Discovery Of Pseudomonas Sp. Esterase And Its Application In Preparation Of Chiral Mandelic Acids

Because of their fascinating features such as high activity, excellent regioselectivity and enantioselectivity, and environmental friendly, esterases have been employed widely in bioprocesses of preparing chiral chemicals, and play more and more important roles in the production of high-value added medicine intermediates and fine chemicals. The production of 2-hydroxy carboxylic acids via stereoselective resolution reactions catalyzed by esterases has been valued in this field. Important chiral 2-hydroxy carboxylic acids, including mandelic acid, o-chloromandelic acid and 2-hydroxy 4-phenylbutyric acid have been used widely in pharmaceutical synthesis and resolution of racemates. Therefore, academia and industrial community have focused on the development of new biocatalysts and new bioprocesses to produce chiral 2-hydroxy carboxylic acids nowadays.This study reviewed current research of producing chiral mandelic acid and its derivatives via chemical and enzymatic methods, especially the stereoselective hydrolysis of mandelic acid esters by microbial esterases. Then the reaction route of hydrolysis of the acylation products of mandelic acid after acetylation of the hydroxyl group was first proposed. The substrate and target reaction directed enzyme screen was conducted, and a strain which could hydrolyze the substrate,2-acetoxylphenylacetic acid enantioselectively was isolated for soil samples, basically realizing the reaction route. Then the fermentation conditions in shake flasks and fermenters were studied deeply to obtain higher enzyme production and biocatalysts with higher specific activity. The immobilization and permeabilization of whole cells biocatalysts improved their apparent activity and stability, and without decrease of enantioselectivity meanwhile. By engineering the catalyst, reaction medium and substrate forms, the substrate range was expand, the reaction efficiency was enhanced and the substrate concentration was elevated. The four main parts as follows composed the paper:In the first part, the substrate-directed enzyme screen of soil samples was conducted. The target substrate is 2-acetoxylphenylacetic acid and mandelate esters, and the target reaction is enantioselective hydrolysis to produce chiral mandelic acid. By diverse screen and selection strategies,259 strains with hydrolysis activity were obtained. Severe degradation of the substrate and the produced mandelic acids were found in the screen of methyl mandelate-hydrolyzing strains, whereas 8 strains with high enantioselectivity were isolated using 2-acetoxylphenylacetic acid as the substrate. In the further screen, a strain with high activity and high enantioselectivity was found to obtain the S-mandelic acid with enantiomeric excess of 98.1%. After 16S rDNA identification, the strain was named as Pseudomonas sp. ECU1011, with a preservation number of CGMCC 2872.In the second part, the fermentation conditions were optimized to enhance the relatively low enzyme production. The medium and fermentation conditions in shake flasks were optimized using single-factor method. It was found that glycerol, yeast extract, NaCl and phosphate were the appropriate components, and the addition of Zn and Mn ions improved the enzyme production. Statistic methods were implemented and yeast extract, NaCl and ZnSO4 has significant effect on the enzyme production. Then the Box-Behnken experiment was used to fix the best level of the three significant components. The optimized medium was verified in a 5-L fermenter. The cultivation process was found to be high oxygen-demanded, pH sensitive and mandelic acid induced. The mandelic acid was added to play a dual functional role of pH regulator and enzyme inducer, and the inhibition of strain growth by pH of higher than 7.5 was removed. Finally the pH-stat and DO-stat process was achieved by feedback control strategies, and the 4166 u/L enzyme production was obtained after 15 h fermentation.In the third part, the whole cell biocatalysts used in the enantioselective deacylation reaction were immobilized and permeabilized. In immobilization tests, lyophilized cells entrapped in calcium alginate with 73% activity recovery displayed high operational stability and reusability after 150 h and 12 batches repetitive reactions. Crude enzyme absorbed by Celite545 with 65% activity recovery exhibited the highest special activity. Severe enzyme inactivation caused by chemistry reaction in crosslink immobilization suggested the enzyme was covalent bonds sensitive. In permeabilization of whole cells biocatalysts tests, the process was optimized and the enzyme activity increased 2.33 folds after 2 h of 10% toluene treatment. The permeabilized cells entrapped in calcium alginate obtained 171% enzyme recovery and 123 h half life. Using the modified biocatalyst with high stability and activity, the repeated production of high value-added o-Cl mandelic acid was successfully conducted.In the fourth part, the enantioselective hydrolysis reaction of Pseudomonas sp. esterase was studied. The optimized reaction conditions were explored. The fittest temperature was 30℃and the fittest pH was 7.0. The highest substrate concentration was 60 mM. We also found that substrates with different substituent groups at benzene ring, different carbon chain of phenyl carboxylic acids and different leaving acyl groups all showed high enantioselectivity. The change of different leaving acyl groups, reaction medium and substrate forms was tested, and it was found that the sodium 2-acetoxylphenylacetate as the substrate could increase the reaction rate 3.75 folds, and the substrate concentration could reach 500 mM.