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Improved thermodynamic stability of subtilisin Carlsberg by covalent modification

Paper ID Volume ID Publish Year Pages File Format Full-Text
18325 42718 2006 7 PDF Available
Title
Improved thermodynamic stability of subtilisin Carlsberg by covalent modification
Abstract

The present work is aimed at improving the kinetic, thermal and thermodynamic stability of subtilisin Carlsberg (SCB) obtained from Bacillus licheniformis by means of simple, inexpensive but effective covalent coupling to oxidized sucrose polymers (OSP) of varying sizes (OSP400 and OSP70) as well as polyglutaraldehyde (PGA). In the presence of 10 mM calcium the half-life of the enzyme at 60 °C increased by 6.06-fold, 5.20-fold and 2.92-fold when coupled with OSP400, OSP70 and PGA, respectively. Even in the absence of added calcium the stability against thermal inactivation was found to be greater for the modified enzymes as evident from the increase in the energy of activation for the inactivation process (Eai). Guanidium thiocyanate-induced unfolding indicated Cm values of 1.3 M, 1.8 M, 1.5 M and 1.4 M for the native and enzymes modified with OSP400, OSP70 and PGA, respectively. Thermally induced unfolding was delayed for the modified enzymes as evident from the shift in Tm of 8.45 °C, 5.91 °C and 4.66 °C for OSP400, OSP70 and PGA modified enzymes. The results indicate that among the modifiers used OSP400 was most effective in stabilizing the enzyme and interestingly the increase in stability reported here is comparable to the most stabilized subtilisin variants obtained by site-directed mutagenesis.

Keywords
Carbohydrates; Chemical modification; Conformational stability; Heat inactivation; Subtilisin
First Page Preview
Improved thermodynamic stability of subtilisin Carlsberg by covalent modification
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 39, Issue 2, 26 June 2006, Pages 301–307
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering