Cloning and sequencing of a high-alkaline pectate lyase gene from an alkaliphilic Bacillus isolate.
Alkaliphilic Bacillus sp. strain KSM-P103 was found to exoproduce a high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2). The gene for this enzyme from the alkaliphile was cloned and sequenced for the first time. The structural gene contained a 1,038-bp open reading frame encoding 345 amino acids. The deduced amino acid sequence of the mature enzyme (302 amino acids, 33,312 Da), designated Pel-103, showed very low similarity to those of known pectate lyases with 28-36% identity: the loop regions were very short and the amino acid usage in the parallel beta-helix core structure was considerably different. Moreover, physicochemical and catalytic properties of Pel-103 were different from those of other enzymes reported so far. Pel-103 was a very basic protein with an isoelectric point close to pH 10.5 and had optimal activity at 60-65 degrees C and at pH as high as 10.5. However, Pel-103 appeared to have a similar core and active site topology to the enzymes of known structure from Erwinia chrysanthemi and Bacillus subtilis. Expression of the gene for Pel-103 in B. subtilis resulted in high pectate lyase activity in the culture broth, concomitant with the appearance of a main protein band on an SDS gel at 33 kDa.
Hatada
Y
Y
Tochigi Research Laboratories of Kao Corporation, Japan.
Higaki
N
N
Saito
K
K
Ogawa
A
A
Sawada
K
K
Ozawa
T
T
Hakamada
Y
Y
Kobayashi
T
T
Ito
S
S
D000468:Alkalies / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000468:Alkalies; D004269:DNA, Bacterial; D011133:Polysaccharide-Lyases; C023911:pectate lyase