An unusual pectate lyase from a Bacillus sp. with high activity on pectin: cloning and characterization.
The gene pelA encoding a pectate lyase from the strain Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1214 bp DNA fragment containing pelA gene was determined, revealing an ORF of 666 nucleotides that encoded a protein of 23233 Da. The deduced amino acid sequence of the encoded enzyme showed homology to pectate lyases A, B, C and D from Fusarium solani, Pel-3 and PelB from Erwinia carotovora and Pell from Erwinia chrysanthemi. Homology was also found to the protein deduced from the Bacillus subtilis yvpA gene, the function of which is unknown. The heterologous expressed enzyme depolymerized polygalacturonate and pectins of methyl esterification degree from 22 to 89%, and exhibited similar activity on polygalacturonate and on 89% esterified citrus pectin. Optimum temperature and pH for enzymic activity were 50 degrees C and pH 10, respectively. Ca2+ was required for activity on pectic substrates, while the enzyme was strongly inhibited by Ba2+.
Soriano
Margarita
M
Department of Microbiology, Faculty of Biology, University of Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain1.
Blanco
Ana
A
Department of Microbiology, Faculty of Biology, University of Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain1.
Dı Az
Pilar
P
Department of Microbiology, Faculty of Biology, University of Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain1.
Pastor
F I Javier
FIJ
Department of Microbiology, Faculty of Biology, University of Barcelona, Avinguda Diagonal 645, 08028 Barcelona, Spain1.
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase