Please use this identifier to cite or link to this item: http://hdl.handle.net/10603/15494
Title: Cloning expression and molecular characterization of CIS epoxysuccinate hydrolase from nocardia tartaricans
Researcher: Vasu V
Guide(s): Meenakshisundaram S
Keywords: Cis-epoxysuccinate hydrolase, cloning, open reading frame, nocardia tartaricans
Upload Date: 30-Jan-2014
University: Anna University
Completed Date: 
Abstract: Cis-epoxysuccinate hydrolase (CESH) gene of about 762 bp was cloned from Nocardia tartaricans ATCC31191, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. On the basis of structural and possible functional similarities between the CESH and other members of the haloacid dehalogenase superfamily, candidate catalytic amino acids (conserved amino acids) in the active site of the CESH were predicted to be crucial for its catalytic activity. In total, 17 different recombinant clones were constructed encoding mutant CESH proteins with a single amino acid residue substitution by the respective mutation primers. Out of 17 mutants, mutant Y192F alone produced insoluble protein and the remaining mutants produced soluble proteins, which were taken for characterization. Interestingly, the mutants D18A, D18E, Q20E, T22A, R55E, N134D, K164A, H190A, H190N, H190Q, D193A and D193E resulted in complete loss of activity whereas the mutants Y58F, T133A, S189A and Y192D retained partial enzyme activity. Furthermore, the active-site residues responsible for the opening of CES were analyzed and the mechanism underlying the catalytic triad involved in L(+)- tartaric acid biosynthesis was proposed. Immobilization of cis-epoxysuccinate hydrolase-containing E. coli for L(+)-tartaric acid production was carried out using entrapment in and#954;- carrageenan gel. The apparent Km and Vmax values for immobilized and free cells were 135 mM and 68 U for free cells (50 mg cells), and 236 mM and 62 U for immobilized cells (1 g immobilized beads contains 50 mg cells). After 10 repeated batches, the conversion rate for immobilized cells was about 95% compared to 10% for free cells, which indicated that the immobilized cells showed good production stability. newline newline newline
Pagination: xix, 157
URI: http://hdl.handle.net/10603/15494
Appears in Departments:Faculty of Technology

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02_certificates.pdf685.33 kBAdobe PDFView/Open
03_abstract.pdf25.8 kBAdobe PDFView/Open
04_acknowledgement.pdf15.34 kBAdobe PDFView/Open
05_contents.pdf69.5 kBAdobe PDFView/Open
06_chapter 1.pdf695.69 kBAdobe PDFView/Open
07_chapter 2.pdf436.35 kBAdobe PDFView/Open
08_chapter 3.pdf3.82 MBAdobe PDFView/Open
09_chapter 4.pdf121.89 kBAdobe PDFView/Open
10_chapter 5.pdf41.47 kBAdobe PDFView/Open
11_appendix 1.pdf4.49 MBAdobe PDFView/Open
12_references.pdf145.68 kBAdobe PDFView/Open
13_publicaitons.pdf48.44 kBAdobe PDFView/Open
14_vitae.pdf23.95 kBAdobe PDFView/Open


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