The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.

The fermentation of an epoxide hydrolase by a newly isolated Trichosporon loubierii strain ECU1040 was optimized. The epoxide hydrolase production was enhanced by 6.9-fold from an initial activity of 45 U/l with the incorporation of substrate induction, optimization of medium composition and other culture conditions, and the implementation of a fed-batch process. It was found that phenyl glycidyl ether (PGE) could efficiently induce biosynthesis of the epoxide hydrolase, though it strongly inhibited the cell growth. To reduce the toxicity of PGE, dibutyl o-phthalate was used in flask cultivation to dissolve PGE, keeping a low concentration of PGE in the broth. Glucose was added to promote cell growth in the presence of PGE, though glycerol was identified as the best single carbon source for the highest specific activity. Trace elements have significant effect on the epoxide hydrolase synthesis.With addition of trace elements and glucose and adjustment of phosphate concentration, the total activity was enhanced by 150%. To further increase the epoxide hydrolase production, a fed-batch culture with PGE in the feed solution was performed in a 5-l jar fermenter. The maximum production of the epoxide hydrolase was 312 U/l, with a specific activity of 23.7 U/g DCW (dry cell weight).

.1%, was sufficient to fully express the catalytic activity of the surfactant-modified enzyme since introduction of additional water led to a sharp decrease in enzyme activity. When the substrate (HMPC) concentration was as high as 1M, the reaction reached 40% conversion in 20h with merely 1.0mg ml−1 of the DGG-lipase, producing (R)-HMPC acetate in nearly 100% ee.

Optically active epoxides can be prepared by kinetic resolution of racemic mixtures using stereospecific epoxide hydrolases. To increase the bio-resolution efficiency of a sparingly water-soluble epoxide (glycidyl phenyl ether, GPE), we investigated the use of organic/aqueous two-phase system.Various conditions were systematically examined and optimized in shake flasks. Isooctanewas found to be the most suitable solvent as the organic phase. The phase volume ratio (φo/w) and biocatalyst concentration were shown to be sensitive parameters affecting both the reaction rate and the enzyme enantiospecificity in the biphase system. An isooctane/aqueous system was developed to overcome the low solubility and instability of GPE in the aqueous phase, resulting in a significant improvement of enatiomeric ratio (E-value) from 39.5 to 94.0 and an average productivity of 18.8mg GPE/(h g) biocatalyst to 48.9mg GPE/(h g) biocatalyst, respectively. Resolution of a 90.1g/l solution of racemic glycidyl phenyl ether in isooctane phase was successfully carried out in a mechanically stirred reactor (120ml), affording (S)-glycidyl phenyl ether in high (100%) enantiomeric excess with a yield of 44.5%.

A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001. The maximum activity of the isolated enzyme was observed at 30℃ and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent. The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40℃ for 6h. Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40℃ and the half-life of the enzyme at 50℃ increased by 2.5 times. The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system.