BBB : Vol. 63 (1999) , No. 6 pp.998-1005

Cloning and Sequencing of a High-alkaline Pectate Lyase Gene from an Alkaliphilic Bacillus Isolate

Yuji HATADA¹⁾, Norihiko HIGAKI¹⁾, Kazuhiro SAITO¹⁾, Akinori OGAWA¹⁾, Kazuhisa SAWADA¹⁾, Tadahiro OZAWA¹⁾, Yoshihiro HAKAMADA¹⁾, Tohru KOBAYASHI¹⁾ and Susumu ITO¹⁾

1) Tochigi Research Laboratories of Kao Corporation

(Received December 7, 1998)
(Accepted February 12, 1999)

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 β-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°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.

Key words:

alkaliphile; Bacillus; trans-elimination; PCR; amino acid sequence

	To cite this article:
	Yuji HATADA, Norihiko HIGAKI, Kazuhiro SAITO, Akinori OGAWA, Kazuhisa SAWADA, Tadahiro OZAWA, Yoshihiro HAKAMADA, Tohru KOBAYASHI and Susumu ITO, “Cloning and Sequencing of a High-alkaline Pectate Lyase Gene from an Alkaliphilic Bacillus Isolate”, Biosci. Biotechnol. Biochem., Vol. 63, 998-1005 (1999) .

	doi:10.1271/bbb.63.998
	JOI JST.JSTAGE/bbb/63.998