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Recovery of active N-acetyl-d-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers

Paper ID Volume ID Publish Year Pages File Format Full-Text
17347 42661 2012 6 PDF Available
Title
Recovery of active N-acetyl-d-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers
Abstract

Overexpression of recombinant N-acetyl-d-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08 ± 0.02 U/mg, was similar to that of the native protein, 2.13 ± 0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41 ± 10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32 ± 5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins.

► Active enzyme can be recovered from inclusion bodies with non-denaturing buffer. ► Structural similarity between recovered and native enzymes is confirmed by CD. ► Inclusion bodies contain native-like proteins. ► Aggregation of proteins into inclusion bodies is reversible.

Keywords
Epimerase; Inclusion bodies; Refolding; Solubilization
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Recovery of active N-acetyl-d-glucosamine 2-epimerase from inclusion bodies by solubilization with non-denaturing buffers
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 50, Issue 1, 5 January 2012, Pages 65–70
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