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Racemization study on different N-acetylamino acids by a recombinant N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264

Paper ID Volume ID Publish Year Pages File Format Full-Text
35481 45092 2009 7 PDF Available
Title
Racemization study on different N-acetylamino acids by a recombinant N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264
Abstract

N-Succinylamino acid racemase (NSAAR) with N-acylamino acid racemase (NAAAR) activity together with a d- or l-aminoacylase allows the total transformation of N-acetylamino acid racemic mixtures into optically pure d- or l-amino acids, respectively. In this work we have cloned and expressed the N-succinylamino acid racemase gene from the thermophilic Bacillus-related species Geobacillus kaustophilus CECT4264 in Escherichia coli BL21 (DE3). G. kaustophilus NSAAR (GkNSAAR) was purified in a one-step procedure by immobilized cobalt affinity chromatography and showed an apparent molecular mass of 43 kDa in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 150 kDa, suggesting that the native enzyme is a homotetramer. Optimum reaction conditions for the purified enzyme were 55 °C and pH 8.0, using N-acetyl-d-methionine as substrate. GkNSAAR showed a gradual loss of activity at preincubation temperatures over 60 °C, suggesting that it is thermostable. As activity was greatly enhanced by Co2+, Mn2+ and Ni2+ but inhibited by metal-chelating agents, it is considered a metalloenzyme. The Co2+-dependent activity profile of the enzyme was studied with no detectable inhibition at higher metal ion concentrations. GkNSAAR showed activity towards both aliphatic and aromatic N-acetylamino acids such as N-acetyl-methionine and N-acetyl-phenylalanine, respectively, with kcat/Km values ranging from 1 × 103 to 9 × 103 s−1 M−1. Kinetic parameters were better for N-acetyl-d-amino acids than for N-acetyl-l-specific ones.

Keywords
Amino acids; N-Acetylamino acids; N-Succinylamino acid racemase; N-Acylamino acid racemase; Enzyme characterization; Geobacillus kaustophilus
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Racemization study on different N-acetylamino acids by a recombinant N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 44, Issue 8, August 2009, Pages 835–841
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