Study On The Expression, Purification And Properties Of Arsenic S-adenosylmethionine Methyltransferase Fused By Two Kinds Of Tags

Arsenic(As)is a highly toxic Metalloid that is widespread in nature.Microorganisms play an important role in the control of arsenic geochemical cycles and arsenic pollution.Microorganisms have a variety of arsenic metabolism mechanisms,among which arsenic methylation can convert inorganic As(?)to less toxic As(V)methylated products and highly volatile As(?)methylated products.This process is considered to be a biological means of effective environmental arsenic remediation and microbial arsenic detoxification.At present,more and more arsenic methylated organisms and new arsenic methylase(ArsM)genes have been discovered.The structure and functional relationships of ArsM and the mechanism of arsenic methylation catalyzed by enzymes have been studied in depth.ArsM is a catalytic arsenic species.The core of methylation,the study of the basic properties of enzymology is the basis for the development and application of ArsM.At present,there are few studies on the enzymatic properties of ArsM except ArsM of Cyyandioschyzon sp.5508 and Arsenicibacter rosenii SM-1.In view of the relatively high expression of CGA009 ArsM clones of Rhodopseudomonas palustris,the genes of the three engineering bacteria with high arsenic volatilization activity are reported from this strain.Therefore,this study cloned and expressed ArsM(ArsM-His)of CGA009 strain and studied the basic properties of the enzyme.Since the elastin-like polypeptide(ELP)has the characteristic of reversible phase change cycle(ITC),as a nonchromatographically-purified protein tag,the purification process is simple and convenient,low in cost,and easy to scale production.Therefore,this study further used the ELP fusion expression method to clone and express ArsM(ArsM-ELP)of CGA009,optimized the expression conditions induced by the fusion enzyme,established its ITC purification system,and studied the enzymatic properties of the enzyme.This lays the foundation for the large-scale preparation and application of ArsM.The main results of this study are as follows:Two recombinant E.coli strains(ArsM-His)were obtained by histidine tag fusion expression.Compared with the control strains,the arsenic resistance of recombinant E.coli increased,and the arsenic sensitive strain AW3110(ArsM-His).The arsenic resistance of the engineering bacteria has greatly increased,and it is initially shown that this ArsM has arsenic methylation function.After purification by nickel affinity column(NI-NTA),the recovery rate of ArsM-His was 10.15 %.The temperature is 25°C-40°C,the enzyme activity is above 97%;the enzyme activity is higher than 60% in the range of pH 5.0-9.0;the optimum temperature and pH are 35°C and 7.5,respectively,indicating that the enzyme has relatively Wide temperature and pH adaptability.At 35°C,45°C,and 55°C,the enzyme activity was maintained at 97%,80%,60%(1 h)and 88%,53%,and 10%(5 h)for 1 h and 5 h respectively,indicating that the enzyme activity Poor thermal stability.Mercaptoethanol(2-Me)had the effect of promoting enzyme activity,SDS,EDTA and Fe2+ had inhibitory effects on enzyme activity,and Na+,Mg2+,Zn2+,Ca2+ and Cu2+ had no obvious effect on enzyme activity.The results of substrate specificity showed that except for As(?),Na+,K+,Ca2+,Fe2+,Fe3+,Cr3+,and As(V)could not detect enzyme activity,indicating that the enzyme's characteristic recognition of inorganic As(?)effect.After 24 h of reaction,the total arsenic in the reaction system was reduced by 11.75%?1.22%,respectively,by the recombinant strains AW3110 and BL21,indicating that the recombinant bacteria AW3110(ArsM-His)and the enzyme ArsM-His have the ability to volatilize arsenic.The recombinant strain BL21 did not show arsenic volatilization ability.Two strains of recombinant E.coli(ArsM-ELP)were obtained using the elastinlike tag(ELP)fusion expression method.Arsenic resistance assay showed that the ArsM has arsenic methylation ability.ArsM-ELP optimal expression conditions: 0.2 mmol/L IPTG,16°C,22 h.The optimal ITC purification system was as follows: 16 mg/mL crude enzyme was incubated with 1.5 mol/L NaCl,incubated at 32°C for 5 min,refolded for 10 min,and cyclically degraded and refolded for 3 times.The recovery rate was 18.84%.The enzymatic properties were basically consistent with that of ArsM-His.The arsenic volatilization ability was reduced by the ELP label,but the acidbase tolerance,thermal stability,and inhibitor tolerance were significantly improved.In conclusion,both the ArsM and E.coli AW3110 genetically engineered bacteria that are fused by the two tags have arsenic volatilization ability,and the basic enzymatic properties of ArsM expressed by these two tags are explored.At the same time,the ITC purification system for purification of ArsM using ELP as a tag was optimized.Compared with His-tagged purification system,ArsM-ELP is inexpensive to purify and easy to prepare in large quantities,which lays the foundation for the large-scale preparation and application of ArsM.