Overproduction and properties of the mannuronate alginate lyase AlxMB.
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 micrograms 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 AlxMA and unnicked AlxMB alginate lyases have identical alginate-degrading activities at high salt concentrations.
Malissard
M
M
Laboratoire de Biochimie Microbienne, Université Claude Bernard, Villeurbanne, France.
Chavagnat
F
F
Duez
C
C
Vacheron
M J
MJ
Guinand
M
M
Michel
G
G
Ghuysen
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase