tailieunhanh - Báo cáo khoa học: Characterization of recombinant prolidase from Lactococcus lactis – changes in substrate specificity by metal cations, and allosteric behavior of the peptidase

TheLactococcus lactisNRRL B-1821 prolidase gene was cloned and over-expressed in Escherichia coli. Under suboptimum growth conditions, recombinant soluble and active prolidase was produced; in contrast, inclu-sion bodies were formed under conditions preferred for cell growth. | ỊFEBS Journal Characterization of recombinant prolidase from Lactococcus lactis - changes in substrate specificity by metal cations and allosteric behavior of the peptidase Soo I. Yang and Takuji Tanaka Department of Food and Bioproduct Sciences College of Agriculture and Bioresources University of Saskatchewan Saskatoon Canada Keywords bitterness metallopeptidase overexpression PepQ proline Correspondence T. Tanaka Department of Food and Bioproduct Sciences College of Agriculture and Bioresources University of Saskatchewan 51 Campus Drive Saskatoon Saskatchewan S7N 5A8 Canada Fax 1 306 966 8898 Tel 1 306 966 1697 E-mail Received 1 August 2007 revised 15 October 2007 accepted 16 November 2007 doi The Lactococcus lactis NRRL B-1821 prolidase gene was cloned and overexpressed in Escherichia coli. Under suboptimum growth conditions recombinant soluble and active prolidase was produced in contrast inclusion bodies were formed under conditions preferred for cell growth. Recombinant prolidase retained more than half its full activity between 30 and 60 C and was completely inactivated after 30 min at 70 C. CD analysis confirmed that prolidase was inactivated at 67 C. The enzyme was active under weak alkali to weak acidic conditions and showed maximum activity at pH . Although these characteristics are similar to those for other reported prolidases this prolidase was distinctive for two kinetic characteristics. Firstly different substrate specificity was observed for its two preferred metal cations zinc and manganese Leu-Pro was preferred with zinc whereas Arg-Pro was preferred with manganese. Secondly the enzyme showed an allosteric response to changes in substrate concentrations with Hill constants of for Leu-Pro and for Arg-Pro. Molecular modeling of this prolidase suggests that these unique characteristics may be attributed to a loop structure near the active site. Fermented foods have significant .