Previous Article | Next Article ![]()
Applied and Environmental Microbiology, April 2000, p. 1360-1368, Vol. 66, No. 4
NIZO Food Research, Ede, The
Netherlands,1 and New Zealand Dairy
Research Institute, Palmerston North, New Zealand2
Received 23 August 1999/Accepted 6 January 2000
The gene encoding the major intracellular tributyrin esterase of
Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to
encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a
canonical promoter sequence was identified. The deduced amino acid
sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled
expression system recently developed for lactic acid bacteria. The
estA gene was inactivated by chromosomal integration of a
temperature-sensitive integration vector. This resulted in the complete
loss of esterase activity, which could then be recovered after
complementation of the constructed esterase-deficient strain with the
wild-type estA gene. This confirms that EstA is the main
enzyme responsible for esterase activity in L. lactis.
Purified recombinant enzyme showed a preference for short-chain
acyl esters, surprisingly also including phospholipids. Medium- and
long-acyl-chain lipids were also hydrolyzed, albeit less efficiently.
Intermediate characteristics between esterases and lipases make
intracellular lactococcal EstA difficult to classify in either of these
two groups of esterolytic enzymes. We suggest that, in vivo, EstA could
be involved in (phospho)lipid metabolism or cellular detoxification or
both, as its sequence showed significant similarity to
S-formylglutathione hydrolase (FGH) of Paracoccus
denitrificans and human EstD (or FGH), which are part of a
universal formaldehyde detoxification pathway.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cloning, Characterization, Controlled
Overexpression, and Inactivation of the Major Tributyrin Esterase
Gene of Lactococcus lactis

*
Corresponding author. Mailing address: NIZO Food
Research, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone:
31-318-659542. Fax: 31-318-650400. E-mail: siezen{at}nizo.nl.
Present address: Departamento de Nutrición y
Bromatología III (Higiene y Tecnología de los
Alimentos), Facultad de Veterinaria, Universidad Complutense de Madrid,
28040-Madrid, Spain.
Present address: Molecular Genetics, Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen, 9750 AA
Haren, The Netherlands.
This article has been cited by other articles:
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»