Glucoamylase, a saccharifying enzyme, is applied in the brewing industry to hydrolyse the dextrins of malted barley into simple sugars which can then be fermented by brewer's yeast. In order to establish the potential of glucoamylase from Lactobacillus amylovorus for application in the brewing industry, the main objectives of this study were: (1) to determine the cultural conditions for growth and glucoamylase production, (2) to purify the enzyme to homogeneity using chromatography and electrophoretic techniques, (3) to study biochemical properties of the purified enzyme, and (4) to clone the gene coding for glucoamylase, and characterize the recombinant glucoamylase. / The actively amylolytic Lactobacillus amylovorus ATCC 33621 produced an intracellular glucoamylase activity. Conditions for growth and glucoamylase production were maximized by using dextrose free MRS medium supplemented with 1% dextrin, at pH 5.5 and 37$ sp circ$C. Enzyme production was maximal during the late logarithmic phase of growth from 16-18 h. Crude cell extract showed optimal activity at pH 6.0 and 55$ sp circ$C. / Native and SDS-PAGE of the purified enzyme showed a monomeric protein of 47 kD. Glucoamylase activity was confirmed by activity staining using a starch/polyacrylamide gel where a zone of clearing was visible on a blue/black background stained with Kl/I$ sb2.$ Optimal pH, pl and temperature of purified glucoamylase were 4.5, 4.39 and 45$ sp circ$C, respectively. The enzyme was rapidly inactivated by temperatures above 55$ sp circ$C and was inhibited by heavy metals, e.g. Pb$ sp{2+}$ and Cu$ sp{2+}$ at 1.0 mM. EDTA did not inhibit the enzyme activity at a final concentration of 10 mM. Enzyme inhibition by 1 mM of p-chloromercuribenzoic acid (pCMB) and iodoacetate suggested that a sulfhydryl group was present in the enzyme active site. Kinetic studies of glucoamylase confirmed that the enzyme reacted preferentially with polysaccharides. HPLC analyses of the end products of enzyme action showed that glucose was the major end product of enzyme action and this glucose was responsible for end product inhibition. / The gene coding for glucoamylase was cloned into Escherichia coli using the STA2 glucoamylase gene of Saccharomyces diastaticus as a probe. Three glucoamylase producing transformants were identified as the insert sizes of about 5.2 Kb, 6.4 Kb and 5.9 Kb, respectively. When the characteristics of both recombinant and purified wild type glucoamylases were compared, both enzymes showed a similar pH range of 3.0-8.0, and temperature optimum of 45$ sp circ$C. The recombinant enzyme pH profiles were broader than that of the wild type and an optimum pH of 6.0 was obtained. This study has shown that glucoamylase from Lb. amylovorus is less heat stable than other bacterial glucoamylases and thus may be suitable for application in the brewing industry. Successful cloning of this gene coding for glucoamylase in brewer's yeast, Saccharomyces cerevisiae, would reap the advantageous properties of the enzyme while eliminating the costs of adding commercial enzymes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.42060 |
| Date | January 1996 |
| Creators | James, Jennylynd Arlene. |
| Contributors | Lee, Byong H. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Food Science and Agricultural Chemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001565582, proquestno: NQ29965, Theses scanned by UMI/ProQuest. |
Page generated in 0.0016 seconds