Cloning, Sequencing and Overexpression in Escherichia Coli of the Alginatelyase-encoding Aly Gene of Pseudomonas Alginovora: Identification of Three Classes of Alginate Lyases

Overview

Journal Biochem J

Specialty Biochemistry

Date 1996 Oct 15

PMID 8912697

Citations 5

Authors

F Chavagnat

C Duez

M GUINAND

P Potin

T Barbeyron

B Henrissat

J Wallach

J M Ghuysen

Affiliations

Soon will be listed here.

Abstract

A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni(2+)-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.

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