Liu Suk Ching. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 123-128.

Recombinant DNA techniques were used to clone and isolate Cellulo- monas fimi cellulase genes. A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant plasmids carrying cellulase genes. The screening procedure is based on binding cellulases and other proteins released from lysed clones to CNBr-acti- vated paper. The paper is treated with anti-cellulase antibody and the antigen-antibody complex is detected by autoradiography using ¹²⁵I-labeled protein A from Staphylococcus aureus. This- immunoassay, was used to identify recombinant plasmids containing strains, carrying at least two different cellulase genes. The enzymes present in extracts of E. coli cellulase clones were active in catalysing the hydrolysis of carboxymethy1 eel 1ulose as indicated by the production of reducing sugars. Osmotic shock treatment of one E. coli cellulase clone revealed that the majority of the cellulase enzyme synthesized by this clone was transported to the periplasmic space. Cellulase encoding plasmids were characterized by the presence of either a 6.6 or a 5.0 kilobase C. fimi DNA gene fragment. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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<p>A nitrile and amide hydrolysing Nesterenkonia sp. was isolated from Antarctic soil and was characterised as a psychrotolerant, halotolerant and alkaliphilic extremophile. Amidases are widely distributed in both prokaryotic and eukaryotic organisms. These enzymes hydrolyze C-N bonds other than peptide bonds and are particularly interesting for their potential industrial application.&nbsp / This study aimed to identify and characterize amidase genes from this novel psychrotolerant microorganism. Using BLAST analysis, two ORFs with conserved amidase sequences were&nbsp / identified from the complete genome sequence of the organism. Two ORFs, AmiF and AmiS, were assigned to two different gene families, the aceta/formamidase family and amidase signature&nbsp / family, respectively. On the genome, the spatial orientation and intergenic distance (1bp overlap) of the ORF‟s suggested that amiF and amiS could possibly be cotranscribed which was confirmed by reverse transcription PCR. A third ORF with a conserved amidase sequence was found &plusmn / 500bps downstream from amiS, suggesting the possible presence of a multi amidase&nbsp / operon. The two genes were cloned and expressed as N-terminal 6x His-Tag fusion proteins. AmiS and Ami F were partially purified using Ni-chelation chromatography. Although both proteins&nbsp / were subjected to activity assay, their activities are yet to be established. Homology modeling of the AmiF and AmiS translated sequences showed that the proteins had the significant similarities&nbsp / to the members of their families. Although the sequence identities between the AmiF and AmiS and their templates were very low (24 % and 25% respectively), the evaluation of the models&nbsp / showed that the quality of the models were good. This study reports the genetic and functional characterisation of amidase genes from the cold adapted microorganisms.</p>

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

No description available.

<p>A nitrile and amide hydrolysing Nesterenkonia sp. was isolated from Antarctic soil and was characterised as a psychrotolerant, halotolerant and alkaliphilic extremophile. Amidases are widely distributed in both prokaryotic and eukaryotic organisms. These enzymes hydrolyze C-N bonds other than peptide bonds and are particularly interesting for their potential industrial application.&nbsp / This study aimed to identify and characterize amidase genes from this novel psychrotolerant microorganism. Using BLAST analysis, two ORFs with conserved amidase sequences were&nbsp / identified from the complete genome sequence of the organism. Two ORFs, AmiF and AmiS, were assigned to two different gene families, the aceta/formamidase family and amidase signature&nbsp / family, respectively. On the genome, the spatial orientation and intergenic distance (1bp overlap) of the ORF‟s suggested that amiF and amiS could possibly be cotranscribed which was confirmed by reverse transcription PCR. A third ORF with a conserved amidase sequence was found &plusmn / 500bps downstream from amiS, suggesting the possible presence of a multi amidase&nbsp / operon. The two genes were cloned and expressed as N-terminal 6x His-Tag fusion proteins. AmiS and Ami F were partially purified using Ni-chelation chromatography. Although both proteins&nbsp / were subjected to activity assay, their activities are yet to be established. Homology modeling of the AmiF and AmiS translated sequences showed that the proteins had the significant similarities&nbsp / to the members of their families. Although the sequence identities between the AmiF and AmiS and their templates were very low (24 % and 25% respectively), the evaluation of the models&nbsp / showed that the quality of the models were good. This study reports the genetic and functional characterisation of amidase genes from the cold adapted microorganisms.</p>

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 55-64).