Lactobacillus 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.
| Identifer | oai:union.ndltd.org:USASK/oai:ecommons.usask.ca:10388/ETD-2014-05-1546 |
| Date | 2014 May 1900 |
| Contributors | Tanaka, Takuji |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text, thesis |
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