Expression, Characterization And Function Of The Alkaline Thermophilic SGNH Esterases

As the earliest life form of the earth, thermophilic bacteria have an amazing ability to adapt to high temperatures. Various thermophilic enzymes of thermophilic bacterias become the focus of industrial and scientific fields due to the good biological stability and unique catalytic activity.In this study, three genes of SGNH esterases K91-Est8, K91-Est19 and Est D1 were selected from two bacterial strains Bacillus sp. K91 and Alicyclobacillus tengchongensis CGMCC1504. The three genes were inserted into pEASY-E2 vector and expressed successfully in Escherichia coli BL21(DE3). The recombinant enzymes K91-Est8, K91-Est19 and EstD1 were purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography. Biochemical characterizations of the three recombinant enzymes and their catalytically active residues were detailed as follows:(1) Purified recombinant K91-Est8 showed optimal activity at pH 9.0 and 50°C, and retained 47.1% of the maximum activity at 80°C and 22.7% of the maximum activity at 95°C. The enzyme activity was stable at 37°C and pH 8.0-9.3 for more than 60 min. It was activated by most metal ions and chemical reagents. The catalytic efficiency(kcat/Km) toward p-NPC2 was approximately 37-fold, 454-fold, 1428-fold higher than toward p-NPC4, p-NPC6, p-NPC8, respectively. No significant activity was observed for the substrates with a chain length>C8. K91-Est8 can remove the acetyl group of the 3-position from cephalosporins 7- aminocephalosporanic acid(7-ACA).(2) Purified recombinant K91-Est19 showed optimal activity at pH 9.0 and 60°C. The enzyme activity was stable at 37°C and pH 8.5-9.5 for more than 60 min. It was not affected by most metal ions and chemical reagents. The catalytic efficiency(kcat/Km) toward p-NPC2 was approximately 17-fold, 3000-fold higher than toward p-NPC4, p-NPC6, respectively. No significant activity was observed for the substrates with a chain length>C6. K91-Est19 can’t remove the acetyl group of the 3-position from cephalosporins 7- aminocephalosporanic acid(7-ACA).(3) Purified recombinant EstD1 showed optimal activity at pH 8.5 and 65°C. The enzyme activity was stable at 37°C and pH 4.0-11.0 for more than 60 min.The half-life of EstD1 was approximately 60 min at 50°C and 65°C. The catalytic efficiency(kcat/Km) toward p-NPC2 was approximately 43-fold higher than toward p-NPC4 and no significant activity was observed for the substrates with a chain length>C4. K91-Est8 can remove the acetyl group of the 3-position from cephalosporins 7- aminocephalosporanic acid(7-ACA).(4) Based on(1)-(3), ative hydrophobic binding pocket more suitable esters with short-chain acyl groups, and more specific for ester with acetyl. The three SGNH esterases also can hydrolysis alpha/beta-naphthyl acetate. Furthermore, They have higher beta-naphthyl acetate catalytic activity than that of alpha-naphthyl acetate. These properties have theoretical significance on substrate specificity study of SGNH hydrolase.(5) Based on amino acid sequence comparisons, homology modeling analysis, and site-directed mutagenesis: S11, D182,H185; S49, D227, H230 and S14, D190, H193 were the catalytically active residues of K91-Est8, K91-Est19 and EstD1, respectively.In summary, the three SGNH esterases K91-Est8, K91-Est19 and EstD1 were expressed and characterized. They not only showed great value for basic research, but also endowed itself with great industrial interest on semi-synthetic β-lactam antibiotics.