Cloning and sequencing of a high-alkaline pectate lyase gene from an alkaliphilic Bacillus isolate

Citation
Y. Hatada et al., Cloning and sequencing of a high-alkaline pectate lyase gene from an alkaliphilic Bacillus isolate, BIOS BIOT B, 63(6), 1999, pp. 998-1005
Citations number
48
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Agricultural Chemistry","Biochemistry & Biophysics
Journal title
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
ISSN journal
0916-8451 → ACNP
Volume
63
Issue
6
Year of publication
1999
Pages
998 - 1005
Database
ISI
SICI code
0916-8451(199906)63:6<998:CASOAH>2.0.ZU;2-Q
Abstract
Alkaliphilic Bacillus sp. strain KSM-P103 was found to exoproduce a high-al kaline pectate lyase (pectate transeliminase, EC 4.2.2.2). 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.