In order to establish the potential of an esterase from Lactobacillus casei for application in the dairy industry, this study was designed with the following objectives: (1) to determine the optimal cultural conditions for growth and enzyme production, (2) to construct a positive selection vector with low copy number for gene cloning, (3) to clone and sequence the gene encoding for esterase, (4) to biochemically characterize the purified recombinant esterase, and (5) to over-express an esterase of L. casei into Escherichia coli as well as into a methylotrophic yeast, Pichia pastoris using strong promoters. / Maximal growth and enzyme production in L. casei were obtained after 20 h in basal MRS medium containing 1% (w/v) lactose at pH 7.0, 30°C. Among various substrates (C2--C16) tested, the highest activity was towards C6 and C8. Although the enzyme was produced constitutively, tributyrin induced the enzyme production by a 2.5 fold. / A new E. coli and lactic acid bacteria shuttle vector with low copy and positive selection termed pCWL70 was constructed. High transformation efficiency and significant vector stability of pCWL70 were found in L. casei and Lactococcus lactis. / An esterase gene (estI) of L. casei CL96 was localized in a 3.3 kb Bam HI DNA fragment that contains an open reading frame of 1,800 bp. The open reading frame estI was isolated by PCR and subcloned into the E. coli expression vector pET29a, and Pichia expression vector pPICZ B, that allows the inducible expression under the control of the T7 promoter and an alcohol oxidase promoter (AOX1), respectively. E. coli BL21(DE3)pLysS containing estI expressed a novel 67.5 kDa protein corresponding to the EstI in N-terminal fusion with the S · tag peptide. / An esterase of L. casei CL96 was successfully over-expressed in E. coli up to 500 folds (about 25% of total cellular protein) as well as in the P. pastoris. In high cell density fed-batch fermentation, the recombinant Pichia strain containing linearized pCESTc was grown at pH 6.5 and 30°C, and the cell density peaked at about 180 h with 468 g wet cell weight per liter. The final yield was 3.7 x 106 units/ml, which is 980-folds higher than that of native L. casei cells. / The amino acid sequence of EstI indicated that the esterase is a member of a novel GHSMG family of lipolytic enzymes and S-formylglutathione hydrolases (FGH). The putative catalytic triad of EstI consists of residues Ser325, Asp516 and His558 as demonstrated by amino acid sequence alignments. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.36892 |
| Date | January 2001 |
| Creators | Choi, Young-Jun, 1967- |
| 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: 001808413, proquestno: NQ69986, Theses scanned by UMI/ProQuest. |
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