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Prolinases from Lactobacillus plantarum WCFS1: Cloning, Purification and Characterization of the Recombinant Enzymes

dc.contributor.advisorTanaka, Takujien_US
dc.contributor.committeeMemberTyler, Roberten_US
dc.contributor.committeeMemberQiu, Xiaoen_US
dc.contributor.committeeMemberNapper, Scotten_US
dc.creatorHuang, Yanyuen_US
dc.date.accessioned2015-10-24T12:01:10Z
dc.date.available2015-10-24T12:01:10Z
dc.date.created2014-05en_US
dc.date.issued2015-10-23en_US
dc.date.submittedMay 2014en_US
dc.description.abstractLactobacillus plantarum WCSF1 has two putative prolinases (PepR1 and PepR2), and they share only 48.5% amino acid sequence identity. To investigate the differences in enzymatic characters between two enzymes, the genes are cloned and expressed in E. coli using non-tagged pKK223-3 and His-tagged pET32b(+) systems. Culture conditions of overexpressed recombinant prolinases (r-PepR1 and r-PepR2) are optimized as pH7.0-7.5 LB media at 16°C with 1 mM IPTG induction. Recombinant prolinases with His-tag give higher yields and are more cost-efficient over non-tagged recombinant prolinases. After purification, these recombinant enzymes show similar hydrolysis activities towards Pro-Gly substrate, proving their nature as prolinases. Structural analyses using CD spectrum and computer modelling show that r-PepR1 and r-PepR2 share structural similarity in their secondary structure having the highest β-sheets over other components; and dynamic light scattering and gel filtration chromatography analyses indicate their quaternary structure being homotetrameric. Structural similarity can be linked to enzyme function feature. The two enzymes have the same enzymatic functionality may be due to their structural similarity. Despite for their structural similarities and the same enzymatic functionality, they show differences in their substrate specificity, optimum temperature and pH, kinetic parameters (Km and kcat values), thermal stability, and proteolysis mode. r-PepR1 has its optimal activity at 25°C pH7.5 against substrate Pro-Met, whereas r-PepR2 is most active at 30°C pH8.0 against Pro-Gly. r-PepR1 has a low thermal stability with a TM (the midpoint temperature of the unfolding transition) at 29°C, whereas r-PepR2 has a higher TM at 48°C. However, r-PepR1 is aggregated and inactivated at near physiological temperature (42°C). The catalytic mode of r-PepR1 could be a metallo-protease since its activity reduces by 38% with a metal-chelating agent EDTA; while the activity of r-PepR2 is inhibited by 47% with a serine protease inhibitor PMSF, suggesting it is a serine protease. These isozymes cooperatively and complementarily work together to hydrolyze proline-containing peptides, showing broader specificity, broader range of working pH and temperature, and higher efficiency, suggesting that the proline recycling are mediated through these two enzymes to adapt a wide rage of environmental conditions.en_US
dc.identifier.urihttp://hdl.handle.net/10388/ETD-2014-05-1546en_US
dc.language.isoengen_US
dc.subjectbitterness, proline, prolidase, isozyme, PepI/PepL/PepR familyen_US
dc.titleProlinases from Lactobacillus plantarum WCFS1: Cloning, Purification and Characterization of the Recombinant Enzymesen_US
dc.type.genreThesisen_US
dc.type.materialtexten_US
thesis.degree.departmentFood and Bioproduct Sciencesen_US
thesis.degree.disciplineFood Scienceen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Science (M.Sc.)en_US

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