Affiliation:
1. Department of Biochemistry and Molecular Biology I, Faculty of Biology, Universidad Complutense de Madrid, Madrid, Spain
2. Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Abstract
ABSTRACT
The
pva
gene from
Streptomyces lavendulae
ATCC 13664, encoding a novel penicillin V acylase (
Sl
PVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (α-subunit) and 60.09 kDa (β-subunit). Based on sequence alignments and the three-dimensional model of
Sl
PVA, the enzyme contains a hydrophobic pocket involved in catalytic activity, including Serβ1, Hisβ23, Valβ70, and Asnβ272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of
pva
in
S. lividans
led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of
Sl
PVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
17 articles.
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