Preparation, Characterization And Application Of Cross-linked Enzyme Aggregates Of Epoxide Hydrolases From Mung Bean

Enzymes are useful tools for performing industrially important reactions in a stereo-,region-and chemoselective manner. As biocatalysts for chemical reactions, they havenumerous advantages including mild reaction conditions, high stereoselectivity with pureenantiomers as products and environmental-friendly process. However, free enzymes areusually fragile to stability, and suffer from reusability. Therefore, immobilized enzyme hasbeen frequently used to overcome these drawbacks.Enantipure chiral vicinal diols have been proven to be valuable and versatileintermediates for fine chemicals, playing a crucial role in the synthesis of medicines,pesticides and cosmetics. Among various biological methods, asymmetric hydrolysis ofepoxides catalyzed by epoxide hydrolases (EHs) can be an efficient and economic option forsynthesis of enantiopure vicinal diols. Recently, two novel EHs capable of effectivelycatalyzing enantioconvergent hydrolysis of racemic p-nitrostyrene oxide to(R)-1-phenyl-1,2-ethanediol(PED) were discovered from Mung bean (Phaseolus radiatus L.),indicating the great potential for synthesis of enantiopure chiral diols. The asymmetrichydrolysis of epoxide carried out in aqueous monophasic system afforded very low yield andproduct e.e., due to poor solubility of epoxide and its pronounced non-enzymatic hydrolysis,and the organic solvent or ionic liquid gave rise to the deactivation of the free EHs to someextent. Also, it was difficult to recycle the free EHs in various reaction systems. Therefore, itis of great interest and need to immobilize the Mung bean EHs.The effects of precipitating and cross-linking agents on the activity recovery ofcross-linked epoxide hydrolases aggregates were examined, and the immobilized epoxidehydrolases (CLEAs) was characterized. The results revealed that the highest activity recoverywas obtained under the concentration of80%ammonium sulfate, glutaraldehydeconcentration of20mM, crosslinking time of12h. Under the optimum conditions, theactivity recovery of CLEAs reached92%. While SO as the substrate, the optimumtemperatures of CLEAs and free enzyme were40oC and35oC, respectively; the optimum pHof CLEAs was7.5, and the free enzyme was6.5. In addition, the CLEAs were superior to thefree enzyme in terms of thermal stability, organic solvent tolerance and pH tolerance. It was found that the catalytic efficiency (Vmax/Km) of CLEAs was higher than that of free enzyme(0.12vs0.08min-1). The CLEAs formed larger clusters into less-defined structures by SEM.A comparative study was made of the biocatalytic asymmetric hydrolysis of SO with freeenzyme and CLEAs of Mung bean EHs in the aqueous. The efficient of hydrolysis of SO withCLEAs have no improved, which may due to the poor solubility of SO and the pronouncednon-enzymatic hydrolysis of SO. To further improve the reaction efficiency, phase transfermethod was used in this work. The second phase was expected to effectively extract substrateand lower the concentration of substrate in aqueous phase, thus relieving the non-enzymatichydrolysis of SO. Organic solvent/buffer biphasic system is adopted for this purpose. Amongall the tested organic solvents, n-hexane was found to be the most suitable one for the reaction.In the n-hexane/buffer biphasic system, the optimal volume ratio of n-hexane to buffer,reaction temperature, buffer pH value, and substrate concentration for the enzymatichydrolysis were found to be1:1,40oC,7.5, and30mM, respectively, under which the initialreaction rate, the product yield and the product e.e. were13.26mM/(L.h),45.8%and93.5%,respectively.It was proved that the organic solvent-containing reaction system showed toxicity toCLEAs to some extent, and could pollute the environment. To solve the aforementionedproblems, the addition ILs into the reaction system was thought to be able to improve theenzyme activity and eliminate the non-enzymatic hydrolysis of SO. Among all the tested ILs,we found water-immiscible ILs were more suitable than the hydrophilic ILs for the reaction.Of the examined water-immiscible ILs,1-butyl-3-methylimidzolum hexafluorophosphate(C4MIM·PF6) was shown to be the most suitable IL phase for the bioreduction. In theC4MIM·PF6/buffer biphasic system, the optimal volume ratio of C4MIM·PF6to buffer,reaction temperature, buffer pH value, and substrate concentration for the enzymatichydrolysis were found to be1:5,40oC,7.5, and120mM, respectively, under which the initialreaction rate, the product yield and the product e.e. value were25.1mM/(L.h),48%and94%,respectively.The operation stability of CLEAs was studied in various systems. The CLEAs stillremained more than88%and50%, respectively, of their initial activity after being usedrepeatedly for four and eight batches (4hours per batch, total32hours) in phosphate buffer, respectively. When the reaction was conducted in n-hexane/buffer biphasic system, theCLEAs maintained about86%of their original activity after5reaction cycles and retainedmore than69%of their initial activity after successive re-use of7batches (10hours per batch,total70hours). In the ILs/buffer biphasic system, the CLEAs still maintained as high as89%activity after3batches (24hours per batch, total72hours), showing excellent operationalstability.In the present study, we report for the first time the preparation and characterization ofcross-linked enzyme aggregates of EHs from Mung bean. The CLEAs could efficientlyasymmetric hydrolysis SO to (R)-PED. This study provides not only a deeper understandingof immobilization technology, but also a novel and efficient route to enantiomerically pureortho-diols.