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Cloning and expression of a thermostable alpha-amylase gene from Streptomyces thermoviolaceus CUB74

Bahri, Sami Mahmoud; (1990) Cloning and expression of a thermostable alpha-amylase gene from Streptomyces thermoviolaceus CUB74. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Streptomycetes are aerobic Gram-positive bacteria whose natural habitat is the soil. They have been of general interest because of their ability to produce a variety of secondary metabolites with antibiotic properties. Apart from antibiotic production, streptomycetes produce many extracellular enzymes including amylases, proteases, cellulases and nucleases. Amylases are widely distributed amongst Streptomyces species and have been coming under increasing scrutiny in the past few years. For example, the α-amylase genes of S. hygroscopicus, S. limosus and S. venezuelae have been cloned and sequenced and their predicted amino acid sequences have been compared. The thesis describes the cloning and expression of a thermostable α-amylase gene from S. thermoviolaceus CUB74 into Escherichia coli and S. lividans. When expressed in E. coli, the Streptomyces α-amylase enzyme was found in the periplasmic space and the supernatant. The extracellular enzyme isolated from the parent Streptomyces strain and the new hosts was further analysed by SDS-polyacry1 amide gel electrophoresis. In addition, the product of starch digestion with this enzyme was analysed by paper chromatography. The α-amylase enzyme was reactivated in the SDS protein gel and its activity was visualized using a starch-agarose gel overlay. Stability of this enzyme at high temperatures was determined in the presence and absence of calcium and substrate. Its production in the parent strain and in S. lividans was also studied in the presence of different carbon sources. The gene specifying this enzyme was sequenced and its predicted amino acid sequence was compared with α-amylases isolated from other species. mRNA produced from this gene in different plasmid constructs was detected by Northern blotting and the transcriptional start site was determined by high resolution S mapping.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Cloning and expression of a thermostable alpha-amylase gene from Streptomyces thermoviolaceus CUB74
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Amylases
URI: https://discovery.ucl.ac.uk/id/eprint/10111006
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