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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 10, Number 6
BKCSDE 10(6)
June 20, 1989 

Irreversible Thermoinactivation Mechanisms of Subtilisin Carlsberg
Dong Uk Kim, Myung-Un Choi*
In order to find the rational methods for improving the thermal stability of subtilisin Carlsberg, the mechanisms of irreversible thermoinactivation of the enzyme were studied at 90℃. At pH 4, the main process was hydrolysis of peptide bond. This process followed first order kinetics, yielding a rate constant of 1.26× 10-1h-1. Hydrolysis of peptide bond of PMS-subtilisin occurred at various sites, which produced new distinct fragments of molecular weights of 27.2 KD, 25.9 KD, 25.0 KD, 22.3 KD, 19.0 KD, 17.6 KD, 16.5 KD, 15.7 KD, 15.0 KD, 13.7 KD, and 12.7 KD. Most of the new fragments originated from the acidic hydrolysis at the C-side of aspartic acid residues. However 25.0 KD, 15.7 KD, and 13.7 KD which could not be removed in purification steps stemmed from the autolytic cleavage of subtilisin. The minor process at pH 4 was deamidation at asparagine and/or glutamine residues and some extend of aggregation was also observed. However, the aggregation was main process at pH 7 with a first order kinetic constant of 16 h-1. At pH 9, the main process seemed to be combination of deamidation and cleavage of peptide bond.
600 - 604
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