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Single-step purification and immobilization of γ-lactamase and on-column transformation of 2-azabicyclo [2.2.1] hept-5-en-3-one

Paper ID Volume ID Publish Year Pages File Format Full-Text
35358 45088 2011 7 PDF Available
Title
Single-step purification and immobilization of γ-lactamase and on-column transformation of 2-azabicyclo [2.2.1] hept-5-en-3-one
Abstract

We prepared two constructs of the γ-lactamase gene from Sulfolobus solfataricus P2 and heterogenously expressed these as two separate proteins. The first construct had His tags fused at both the N and C terminals of the γ-lactamase gene (double fusion enzyme). The second had a single His tag fused at the N terminal of the gene (single fusion enzyme). Enterokinase treatment of the two fusion enzymes produced two additional proteins. The kinetic parameters, absorption capacity on nickel-chelating agarose, and immobilization stability of the four proteins were compared. The double fusion enzyme was chosen as the on-column transformation catalyst. It was purified to electrophoretic homogeneity on nickel-chelating agarose and immobilized on the same matrix in the meantime. The optimal temperature of the immobilized enzyme was 10 °C higher than that of the free enzyme. The optimal pH was also 2.0 units higher. The immobilized enzyme maintained approximately 80% of its maximum activity at pH 5–11. The activity of the free enzyme decreased rapidly at pH values below 6.0 or above 8.0. The immobilized enzyme was used for on-column transformation reactions for 5 h each day. It retained approximately 75% of its original activity after repeated transformation cycles over a period of 30 days.

Keywords
Amidase; γ-Lactam; γ-Lactamase; Immobilization; On-column transformation; Sulfolobus solfataricus P2
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Single-step purification and immobilization of γ-lactamase and on-column transformation of 2-azabicyclo [2.2.1] hept-5-en-3-one
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 46, Issue 1, January 2011, Pages 81–87
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