Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase.
The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively. The activity of purified PelAHis was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.
Berensmeier
S
S
Department for Carbohydrates, Technical University Braunschweig, Langer Kamp 5, 38106 Braunschweig, Germany.
Singh
S A
SA
Meens
J
J
Buchholz
K
K
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D016974:Dickeya chrysanthemi / Q000235:genetics; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010368:Pectins / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013400:Sugar Acids / Q000302:isolation & purification; D013696:Temperature; D014312:Trisaccharides / Q000302:isolation & purification
D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013400:Sugar Acids; D014312:Trisaccharides; C065008:O-galactopyranosyluronic acid-(1-4)-O-galactopyranosyluronic acid-(1-4)-galactopyranosyluronic acid; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; C003181:polygalacturonic acid