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Chemically stabilized subtilisins in peptide synthesis

Colleary, Sandra and Ó Fágáin, Ciarán (2008) Chemically stabilized subtilisins in peptide synthesis. In: Flynne, William G., (ed.) Biotechnology and bioengineering. Nova Science Publishers, Hauppauge, NY, pp. 75-97. ISBN 978-1-60456-067-1

We have stabilized alcalaseTM and subtilisin Carlsberg (SC) against heat by chemical modification with ethylene glycol bis-succinimidyl succinate (EGNHS), a procedure not previously reported for subtilisins. The increases in thermal stability at 65oC were 1.8-fold and 4.7-fold respectively. Caseinolytic activity of alcalase in aqueous buffer was unchanged following modification but apparent Km of SC decreased 2.5-fold. Native and modified forms of both enzymes synthesized the tripeptide Z-Tyr-Gly-Gly-NH2 under kinetic control in mixtures based on 0.2M barbitone buffer, pH 9.0 and 50% v/v dimethyl formamide/ barbitone buffer. Native enzymes gave faster rates of product formation than their modified counterparts in buffer but differences were much less pronounced in the mixed solvent. We also compared native alcalase and SC in terms of thermal stability, tolerance of organic solvents and autolysis. Alcalase was approx. 4.6-fold more stable than SC at 65oC and was more tolerant of acetone, acetonitrile and 1,4-dioxane. Alcalase underwent autolysis at approx. half the rate of SC. Against succinyl- Ala-Ala-Pro-Phe substrates, alcalase showed a much higher esterase/ amidase ratio (567) than SC (29) in aqueous buffer but this was reversed in 50% v/v dimethylformamide, where the esterase/ amidase ratios were 43 and 113 respectively.
Item Type:Book Section
Uncontrolled Keywords:Alcalase; subtilisin Carlsberg; stabilization; chemical modification; peptide synthesis;
Subjects:Biological Sciences > Biochemistry
Biological Sciences > Enzymology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Institutes and Centres > National Centre for Sensor Research (NCSR)
Publisher:Nova Science Publishers
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 License. View License
ID Code:575
Deposited On:18 Sep 2008 13:00 by DORAS Administrator . Last Modified 19 Jul 2018 14:41

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