Pro mutation affecting the interaction between Arg339 with catalytic residue His323,while Leu287Ile and Leu287Asn mutation changing the interaction networks among residues in catalytic pocket.After digestion with 10U·mL^-1 purified L287N and L253P/L287N at 20^oC for 50 h,30.9 and 37.1%of EC were removed from rice wine,respectively,which should be ascribed to the enhance of EC affinity.（5）BpUrease was over-expressed in B.subtilis.We achieved expression of BpUrease in a food-grade B.subtilis TEA/pTTB expression system.After reassembling Bpurease gene cluster with inserting RBS（AGGAGG）,the expression was increased from 38 to 187 U·L^-1.Furthermore,by altering the position of ureC in front of ureAB,the expression was further increased to 530 U·L^-1,which was a 183%increase.Additonaly,after strengthening the expression of iron transporter encoding gene BpureH,the activity was further increased to1307 U·L^-1,which was a 147%increase.Eventually,by applying a fed-batch fermentation with glucose feeding,the urease expression was improved to 21,964 U·L^-1 in 3-L fermentor.To the best of our knowledge,this is the highest acid urease activity reported.