Carboxypeptidase Y (E.C. 3.4.12.1) was produced from Kluyveromyces fragilis ATCC 28244. The maximum growth and enzyme production were obtained during 24 hr of growth at the late logarithmic phase with optimized conditions (25$ sp circ $C, 300 rpm, pH 5) using YPD (1% yeast extract, 2% peptone, 2% dextrose, w/v) broth medium. A Fast Protein Liquid Chromatography (FPLC) was used for the enzyme purification. The enzyme was purified to 216 fold over the crude extract with a recovery of 18%. The apparent molecular weight of the purified enzyme was estimated to be 120 kDa on Native-PAGE and 56 kDa on SDS-PAGE suggesting that carboxypeptidase Y from Kluyveromyces fragilis consists of two subunits. The pH and temperature optima of the enzyme were pH 6.0 and 35$ sp circ$C, respectively. The enzyme activity was strongly inhibited by diisopropylphosphofluoridate (DIPF) and phenylmethylsulfonylfluoride (PMSF), and caused an average 50% loss of activity when incubated with various metal cations. / The apparent K$ sb{ rm m}$ and V$ sb{ rm max}$ values obtained for n-benzoyl-L-tyrosine-p-nitroanilide (BTPNA) and carboxybenzoxyphenylalanylalanine (Cbz-Phe-Ala) were 5.1 mM and 13.4 $ mu$mole/min/mg and 2.98 mM and 22.58 $ mu$mole/min/mg, respectively. Carboxypeptidase Y hydrolysis on the tryptic digests of $ alpha sb{ rm s1}$- and $ beta$-casein showed that the enzyme randomly removed five and three hydrophobic peptides, respectively and greatly reduced the size and heights of the other peptides analysed on Reversed Phase-High Performance Liquid Chromatography (RP-HPLC).
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.22527 |
| Date | January 1994 |
| Creators | Transfiguracion, Julia de la Cruz |
| 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 | Master of Science (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: 001443083, proquestno: MM00061, Theses scanned by UMI/ProQuest. |
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