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Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

Published online by Cambridge University Press:  01 June 2009

Leszek Stepaniak  and
Patrick F. Fox
Leszek Stepaniak
Affiliation:
Department of Dairy and Food Chemistry, University College, Cork, Irish Republic
Patrick F. Fox
Affiliation:
Department of Dairy and Food Chemistry, University College, Cork, Irish Republic
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Summary

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0·1 m-phosphate buffer, pH 6·6, being completely denatured above 70 °C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 °C (minimum at 55 °C), but stability was very high above 80 °C in this buffer. D-values were determined at 10 °C intervals in the range 70–150 °C in SMS from which a Z value of 31·9 °C and an Ea of 8·82 × 104 J mol-1 were calculated; the half-life at 150 °C was 9 s.

Instability at 55 °C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 °C was inversely related to the rate of heating to 80 °C, i.e. length of time spent in the neighbourhood of 55 °C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 °C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 °C by addition of EDTA or by adjusting the reaction pH to 4·2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented.

Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 °C from 80, 100 or 150 °C, but did regain autolytic activity on cooling to below 50 °C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to ∼ 55 °C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 °C.

Type
Original Articles
Information
Journal of Dairy Research , Volume 50 , Issue 2 , May 1983 , pp. 171 - 184
Copyright
Copyright © Proprietors of Journal of Dairy Research 1983

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