Enhanced Thermostability Of Sterol Esterase From Ophiostoma Piceae By Computer Aided Rational Design

Ophiostoma piceae sterol esterase(EC 3.1.1.13),which catalyzed the hydrolysis of sterol ester and esterification of sterol in non-aqueous phase,has important applications in food processing,pharmaceuticals(synthesis of nucleoside or steroidal hormones),and the control of pulp resin barriers.While the solubility of sterol ester is poor and the substrate concentration is low,which seriously reduces the catalytic efficiency of enzyme.While O.piceae sterol ester is not thermostable enzyme that can be operated at higher temperatures or co-solvent concentrations,with ensuing reduction in microbial contamination,better solubility,as well as in many cases more favorable equilibrium.we develop guidelines for selecting appropriate mutations by engineering the flexible?-turns,adding salt bridges and proline effect.The kinetic parameters of catalytic hydrolysis reaction of positive thermostable mutants were determined.The specific experimental results are as follows:1.The characters and activity of three esterases(Bacillus subtilis lipase A(BSA),Burkholderia sp.lipase(BCL)and Ophiostoma piceae sterol esterase(OPE)hydrolyzing degradable polyester polymers(polycaprolactone and polyvinyl acetate as model substrates)was determined.The relative molecular weights of BSA,BCL and OPE are 24k Da,34 k Da and 76 k Da,respectively.The optimum temperatures for BSA,BCL and OPE catalytic hydrolysis reactions are 40?,40?and 35?respectively.The optimal p H values were 8.0,8.0 and 7.5,respectively.All three esterases showed preference for medium chain fatty acid esters(C8-C10).The initial rates of BSA and BCL on the hydrolysis of polycaprolactone were respectively 57 and 80?g min^-1U^-1.And that of polyvinyl acetate hydrolyzed by OPE is 4.4×10^-3?mol min^-1U^-1.2.The electronic library of OPE thermostability mutants was constructed,evaluation and screened by means of computation;Based on the possible mutation effects,the mutants in the electronic library were classified after being evaluated and screened one by one.An electronic library containing potential 1675 thermostability mutants was constructed by Fold X,Rosettadd G and Po PMu Si C.The screening criteria for mutant libraries are as follows:(1)Excluding mutants in catalytic triplets;(2)Excluding mutants with unstable protein structure after mutation,such as steric structural conflict,exposure of aromatic amino acids and methionine to molecular surface,reduction of hydrogen bond and salt bridge force,and destruction of alpha helix;(3)The screening method for?turn is as follows:(1)Select the 5 most flexible turns after the flexible analysis and sequencing of all?turns of OPE proteins;(2)The?turn type and amino acid composition preference of thermostable protein were analyzed statistically and the mutated amino acids conforms to the above statistical law of amino acid preference of thermostable protein?turn;(4)Proline screening is according to the following:(1)Proline should locate in the region of the Ramachandran plot(-90<?<-40°,120<?<180°or-50<?<10°);(2)RSA>50%relative solvent surface area;(3)?z?RMSF(370K to 300K)>0;(5)Salt bridge screening includes the following two points:(1)The putative salt bridge pairs of a position were all at an interval of>5 residues of the primary sequence;(2)RSA of one residue forming the salt bridge is greater than 50%at least.There are 25 candidate mutants in the electronic library of OPE thermal stability mutants with the above three effects by further screening.In the optimized library of mutants,6 mutants located at the?-turn,9 mutants with salt-bridge effect,10 mutants with proline effect.The above 25candidate thermostable mutants will be verified by subsequent experiments.3.Those 25 candidate thermostable OPE mutants were evaluated experimentally by using site-specific mutation technique.These mutant genes were heterologous expression in P.pastoris and purified by affinity chromatography column;T₅₀and t_1/2of mutants were measured and compared with wild type OPE.Experimental results show that:The experimental results showed that the thermal stability of the dual-point mutant S109P/S271P was significantly improved compaired to the single-point mutation.The T₅₀³⁰is increased by 8.9?,and the half-life t_1/2was 3.6 times than that of the wild-type mutant.Among the thermostable mutants screened by salt-bridge effect,the single-point mutation OPE-A231E showed the most obvious increase in thermal stability,with the T₅₀³⁰increased by 11.8?,the t_1/2being 6.4 times than that of the wild type.The T₅₀³⁰of the optimal double-mutant T383E/A355E increased by 16.7?,the t_1/2was 10.5 times than that of the wild type.T₅₀³⁰of OPE-D136P/V252P,the optimal two-point mutant,increased by 11.6?,and the half-life t_1/2was 4.5 times that of the wild type.