[논문]Biotransformation of Amides to Acids Using a Co-Cross-Linked Enzyme Aggregate of Rhodococcus erythropolis Amidase

Park, Hyun-Joo; Uhm, Ki-Nam; Kim, Hyung-Kwoun

doi:10.4014/jmb.0909.09009

Biotransformation of Amides to Acids Using a Co-Cross-Linked Enzyme Aggregate of Rhodococcus erythropolis Amidase 원문보기

Journal of microbiology and biotechnology, v.20 no.2, 2010년, pp.325 - 331

Park, Hyun-Joo (Division of Biotechnology, The Catholic University of Korea) , Uhm, Ki-Nam (Equispharm Ltd.) , Kim, Hyung-Kwoun (Division of Biotechnology, The Catholic University of Korea)

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Rhodococcus erythropolis amidase was expressed in Escherichia coli cells. The crude amidase in the cell-free extract was immobilized using the cross-linked enzyme aggregate (CLEA) method. The crude amidase was mixed with bovine serum albumin and then precipitated with ammonium sulfate. The resultant precipitant was subsequently cross-linked with glutaraldehyde. Scanning electron microscopy revealed that this co-CLEA had a ball-like shape with a diameter of approximately $1\;{\mu}m$. This co-CLEA evidenced hydrolytic activity toward a variety of amide substrates. The amidase co-CLEA evidenced an optimum temperature of $60^{\circ}C$ and an optimum pH of 8.0, results that were similar to those of the soluble amidase. The reaction stability of the co-CLEA was increased. That is, it was stable up to $50^{\circ}C$ and in a pH range of 5.0-12.0. Additionally, the co-CLEA could be recovered by centrifugation, and retained 96% activity after 3 repeated cycles. This amidase co-CLEA may prove useful as a substitute for soluble amidase as a biocatalyst in the pharmaceutical and chemical industries.

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이론/모형

The amidase activity assay was performed by measuring the reaction product, NHS, using the method developed by Fawcett and Scott [7]. A small amount of amidase CLEA was added to 1 ml of 10 mM potassium phosphate bufler (pH 7.
5) containing 5 mM isobutyramide, and the reaction mixtures were incubated for 30 min at various temperatures (10~70℃), after which the amount of NH₃ produced was measured. To determine the temperature stability, amidase co-CLEA was incubated for 30 min at various temperatures (10-70℃) and its residual activity was measured by the standard assay method.

참고문헌 (21)

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Biotransformation of Amides to Acids Using a Co-Cross-Linked Enzyme Aggregate of Rhodococcus erythropolis Amidase 원문보기

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Biotransformation of Amides to Acids Using a Co-Cross-Linked Enzyme Aggregate of Rhodococcus erythropolis Amidase 원문보기

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