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Identification and Characterization of a Gamma-Glutamyl Transpeptidase from a Thermo-Alcalophile Strain of Bacillus pumilus

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Abstract

A γ-glutamyltranspeptidase (GGT, E.C. 2.3.2.2) was isolated from a strain (A8) originating from Lake Bogoria (Kenya) and homologous with Bacillus pumilus. This GGT shows an optimal activity at pH 8.9 and 62°C. The enzyme is thermostable up to 43°C. The best reagent among the potential inhibitors was shown to be DON, which is an inhibitor highly specific for GGTs. Gly-Gly-Ala, Gly-Gly-Gly and Gly-Gly were identified as the best acceptors for the transpeptidation reactions catalyzed by the enzyme. The SDS-PAGE study revealed that the enzyme consists of two non-identical subunits (38,000 and 23,000). Only the large subunit was active when the enzyme was dissociated under denaturing conditions. The behavior of the native enzyme suggests that the active site of the large subunit is masked by the small subunit.

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Abbreviations

DCI:

3,4-Dichloroisocoumarin

DFP:

Di-isopropylfluorophosphate

DON:

6-Diazo-5-oxo-l-norleucine

E64:

Trans-epoxysuccinyl-l-leucylamido-(4-guanidino)-butane

EDTA:

Ethylenediaminetetraacetic acid disodium salt

EGTA:

Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid

GGT:

Gamma-glutamyltranspeptidase

PAGE:

Polyacrylamide gel electrophoresis

PMSF:

Phenylmethanesulfonyl fluoride

SDS:

Sodium dodecylsulfate

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Acknowledgments

The authors would like to thank Dr F. Mulaa and Prof. D. Makawiti (University of Nairobi) for their help in harvesting sediment samples around Lake Bogoria, Dr M. Dion and Dr A. Defontaine (CNRS-UMR 6204, Nantes) for isolating and identifying the bacterial strains and J. C. Huet (INRA, Jouy-en-Josas) for determining the amino acid sequences.

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Authors and Affiliations

  1. Laboratoire de Biochimie, Unité de Biotechnologie, Biocatalyse et Biorégulation, CNRS-UMR 6204, Faculté des Sciences et Techniques, 2 rue de la Houssinière, BP 92208, F44322, Nantes Cedex 3, France

    Claire Moallic, Soumaila Dabonné, Bernard Colas & Jean-Pierre Sine

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  1. Claire Moallic
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  2. Soumaila Dabonné
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  3. Bernard Colas
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  4. Jean-Pierre Sine
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Correspondence to Jean-Pierre Sine.

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Moallic, C., Dabonné, S., Colas, B. et al. Identification and Characterization of a Gamma-Glutamyl Transpeptidase from a Thermo-Alcalophile Strain of Bacillus pumilus . Protein J 25, 391–397 (2006). https://doi.org/10.1007/s10930-006-9025-4

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  • DOI: https://doi.org/10.1007/s10930-006-9025-4

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