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Biotechnol Bioeng. 2015 Jul;112(7):1365-75. doi: 10.1002/bit.25565. Epub 2015 Feb 25.

Characterization of the activity of the spore cortex lytic enzyme CwlJ1.

Author information

1
Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York, 12180.
2
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180.
3
U.S. Army Engineer Research and Development Center, Construction Engineering Research Laboratory, Champaign, Illinois.
4
Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York, 12180. kaner@rpi.edu.
5
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180. kaner@rpi.edu.
6
Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York, 12180. dordick@rpi.edu.
7
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, 12180. dordick@rpi.edu.
8
Department of Materials Science and Engineering, Department of Biology, Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York, 12180. dordick@rpi.edu.

Abstract

The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of Bacillus anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report for the first time a detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B. anthracis when added exogenously. CwlJ1 was active on both decoated spores and spore cortical fragments. Through liquid chromatography-mass spectrometry analysis of the digested cortical fragments, we determined that CwlJ1 was a thermostable N-acetylmuramoyl-L-alanine amidase. CwlJ1 mainly recognized large segments of glycan chains in the cortex instead of the minimal structural unit tetrasaccharide, with specificity for muramic acid-δ-lactam-containing glycan chains and preference for the tetrapeptide side chain. Unlike most amidases, CwlJ1 did not appear to contain a divalent cation, as it retained its activity in the presence of EDTA. This study shines some light on the mechanism of spore germination, and provides increased insight into the development of sporicidal enzyme systems for decontamination of B. anthracis and other related bacteria.

KEYWORDS:

Bacillus anthracis; CwlJ1; amidase; cortex; germination

PMID:
25676066
DOI:
10.1002/bit.25565
[Indexed for MEDLINE]

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