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Properties of Rhodotorula gracilisd-Amino Acid Oxidase Immobilized on Magnetic Beads through His-Tag

Paper ID Volume ID Publish Year Pages File Format Full-Text
21933 43244 2008 6 PDF Available
Title
Properties of Rhodotorula gracilisd-Amino Acid Oxidase Immobilized on Magnetic Beads through His-Tag
Abstract

d-Amino acid oxidase catalyzes one of the key steps in the production of semisynthetic cephalosporins. We expressed and purified recombinant Rhodotorula gracilisd-amino acid oxidase with C-terminal his-tags. This engineered enzyme was immobilized onto Ni2+-chelated nitrilotriacetic acid magnetic beads through the interaction between his-tag and Ni2+. The kinetic constants, storage properties, and the reusability of the immobilized d-amino acid oxidase were determined. The effects of temperature, pH, and hydrogen peroxide on the activity of immobilized d-amino acid oxidase were also studied. The highest activity recovery was 75%. Thermal stability was improved after immobilization; the relative activity of the immobilized enzyme was 56% whereas the free enzyme was completely inactivated after incubation at 50°C for 1 h. In the presence of 10 mM hydrogen peroxide, the immobilized enzyme did not show a rapid loss of activity during the first 2 h of incubation, which was observed in the case of the free enzyme; the residual activity of the immobilized enzyme after 9 h was 72% compared with 22% of the free form. The long-term storage stability was improved; the residual activity of the immobilized enzyme was 74% compared with 20% of the free enzyme when stored at room temperature for 10 d. The immobilized form retained 37% of its initial activity after 20 consecutive reaction cycles.

Keywords
d-amino acid oxidase; his-tag; magnetic beads; Rhodotorula gracilis; thermal stability
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Properties of Rhodotorula gracilisd-Amino Acid Oxidase Immobilized on Magnetic Beads through His-Tag
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 105, Issue 2, February 2008, Pages 110–115
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us