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Cloning, DNA shuffling and expression of serine hydroxymethyltransferase gene from Escherichia coli strain AB90054

Paper ID Volume ID Publish Year Pages File Format Full-Text
18011 42710 2007 9 PDF Available
Title
Cloning, DNA shuffling and expression of serine hydroxymethyltransferase gene from Escherichia coli strain AB90054
Abstract

A gene (glyA) encoding serine hydroxymethyltransferase from Escherichia coli strain AB90054 was cloned, sequenced and expressed in E. coli. The gene consists of 1254 bp and encodes a protein of 417 amino acids. Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and high throughput screening of the resultant mutant libraries) have been used to enhance the catalytic activity of serine hydroxymethyltransferase. After three rounds of error-prone PCR and three sequential generations of DNA shuffling were performed on the glyA gene and recombinants were screened using a genetic complement coupled assay, a mutant 3E7 with approximately 8-fold increased enzyme activity and 41-fold increased enzyme productivity compared with its wild-type parent was obtained. Mutant 3E7 contains eight amino acid substitutions. The purified 3E7 and wild-type serine hydroxymethyltransferase each showed a single band at about 45 kDa by SDS-polyacrylamide gel electrophoresis. Some properties of the purified WT enzyme and evolved enzyme were also determined in this work. Homology model structure of 3E7 indicated that all the mutations do not interact directly with substrate but S355N exert its effect by forming H-bonds with THF and contributing to transition state stabilization.

Keywords
SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; PLP, pyridoxal-5′-phosphate; C1, one-carbon; WT, wild type; PCR, polymerase chain reactions; SDS-PAGE, SDS-polyacrylamide gel electrophoresisSerine hydroxymethyltransferase; Protein engineering
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Cloning, DNA shuffling and expression of serine hydroxymethyltransferase gene from Escherichia coli strain AB90054
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 4, 5 March 2007, Pages 569–577
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