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 Km and Vmax of the
fusion protein were 0.56 g/l and 51 μmol/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.