| Esterase?Esterase,EC,3.1.1.X?is one of hydrolases.It can be able to hydrolyze the esters into alcohols and acids,or to catalyze the reverse reaction to produce various esters.Esterase is widely used in the fields of industry,agriculture,food and pharmaceuticals,for its ability to catalyze ester hydrolysis and transesterification.Cold-active enzyme can be used for synthesizing special compound and save energy for its high catalytic efficiency and thermal instability.Therefor,EstA was studied and the results were as follows.In this study,a esterase gene estA was cloned from Enterobacter sp..The gene est A contained an open reading frame of 717 bp,which encoded a protein of 238 amino acids with an estimated molecular mass of 26 kDa and EstA had not reported before through sequence alignment.Additionally,sequence alignment revealed that est A sequence had a typical structure of hydrolase-catalytic triad:Ser-Asp-His.It also has the conserved structure Gly-Ala-Ser-Met-Gly of Ser containing.Protein EstA was induced,expressed and purified in Escherichia coli BL21?DE3?,and its enzymatic properties was characterized.Est A showed highest activity to nitrobenzene acetate.The activity of EstA decreased with the increase of substrate side chain length and showed lowest activity to p-nitrophenyl palmitate.The optimum reaction temperature of EstA is 40°C,and the optimum reaction pH of EstA is 9.0.The Km,Kcat and Kcat/Km value of EstA for p-nitrophenyl acetate were 393.75 ?mol/L,905.6 min-1 and2.30×10^3 min-1·?M-1,respectively.EstA remained more than 70% activity between 0°C and 20°C,indicating its cold adaptation.EstA retained more than 60% of its initial activity in the present of 3 M NaCl and it also remained about 100% activity after incubation in 4 M NaCl at 4°C for 24 h,indicating its salt tolerance.Ethylene glycol,30% isopropanol and 30% acetone had obvious inhibition to EstA.But 10% and 20% isopropanol,acetone,ethanol,methanol and DMSO had less effect to EstA,indicating its organic solvent tolerance.Morever EstA remained more than 60% activity at 0.5% and 1% Triton-100,tween-20,tween-80,CTAB,10% Triton-100 and 10% tween-20.EstA was not stable under high temperature,retaining little activity of its originalactivity after incubation at 45°C for 120 min or at 50°C for 30 min.In order to improve the thermal stability,a variant A92 P was gained by random mutation,which showed better thermal stability than EstA,remaining 95% of its original activity after incubation at 45°C for 120 min,19 times than the wild type EstA.Morever,mutant A92 P still remained 55% of its original activity after incubation at 50°C for 30 min,2 times than the wild type EstA.A variant A92 D was obtained by saturation mutation which showed good thermal stability.The incubation after 45°C for 120 min had little effect on the activity of mutant A92 D and it remained 85% activity after incubation at 50°C for 30 min,approximately 3.4 times than EstA.Homology models of EstA and A92P/A92 D were built to understand the relationship between structure-function and thermal stability.The analysis results showed that the improved thermostability of A92 D may be due to the hydrophilic Asp instead of the hydrophobic Ala to increase the interaction between it and solvent as well as the surrounding areas.The improvement A92 P of thermostability may be due to Pro with annular structure limitting the rotation of Secondary Structure,increasing protein rigid structure.These results providing improtant informations for further reseaching the ralationship between esterase structuer and function and the reseach of the rmostability. |
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