Alkaliphilic Bacillus sp. strain KSM-P103 was found to exoproduce a high-al
kaline pectate lyase (pectate transeliminase, EC 188.8.131.52). The gene for thi
s 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 am
ino 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 sh
ort 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 e
nzymes 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 m
ain protein band on an SDS gel at 33 kDa.