Study On The Stereoselectivity Of Hyperthermophilic Esterase APE1547

Hyperthermophilic esterase APE1547 was the gene from archaeonAeropyrum pernix K1 and has been cloned and transformed the recombinantplasmid into E. coli BLP (DE3) strain to express hyperthermophilic esterase.The hyperthermophilic esterase has high activity, high thermostability andresisting denaturant. The stereoselectivity, especially enantioselectivity is oneof the characteristics of the esterase. Enzymes are important and usefulcatalysts to synthesize pure enantiomers for bioactive materials andpharmaceutical, especially for chiral medicine development and utilization.Hyperthermophilic esterase shows enantioselectivity. It has expandingapplications of esterases in stereo-specific hydrolysis, transesterfication, andester syntheses. Its application in biotechnology is increasing rapidly anddesirable for wide range of applications. The hyperthermophilic esterase hasthe potential uses in industrial processes, especially in production of pureenantiomers for pharmaceutical, agrochemical and food.Rac-2-octanol acetate was used as intermediate substrate to produce purechiral 2-octanol which is an important ingredient of Liquid Crystal Display. Itis also a critical intermediate substrate material in production and synthesis ofsteroid, vitamin E and pesticide. Because the E-value of hyperthermophilicesterase APE1547 in the hydrolysis of 2-octanol acetate was low, we mustchange the enzyme molecules to improve its enantioselectivity for theindustrial processes.The methods of direction evolution, sited-directed mutagenesis andacetone treatment were used to improve the enantioselectivity ofhyperthermophilic esterase APE1547.1. Directied evolution of the hyperthermophilic esterase APE1547 andconstruction of the screening methodWith the development of biotechnology, especially the application ofDNA cloning technology in biological research, it is now possible to build alarge number of gene mutant clones library for directed evolution of enzymemodification. The key step in directed evolution of enzyme is the effectivescreening method to identify the improved variants. Highly developedbiocatalysis technology requires effective methods that quickly detect the highenatioselective mutants and discard the negative mutants. This method buildsa high throughput screening model that can screen large amounts of mutantsand can be applied to various substrates, it has the following merits: 1 Highscreening efficiency, use of target substrates to design selective culturemedium, which can screen large numbers of colonies (300-600 colonies perplate);2 pH indicator methods combined with 96-hole plate make screeningof positive easier;3 The first round of screening makes positive mutantscontaining less than 3.8% which reduces the screening task;4 Use chiral andracemic substrate instead of both kinds of chiral substrates in theenatioselectivity screening can save the cost;5 Because they don't needcomplex treatment of substrates such as decompress distillation, columnchromatography to purify substrate. Using this method we just need to extractthe substrate from reaction solution to measure E value. All these advantagesmake this method reliable and easy-to-use viable on exploration of newenzymes.To enhance the enantioselectivity of APE1547, a directed evolutionapproach is employed to generate mutant library from the wild enzyme. Theenantioselectivity of mutation is increased up to 2.6-fold compared to that ofwild type enzyme after one round of epPCR. The enantioselectivity ofmutation is increased up to 6.3-fold by DNA shuffling.2. Screening the substrate with computer softwareThe crystal structure of hyperthermophilic esterase APE1547 wasresolved. The substrate was docked to activite site of APE1547 for buildingmodeling. The optimum substrate that was catalyzed by hyperthermophilicesterase APE1547 was selected.3. Improving the enantioselectivity of a hyperthermophilic esterase withsited-directed mutagenesisThe methods of sited-directed mutagenesis were used. The small mutationlibrary was built using four nucleotide acids. There are four mutations wereselected with high enantioselectivity through screening method built by us.The E value was increased 1.4, 2.8, 3.8, 10 folds, respectively. The enzymeactivity has improved 1.27, 1.45 and 0.15 folds. One mutant's activitydeclined 49%. The advantage of the method was that it reduced 75% the costin synthesizing primer, the times and PCR.4. Study on the relationship between enantioselectivity and structure ofhyperthermophilic esteraseThe structure of mutants which created by direction evolution andsited-directed mutagenesis were analyzed. The molecule structure forimproving enantioselectivity was speculated. We find that the reason thatcaused the change of enzymatic configuration in different distance. Gly, Proand Arg were at different site led to the change of enzymatic configuration. Itwas more likely to lead substrate to enter the active center. This effectivemethod that can improve the enatioselectvity of enzyme was proved.5. Study on improving enantioselectivity with acetoneThe APE1547 was a hyperthermophilic esterase genetically recombinedfrom archaeon Aeropyrum pernix K1. The character of modified APE1547mutant changed slightly while the mutant was obtained by directed evolutionand was purified with Ni-chelating column. However, the results showed thatthe enzymes, with the treatment of acetone and mutant respectively, achievedmore improved activity and higher enantioselectivity in compare with theenzyme treated by Ni-chelating column. The total activity yield of thewild-type and mutant increased 7 and 15.6 folds, respectively. The relativiteactivity increased 5.7 and 6.9 folds compare crude enzyme, respectively.Other mutant also had changed in both the total activity yield and relativiteactivity. In addition, some characters of the mutant changed. It showed thatthe change of mutant structure led to the changes of the characters, forexample, the changes of enantioselectivity etc. In the hydrolysis of 2-octanolacetate, the E-value of mutant A had a 6-fold increase over the enzymepurified with Ni-chelating column, indicating a better enantioselectivity forthe mutant.In conclusion, the purification method was very important. A goodpurification method improved the enzymatic activity and the other characters.The scope of enzymatic application was enlarged. Many products wereobtained in pharmaceutical, agricultural, or organic chemistry. The acetonetreatment had effectively improved the activity and enantioselectivity ofAPE1547 esterase and mutants. The advantage of the method was itsconvenience and efficiency. It is a powerful method for further research.