Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst

R.V. Ulijn, M. Erbeldinger, P.J. Halling

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH2 was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the solid-to-solid and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied
LanguageEnglish
Pages633-638
Number of pages5
JournalBiotechnology and Bioengineering
Volume69
Issue number6
DOIs
Publication statusPublished - 20 Sep 2000

Fingerprint

Thermolysin
Acetonitrile
Peptides
Enzymes
Catalysts
Reaction rates
Suspensions
Diatomaceous Earth
1-Propanol
Immobilized Enzymes
Polypropylenes
Propanol
Silicon Dioxide
Powders
Precipitates
Hydrolysis
Silica
Water
acetonitrile

Keywords

  • peptide synthesis
  • low water media
  • organic media
  • solid-to-solid
  • thermolysin

Cite this

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abstract = "This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH2 was synthesized in acetonitrile containing 5{\%} water (with various catalyst preparation methods) as well as by the solid-to-solid and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10{\%} water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60{\%} in low water organic solvent, whereas they were found to >80{\%} in the aqueous systems studied",
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Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst. / Ulijn, R.V.; Erbeldinger, M.; Halling, P.J.

In: Biotechnology and Bioengineering, Vol. 69, No. 6, 20.09.2000, p. 633-638.

Research output: Contribution to journalArticle

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AU - Halling, P.J.

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AB - This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH2 was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the solid-to-solid and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied

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