Study On The Recombinant Expression And Properties Of N-CH₃ Removal Enzyme

Histone lysine demethylase and nicotine N-demethylase are oxidases that can catalyze the degradation of N-CH₃ compounds toproduce formaldehyde,demethylated products and other by-products through demethylation.The above enzymes have been applied widely in areas such as pharmaceutical manufacturing and environment treatments.In this study,we selected the histone lysine demethylase 1(Histone lysine demethylase 1,Lsd1)from zebrafish,the histone lysine demethylase(Jumonji C,Jmjc)from coffee and the nicotine N-demethylase(Nnd)from tobacco for expression and property characterization.The selected enzymes were firstly soluble expressed in the E.coli(Escherichia coli)expression system,and the corresponding enzymatic properties were further studied.According to the identified properties of each enzyme,methods including structure modification and fermentation optimization were carried out to improve the enzyme activity.The details and results are described as follows:(1)Through the screening of vector plasmids,the recombinant bacteria E.coli BL21(DE3)/p TIG-TRX-ls D1;E.coli BL21(DE3)/p TIG-TRX-jmj C and E.coli BL21(DE3)/p ET28a-nn D were successfully constructed.Under the fermentation temperature of 25?,the soluble target protein could be obtained by IPTG induction with the concentration of 0.1 mmol·L^-1.After verifying the capability of each enzyme to catalyze the corresponding substrate,Lsd1 and Jmjc were identified with the catalytic ability except Nnd.(2)In terms of reaction activity,the corresponding optimum temperature and p H for Lsd1were 35?and 7.0,while for Jmjc were 20?and 8.0.Comparing Lsd1 with Jmjc in stability,Lsd1 showed better thermostability,but Jmjc was found to have better tolerance to the change of p H.Both of them were suitable for preservation in neutral and alkaline conditions.In consideration of the effects of different metal ions(Cu²⁺?Mn²⁺?Co²⁺?Mg²⁺?Ca²⁺?Zn²⁺?Ni²⁺),the impacts brought on the two enzymes vary.Among which,Cu²⁺was observed to have a significant effect on the activity of Lsd1.According to the kinetic study,the catalytic coefficient of these two enzymes was relatively low since the overall reaction time was long.The K_m of Lsd1 and Jmjc were 1.05 mmol·L^-1 and 1.54 mmol·L^-1,respectively.The V_max of Lsd1 and Jmjc were 7.9×10^-7 mmol·(L·min)^-1 and 1.2×10^-6 mmol·(L·min)^-1,respectively.(3)Through the simulation analysis of the structure of Jmjc,some of the structures that may block the“active pocket”of Jmjc were screened out and removed.It was found that the temperature stability of the enzyme was improved after modification.Compared to the optimal condition,100%of the enzyme activity could be remained at 50?,while the remaining enzyme activity of the pre-modified Jmjc was only 50%at the same temperature condition.In addition,although there was a decrease of enzyme activity after modification(V_max=2×10^-8mmol·(L·min)^-1),the affinity of the modified enzyme to the substrate was observed to be greatly improved(K_m=0.17 mmol·L^-1).(4)Taking the enzyme activity as an index,the enzyme activity of Lsd1 was further improved by fermentation optimization.After the single factor optimization experiment,rather than to enhance the growth of bacteria,lactose was found to improve the enzyme activity of Lsd1 when it was provided as the carbon source.When the compound nitrogen source was used as the nitrogen source,both the growth rate of bacteria and enzyme activity were found to be the greatest.Compared with other metal ions,the addition of Cu²⁺to the medium promoted the enzyme activity most significantly and greatly shortened the reaction time.Via the comparison of different inorganic salts,sodium chloride was verified as the optimum inorganic salt for fermentation.After the analysis of orthogonal test and response surface experiment,the formula of the fermentation medium was determined to be Cu²⁺(2.9 mmol·L^-1),compound nitrogen source(14.5 g·L^-1)and sodium chloride(9.7 g·L^-1).