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A simple and effective method to prepare immobilized enzymes using artificial oil bodies

Paper ID Volume ID Publish Year Pages File Format Full-Text
18678 42732 2006 7 PDF Available
Title
A simple and effective method to prepare immobilized enzymes using artificial oil bodies
Abstract

Overproduction of heterologous proteins in Escherichia coli commonly results in the formation of insoluble aggregates. Prior to the purification and immobilization of insoluble aggregates, an indispensable need calls for their renaturation. However, this process is usually laborious, inefficient, and costly. In this study, a new method based on artificial oil bodies (AOBs) was developed to complete protein refolding and immobilization in one step. To illustrate the proposed method, the d-hydantoinase gene fused to the C terminus of oleosin, a storage protein of plant oil body, was constructed and overexpressed in E. coli. As a result, the hybrid protein was largely produced in the form of inclusion bodies, and the protein yield reached 30% of total cell protein content. Without the use of denaturant, the mixture containing the insoluble fusion protein and plant oil was subjected to sonication to form AOBs. Subsequent analysis by the protease accessibility and enzyme activity assay confirmed the presence of active d-hydantoinase on the surfaces of AOBs. In comparison with its free counterpart, the immobilized d-hydantoinase exhibited higher tolerance to heat. Furthermore, the immobilized enzyme could be reused for seven cycles to give a conversion yield exceeding 80% when applied in a bioconversion process. It clearly indicates that the AOBs-based system is featured with simplicity as well as efficiency and is practically useful for direct immobilization of enzymes with low solubility.

Keywords
Bioconversion; d-Hydantoinase; Enzyme immobilization; Oil body; Oleosin
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A simple and effective method to prepare immobilized enzymes using artificial oil bodies
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 39, Issue 5, 4 September 2006, Pages 1152–1158
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