A Strategy For Constructing A Mutant Of Lipase With Highly H₂O₂-resistance And Its Application In Epoxidation

Enzymatic oxidation reactions bearing the promise of being more environmentally benign are considered economically alternatives to the chemical methods.Unpleasantly,applied enzymes are prone to oxidation caused by oxidative substrates H₂O₂ or inactivation.Lipases are a kind of promsing catalyst for epoxidation.Recently,more and more researches focus on improving the H₂O₂-resistance of enzymes,but efficient methods are still absent.Thus the applications of lipases are limited.In this study,we provided a new stategy for engineering the lipase from Penicillium camembertii?PCL?used as template by investigating the binding conformation between the catalytic pocket of PCL and H₂O₂,and efficiently improved the H₂O₂-resistance of PCL.Our study offered new ideas for improving the oxidation stability of industrial enzymes by rational design.?1?Bioinformatics analysis of PCL.We built the open form of PCL by homology modelling and constructed the PCL with the two substrates pentanoic acid and H₂O₂ by molecular modelling.The docking result furtherly analyzed the binding conformation between the catalytic pocket of PCL and H₂O₂ and determinated the key residues,Y21 and H144,for substrate binding.?2?Study on the reason for oxidation of enzymes by mutagenesis experiments.We constructed the mutants Y21F and H144W for imitating the oxidized state of Y21 and H144which lost their polar groups.Results showed that the enzyme activity of the mutants Y21F and H144W significantly decreased and lost about 50%and 80%of their initial activity after2 h incubation in 1 M H₂O₂ whereas the wide-type of PCL lost about only 15%,indicating that the H₂O₂ binding site Y21 and H144 played an important role in stabilizing the substrate binding pocket and remaining the catalytic efficiency of the enzyme.In addition,hydrogen bonds were observed among Y21,H144 and H259 through structure analysis,which might contribute to stabilizing the catalytic pocket of the enzyme and remaining H259 in a suitable structure form.?3?Study on engineering PCL to improve the H₂O₂-resistance.We designed to introduce hydrogen bond donors above Y21 and H144,so the substitutions of the non-polar amino acids in H₂O₂ binding pocket of PCL by polar amino acids were conducted.Combined with traditional engineering ways,we totally constructed eight mutants.The docking results showed that the mutants Y84R,I260E and I260R with obviously improved H₂O₂-resistance all formed hydrogen bonds with H₂O₂,which stabilized H₂O₂ above Y21 and H144 and avoided the direct contact between essential amino acids and H₂O₂.The mutants Y84R,I260E and I260R showed significantly enhanced thermal stability.The half-lives(t_1/2,45°C)of the mutants Y84R,I260E and I260R were about 7.0,3.3 and 15.7-fold higher than that of PCL,and T_m of these three mutants displayed increments of 2.5,0.9 and 3.2°C,which demonstrated that the structure stability of them was improved.Structure analysis indicated that R84 and R260 formed hydrogen bonds or salt bridges with animo acids around,thus stabilizing the structure and enhancing the thermostability of the enzymes.?4?Advantages and applications of epoxidation of alkenes catalyzed by the mutant I260R with high-tolerance towards H₂O₂.It has been proven that the mutant I260R with high-tolerance versus H₂O₂ can not only maintain activity under H₂O₂ condition for longer time but also tolerate higher concentration of H₂O₂ than the wide-type of PCL.When the concentration of H₂O₂ was 3.33 M,the epoxidation frequency of 1-octadecene catalyzed by the wide-type of PCL reached the maximum.However the epoxidation frequency of the mutant I260R reached the maximum at 4.44 M H₂O₂.The results indicated that compared with the wide-type of PCL,the tolerance limitation towards H₂O₂ of the mutant I260R enhanced by more than 1 M,and its maximum turnover frequency also increased by 57%.In enzyme-catalyzed epoxidation of different kinds of alkenes,we found that the mutant I260R with high-tolerance versus H₂O₂ can efficiently catalyzed epoxidation reactions in solvent free system.