Study On Molecular Modification Of GDSL Family Esterases

Cephalosporins are a broad-spectrum antibiotic whose source is mainly dependent on synthetic or semi-synthetic.7-Aminocephalosporanic acid?7-ACA?is the common used material for production of cephalosporins.With the emergence of bacterial resistance and other issues,the development of new antibiotics has become an inevitable trend.Deacetylated 7-ACA?D-7-ACA?can form a hydroxyl group at the 3-position,which is highly reactive and susceptible to be attacked by nucleophiles.So,the hydroxyl group at 3-position can be replaced by various of functional groups and then produce new cephalosporins.Now,D-7-ACA is mainly obtained by the method of chemical treatment,which can cause adverse environmental pollution.Therefore,the use of environmentally friendly enzymatic hydrolysis method will be the main way to obtain D-7-ACA in the future.The GDSL esterase family was named by Upton and Buckley in 1995 because of a conserved sequence of GDSL at its N-terminus.GDSL family esterases are widely distributed in nature.They are closely related with the growth and development in plants.However,although the GDSL esterases are also widely distributed in microorganisms,little is known about their function in microorganisms.In previous study,we identified and characterized two new esterases Est8 and EstD1 which both belong to GDSL esterase family.Both of them have deacetylation activity on 7-ACA,but the activity is not high.In order to improve the deacetylation activity of 7-ACA by Est8 and EstD1,the methods of directed evolution and rational design based on three-dimensional structure of the two enzymes were used in this project.The main results are as following:?1?Using the Est8 and EstD1 genes as templates,mutations were made by error-prone PCR.470 mutants were randomly selected from solid plates,and high-throughput screening was performed by a colorimetric method depending on the color change of the pH indicator.The mutant Est8-G45R which has a 2.7-fold as much as the original enzyme in the deacetylation activity of 7-ACA was obtained.?2?Est8-G45R was heterologously expressed and purified,and the enzymatic properties were studied,the results are as following:a.The optimum pH of Est8 and Est8-G45R was pH 8.5,and both can maintain more than 50%of the enzyme activity in the range of pH 7.5-9.0.To determine the pH stability,esterase Est8 can maintain more than 50%activity after treatment with pH 3.0-10.0 buffer for 1 h.However,less than 40%activity was detected after incubated in pH 2.2-6.0 buffer for 1 h.b.The optimum temperature of Est8 and Est8-G45R are 40°C and 35°C,respectively.Both can maintain more than 50%original activity when incubated in the temperature range from 20-45°C.The half-life of Est8 was more than 1 h when incubated at 37°C,50°C and 60°C,respectively.However,the half time of Est8-G45R was less than 10 min when incubated at 50°C and 60°C,respectively.c.1 mM Na⁺,K⁺,Ca²⁺,Al³⁺,Fe³⁺have little effect on the activity of esterase Est8,while Ag³⁺and SDS inhibited about 30%activity of Est8.Ca²⁺inhibited about30%original activity of Est8-G45R and the inhibitory effect Ag³⁺reached more than50%of original acivity.d.The Km,Vmax and Kcat of the esterase Est8 to the substrate pNPC₂ were 3.06mM,297.3?mol/min/mg and 154.5 s^-1,respectively;The Km,Vmax and Kcat of Est8-G45R to the substrate pNPC₂ were 14.44 mM,288.8?mol/min/mg and 601.7 s^-1,respectively.?3?To obtain the protein crystals of Est8 and EstD1 which were suitable for X-ray diffraction,several crystallization conditions were screened and optimized.At last,X-ray diffraction data were collected to resolutions of 2.3?and 2.5?for Est8and EstD1,respectively.?4?The deacetylation activity of 7-ACA was determined by esterase Est8 and four mutants of esterase?Est8-G45K,Est8-G45D,Est8-G45E and Est8-G45A?.The results showed that the band electricity of amino acid at position 45 of esterase Est8was correlated with enzyme activity.And the positive charge effect is better.In this study,the three-dimensional structure of the Est8 and EstD1 were got,and a mutant Est8-G45R with improved deacetylation activity toward 7-ACA was screened by the method of directed evolution.Based on the protein structure of Est8,the catalytic mechanism of Est8-G45R and 7-ACA substrate will be further analyzed.Meanwhile,the subsequent rational design based on the structure of Est8 and EstD1protein will better help us understand the catalytic mechanism toward 7-ACA.After this research,the family of GDSL esterases can be better understood,it also could provide theoretical basis for the new functions excavated of GDSL family esterases.