Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens

Citation
Re. Vega et al., Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens, INT BIO BIO, 43(1-2), 1999, pp. 49-55
Citations number
27
Categorie Soggetti
Environment/Ecology
Journal title
INTERNATIONAL BIODETERIORATION & BIODEGRADATION
ISSN journal
09648305 → ACNP
Volume
43
Issue
1-2
Year of publication
1999
Pages
49 - 55
Database
ISI
SICI code
0964-8305(199901/03)43:1-2<49:CAEIEC>2.0.ZU;2-B
Abstract
A polyester polyurethane (PU)-degrading enzyme, PU esterase, derived from P seudomonas fluorescens, a bacterium that utilizes polyester PU as the sole carbon source, was purified to homogeneity as indicated by sodium dodecyl s ulfate-polyacrylamide gel electrophoresis. This enzyme was a soluble, extra cellular protein with a molecular mass of 48 kDa and was inhibited by pheny lmethylsulfonylfluoride (PMSF). A genomic library of Ps. fluorescens was co nstructed using the Escherichia coli bacteriophage lambda vector lambda ZAP II. A recombinant phage exhibiting activity against Impranil DLN was isola ted. The gene encoding the polyurethanase (PUase) protein was subcloned int o a plasmid expression vector pT7-6 and expressed in E, coli. Upon expressi on, the PUase was secreted by the host, displayed esterase activity which w as inhibited by PMSF, and in vivo S-35-methionine labeling of the gene prod uct encoded by the open reading frame of the clone insert revealed a single polypeptide with a molecular mass of 48 kDa. (C) 1999 Elsevier Science Ltd . All rights reserved.