Cross-linked enzyme aggregates (CLEAs) of halohydrin dehalogenase from Agrobacterium radiobacter AD1: Preparation, characterization and application as a biocatalyst

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential to produce valuable optically pure epoxides and beta-substituted alcohols. However, this enzyme has been reported to be very sensitive and less stable under oxidative conditions. Enzyme immobilization represents a powerful means to overcome this limitation and provides the enzyme characteristics of a biocatalyst. In this study, the crude extract of HheC was directly subjected to enzyme immobilization using a carrier-free cross-linked enzyme aggregates (CLEAs) method. The results showed that under the optimized conditions, the obtained HheC-CLEAs retained more than 90% activity of the free enzyme; preserved more than 50% of their original activity after storage at 4 degrees C for 2 months, even in the absence of a reducing agent; displayed a strong tolerance to organic solvents with fully active after incubation in the presence of 50% cyclohexane and n-hexane for 5 h; the presence of organic solvents could minimize the negative effect of enzyme immobilization on the enzntioselectivity of HheC. Most importantly, HheCeCLEAs maintained more than 70% activity after 10 reusability cycles. The utility of HheCeCLEAs as a valuable biocatalyst was exhibited by the kinetic resolution of azide-mediated ring-opening reaction of rac-1,2-epoxy-2-methylbutane. These results indicated that HheCeCLEAs overcame some disadvantages of free enzymes to become biocatalysts. Together with further engineering of the enzyme, HheCeCLEAs could become a promising biocatalyst for the synthesis of valuable chiral compounds.