Improve The Enantioselectivity Of Hyperthermophilic Esterase APE1547 By Whole-plasmid PCR Mutagenesis

Chiral 2-octanol is an important ingredient of Liquid Crystal Display and also a critical intermediate substrate material in synthesis of steroid, vitamin E and pesticide .The chemical resolution method of 2-octanol allways should process in high temperature and high pressure with multiple steps .Enzymes can show high chemo-, regio- and stereo selectivity, enzyme-catalyzed reactions carried out at ambient temperature and exhibit high activity in aqueous solution and even in organic solvents, which makes them in some cases superior to chemical catalysts. Although the potentials of enzyme are fully evaluated, low stability and activity often limit general use of enzymes as industrial catalysts.Hyperthermophilic esterase APE1547 from the thermophilic archaeon Aeropyrum pernix K1 has been cloned and over-expressed in Escherichia coli. The enzyme shows extremely high thermo stability and shows a wide range of substrate specificity. In order to take the advantage of its thermostability, we applied APE1547 on chiral resolution of 2-octanel.However the enantioselectivity of APE1547 toward 2-octanol (E=1.98) doesn't offer satisfactory results. In this context, Hyperthermophilic esterase APE1547 was subjected to directed evolution to generate mutants with increased enantioselectivity.Errror–prone PCR and DNA shuffling was applied on mutation library construction and high through-put screening method based on BTB pH indicator was employed on selecting positive mutant.As a complementary part of directed evolution of Hyperthermophilic esterase APE1547 We carried out site-directed mutangensis on the esterase.Based on 3D structure of APE1547 we choose 3 amino acids which would like to affect the enantioselectivity of Hyperthermophilic esterse APE1547 as site-directed mutant site. Whole plasmid PCR was appllied to created sitei-directed mutant。As we choose low-fidelity polymerization conditions and TaqDNA polymerase in PCR reaction, some random mutants was employed in the target gene together with site-directed mutants.The amplification products were digest by Dpn I and transformed into host strain.After screening the mutant library with BTB high-throughput screening method we found 12 positive mutants and the best mutant DT28's enantioselectivity performed with 9.5 times better than wide type esterase. After DNA sequencing we found the amino acid substitution of each mutant: DT15(P552L)E =7.5,K319(N523S) E=5.6,DT23(L317I,318R) E=2.7 DT28 E=19.6.Each of mutated esterase was purified and their express level was studied, mutant K319 increase protein express ability about 1.7 fold than wide type APE1547. All the purified enzymes were used for enzymatic charcteristics identification study.Mutant DT15's optimal temperature has decrease10℃compared with wide type with one amino acid substitution (P552L) .Mutant DT23 with L317I and 318R injected in the protein greatly improve the thermal stability.Through Structural modeling we found this new amino acid 318R form a new hydrogen bond with 23Glu which strengthen the stability ofβ-propeller domain and in turn improve the thermostability of APE1547 but as this mtant is far away from catalytic site it dosen't effluence mutatnt DT23's enzymatic activity. Mutant K319(N523S) with its amino acid 523S form a new hydrogen bond with 554 Ala induced a stronger thermostability .This amino acid substitution also increase the catalytic turn over number of p-nitrophenyl capryiate .With the alligment of the similar sequence we found the unique position 523N in APE1547 was highly conserved. Mutant K319's enzymatic characteristics has obviously changed with only one amino acid mutagenesis means 523N plays an important role on APE1547's activity and enantioselectivity and this discovery provides new clues on next step's rational mutagenesis on hyperthermophilic esterase APE1547.All the positive mutants selected from this library can be used for further directed evolution.In conclusion, whole plasmid PCR amplification was successfully applied on the moleculer modification of hyperthermophilic esterase APE1547.We composed the site-directed mutagenesis and random mutagenesis in one PCR reaction by using site-directed mutagenesis primer and low-fidelity polymerization condition. Several positive mutants with improved enantio- selectivity were selected and one amino acid site which would like to affect hyperthermophilic esterase APE1547's enzymatic property was identified .