Cloning, expression, and characterization of lactic acid bacteria recombinant prolidases

Lactobacillus plantarum (Lb. plantarum) NRRL B4496 and Lactococcus lactis (Lc. lactis) NRRL B1821 prolidase genes were isolated, cloned, and sequenced. The sequence-confirmed genes were subcloned into the expression systems. The recombinant prolidases from the pKK223-3 systems were purified through ammonium sulphate precipitation and anion-exchange column chromatography. Recombinant Lb. plantarum prolidase, however, demonstrated a loss of activity during the purification. The following characterization work was performed on purified recombinant Lc. lactis prolidase. The mass spectroscopic result and the molecular modelling suggested a 80 kDa homodimer with two metal cations at the catalytic centre of the prolidase. The optimum temperature was 50 ºC and showed more than 50% activities between 40 and 55 ºC. The enzyme was most stable at 30 ºC and withstood 20 min of heat-treatment up to 60 ºC, however, lost activity over 70 ºC. Circular dichroism indicated a denaturation temperature of 67 ºC. The optimum pH was 6.5 for hydrolyzing Leu-Pro and the enzyme did not display any activity below pH 5.5 nor above pH 7 with this peptide. However, Phe-Pro was hydrolyzed the fastest at pH 7 and Arg-Pro had a maximum rate at pH 9. This metallopeptidase exhibited a broad range of metal cation preference, hydrolyzing Leu-Pro with Mn++, Co++, Zn++, Ca++, and Mg++. Further kinetic analysis showed unusual allostery of the enzyme (Hill coefficient: 1.3). The unique substrate intakes onGlu-Pro and tripeptides were observed while Val-Pro was not hydrolyzed. The molecular modelling of this prolidase suggested a difference in the substrate specificity resulting from a loop structure, L33 to R40, near the substrate binding site.

Name thesis:degree_name

Master of Science (M.Sc.)

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Applied Microbiology and Food Science

Grantor

University of Saskatchewan

Year dc:date.issued

2007

Author dc:creator

• Yang, Soo In

Advisor dc:contributor.advisor

• Tanaka, Takuji

Committee members dc:contributor.committeemember

• Shand, Phyllis J.
• Korber, Darren R.

• Lactic acid bacteria
• PepQ
• prolidase

Language dc:language.iso

en_US

Handle dc:identifier.uri

https://hdl.handle.net/10388/etd-04142007-123803

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/etd-04142007-123803