[en] In previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 mu g per litre of culture. The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge. AlxM has now been overproduced in E. coli BL21(DE3)/pAL-Sur/pLysS. Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture. It has been purified to protein homogeneity using a one-step procedure. The nicked ALxM(A) and unnicked ALxM(B) alginate lyases have identical alginate-degrading activities at high salt concentrations.
Gacesa, Russel (1990) The structure and properties of alginate. Pseudomonas Infection and Alginates: Biochemistry, Genetics and Pathology , P. Gacesa, N.J. Russel, Chapman and Hall, London; 29-49.
Gacesa (1992) Enzymic degradation of alginates. Int. J. Biochem. 24:545-552.
Skjäk-Braek, Grasdalen, Larsen (1986) Monomer sequence and acetylation patterns in some bacterial alginates. Carbohydr. Res. 154:239-250.
Yonemoto, Tanaka, Hisano, Sakaguchi, Abe, Yamashita, Kimura, Murata (1993) Bacterial alginate lyase gene: nucleotide sequence and molecular route for generation of alginate lyase species. J. Ferment. Bioeng. 75:336-342.
Maki, Mori, Fujiyama, Kinoshita, Yoshida (1993) Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp. OS-ALG-9. J. Gen. Microbiol. 139:987-993.
Schiller, Monday, Boyd, Keen, Ohman (1993) Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning sequencing and expression in Escherichia coli. J. Bacteriol. 175:4780-4789.
Boyd, Ghosh, May, Shinabarger, Keogh, Chakrabarty (1993) Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product. Gene 131:1-8.
Malissard, Duez, Guinand, Vacheron, Michel, Marty, Joris, Thamm, Ghuysen (1993) Sequence of a gene encoding a (poly ManA) alginate lyase active on mucoid Pseudomonas aeruginosa alginate. FEMS Microbiol. Lett. 110:101-106.
Baron, Wong, Hicks, Gacesa, Willcock, McPherson (1994) Alginate lyase from Klebsiella pneumoniae, subsp aerogenes: gene cloning, sequence analysis and high-level production in Escherichia coli. Gene 143:61-66.
Brown, Preston, Ingram (1991) Cloning of alginate lyase gene (alxM) and expression in Escherichia coli. Appl. Environ. Microbiol. 57:1870-1872.
Conway, Osman, Konnan, Hoffmann, Ingram (1987) Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. J. Bacteriol. 169:949-954.
Innis, Gelfand (1990) Optimization of PCRs. A Guide to Methods and Applications , 2nd edn., M.A. Innis, D.H. Gelfand, J.J. Sminsky, T.J. White, Academic Press, San Diego, CA; 3-12.
Maniatis, Fritsh, Sambrook Molecular Cloning: A Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; 1982.
Marty, Dournes, Chabanon, Montrozier (1992) Influence of nutrient media on the chemical composition of the exopolysaccharide from mucoid and non mucoid Pseudomonas aeruginosa. FEMS Microbiol. Lett. 98:35-44.
Karkhanis, Zeltner, Jackson, Carlo (1978) A new and improved microassay to determine 2-keto-3-deoxy-octonate in lipopolysaccharides of Gram-negative bacteria. Anal. Biochem. 85:595-601.
Bradford (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.
