Journal
JOURNAL OF BIOTECHNOLOGY
Volume 272, Issue -, Pages 48-55Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2017.12.014
Keywords
Halohydrin dehalogenase; Enzyme immobilization; Cross-linked enzyme aggregates; Enzyme characterization
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Funding
- National Natural Science Foundation of China [21673034]
- National Science and Technology Major Project on Water Pollution Prevention and Control [2012ZX07203-003]
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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.
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