The cloning and characterization of an α-amylase and a branching enzyme from Butyrivibrio fibrisolvens H17c and their expression in Escherichia coli

Doctoral Thesis


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University of Cape Town

Butyrivibrio fibrisolvens H17c is an important anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to establish a genebank of B. fibrisolvens H17c DNA in E.coli and to isolate and characterize genes encoding enzymes involved in the degradation of the major plant polysaccharides. A library of chromosomal DNA fragments from B. fibrisolvens was established in the E. coli-Bacillus subtilis shuttle vector pEBl. The library was screened for the expression of B. fibrisolvens genes in E. coli. E. coli clones expressing glutamine synthetase, carboxymethylcellulase, β-glucosidase and amylolytic-type activities were isolated. A gene (amyA) expressing amylolytic activity and encoding an α-amylase was located on a 5.0 kb DNA fragment and expressed from its own promoter in E. coli. It was shown that more than 86% of the amylolytic actvity was located in the periplasm of the E.coli host and TnphoA mutagenesis indicated the presence of a functional signal peptide. The nucleotide sequence of amyA was determined and encoded a protein of 976 amino acids with a calculated Mr of 106,964. High sequence similarity was demonstrated between the B. fibrisolvens α-amylase and other α-amylases in the three highly conserved regions which constitute the active centre. Conserved regions were all located in the N-terminal half of the B. fibrisolvens amylase and no homology to other amylases was detected for the remainder of the protein. Approximately 40% of the C-terminal region of the protein could be deleted without loss of enzymatic activity. The B. fibrisolvens α-amylase degraded amylose, amylopectin and soluble starch with maltotriose as the major initial hydrolysis product. A gene (glgB) encoding a glycogen branching enzyme, the activity of which produced clearing on starch azure plates, was isolated. The glgB gene was expressed from its own promoter in the host E.coli and encoded a protein of 639 amino acids with a calculated Mr of 73,875. The deduced amino acid sequence of the glgB gene showed high sequence homology (46-50%) to other branching enzymes. The branching enzyme was purified to homogeneity and the properties of the purified enzyme were investigated. Optimal activity of the branching enzyme was at pH 7.2 and 37°C. The branching enzyme was shown to transfer chains of between 5 to 10 glucose units using α-1,4 glucans as substrates, and to stimulate the "de novo" synthesis of a polysaccharide similar to glycogen.

Bibliography: pages 145-172.