Purification, characterization, and overexpression of thermophilic pectate lyase of Bacillus sp. RN1 isolated from a hot spring in Thailand.
A thermophilic pectate lyase, Pel SWU, was isolated from a culture filtrate of Bacillus sp. RN1 isolated from a hot spring in Ranong Province, Thailand. The enzyme was purified to homogeneity using cation-exchange and hydrophobic column chromatographies. The molecular mass of Pel SWU was estimated to be 33 kDa. The specific substrate was demethylated galacturonic acid. The enzyme was stable at pH 4.0-10.0 and at temperatures up to 70 degrees C in the presence of calcium and polygalacturonic acid (PGA). The optimum pH and temperature were 10.0 and 90 degrees C. The pel gene encoding Pel SWU was 1,023 bp, which corresponds to 341 amino acids. The properties of the recombinant enzyme was similar to those of Bacillus Pel SWU. Unsaturated di- and trigalacturonic acids were formed mainly as the final products of degradation by Pel SWU, as revealed by high-performance anion-exchange chromatography (HPAEC) and electrospray ionization mass spectrometry (ESI-MS) analyses. This thermophilic pectate lyase should be useful in the degradation of pectin networks at high temperature.
Sukhumsiirchart
Wasana
W
Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand. wasanas@swu.ac.th
Kawanishi
Sakiko
S
Deesukon
Warin
W
Chansiri
Kosum
K
Kawasaki
Haruhiko
H
Sakamoto
Tatsuji
T
D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D045482:Hot Springs / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013785:Thailand
D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase