Isolation, expression and purification of the hydantoin hydrolysing enzymes of agrobacterium tumefaciens

Clark, Sally-Ann (2003) Isolation, expression and purification of the hydantoin hydrolysing enzymes of agrobacterium tumefaciens. Masters thesis, Rhodes University.

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Abstract

The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into enantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolysing enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding the hydantoin hydrolysing enzymes are linked on the genome. A gene encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefaeiens RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. In this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase was used to re-isolate the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A. tumefaeiens RU-OR. In this study, the heterologous expression of neaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoylamino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR 1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR1-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.

Item Type:Thesis (Masters)
Uncontrolled Keywords:Agrobacterium tumefaciens, Amino acids
Divisions:Faculty > Faculty of Science > Biochemistry, Microbiology & Biotechnology
ID Code:22
Deposited By: Rhodes Library Archive Administrator
Deposited On:20 Oct 2005
Last Modified:06 Jan 2012 16:17
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