Heterologous Expression Of Leucine Dehydrogenase From Bacillus Cereus And Enzymology Research

Leucine dehydrogenase?LeuDH;EC 1.4.1.9?belongs to the oxidoreductase family of enzymes which catalyzes the reversible oxidative deamination reaction of L-Leucine and other branched chain amino acids to their respective?-ketoacids and analogues.LeuDH has been isolated from Bacillus species,a homopolymer which was composed of 4-8 subunits.Producting LeuDH in industry by single subunit to construct E.coli recombinant bacterias.Based on the application of enzymes in medical field,it was urgent to select a safer expression host to ensure the safety of enzymes.In this research,the leucine dehydrogenase from Bacillus cereus ATCC14579 was heterologously expressed in Bacillus subtilis 168.Screening Sec and Tat pathway signal peptides to promote the secretion of LeuDH.According to the different N-terminal positive charges,seven signal peptides?Sec pathway:AmyQ,SacB,LipB,NprE;Tat pathway:PhoD,YwbN,and LipA?were selected for secretory expression of LeuDH.Tat pathway signal peptide PhoD with the N-terminal charge number of 6 performed the highest exogenous activity,reaching 20.25 U×mL^-1,and its secretion efficiency was 38.06%,which was 1.1 times that without signal peptide;the secretion efficiency of the Sec pathway signal peptides AmyQ and SacB were followed by 35.01%and 32.64%,respectively.Six different promoters(inducible promoters:P_grac,P_glv-M1 and constitutive promoters:P₄₃,P_laps,P_Hpa?,P_amyQ)were inserted downstream of P_Hpa? to investigate the effect on LeuDH expression.The constitutive promoter P_amyQ had the best performance.The activity was 31.24U×mL^-1,which was 3.4 times that of the single promoter P_Hpa?;Inducible promoters P_grac,P_glv-M1 were induced with 0.8 mmol×L^-1 IPTG and 2%maltose for 24 h,32 h were correspond to 1.87 U×mL^-11 and 0.82 U×mL^-1,respectively.The four signal peptides AmyQ,SacB,PhoD,and YwbN were fused to behind the best P_Hpa?-P_amyQ promoter,respectively.The activity of the B.subtlis 168/pW6IV(P_Hpa?-P_amyQ,YwbN)was 21.76 U×mL^-11 and its secretion efficiency reached 40.78%,which was the most favorable for the secretion of LeuDH.Fermentation,purification and enzymatic properties of the strain B.subtlis 168/pW6 with the best activity of producing leucine dehydrogenase were analyzed.In 7.5 L fermenter,the enzyme activity reached 217.96 U×mL^-1,which was 5.19 times higher than the shake flask level.The purified enzyme expression level was 13 U×mg^-1.Enzymatic properties indicated that activity toward some the aliphatic amino acids,the Michaelis constants K_m and V_max were6.17 mmol×L^-1 and 14.49?mol L^-1 min^-1 for the best affinity with substrate L-Leucine,separately;the stability was maintained at pH 5.0-11.0;the optimum reaction temperaturewas55°C;Denaturation temperature?T_m?was measured by DSC,which was 64.13°C;the changed of the secondary structure at different temperatures by circular dichroism proved that the?-helical content gradually decreased with increasing temperature.