A non-modular endo-beta-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa.
Pseudomonas fluorescens subsp. cellulosa when cultured in the presence of carob galactomannan degraded the polysaccharide. To isolate gene(s) from P. fluorescens subsp. cellulosa encoding endo-beta-1,4-mannanase (mannanase) activity, a genomic library of Pseudomonas DNA, constructed in lambda ZAPII, was screened for mannanase-expressing clones using the dye-labelled substrate, azo-carob galactomannan. The nucleotide sequence of the pseudomonad insert from a mannanase-positive clone revealed a single open reading frame of 1257 bp encoding a protein of M(r) 46,938. The deduced N-terminal sequence of the putative polypeptide conformed to a typical prokaryotic signal peptide. Truncated derivatives of the mannanase, lacking 54 and 16 residues from the N- and C-terminus respectively of the mature form of the enzyme, did not exhibit catalytic activity. Inspection of the primary structure of the mannanase did not reveal any obvious linker sequences or protein motifs characteristic of the non-catalytic domains located in other Pseudomonas plant cell wall hydrolases. These data indicate that the mannanase is non-modulator, comprising a single catalytic domain. Comparison of the mannanase sequence with those in the SWISSPROT database revealed greatest sequence homology with the mannanase from Bacillus sp. Thus the Pseudomonas enzyme belongs to glycosyl hydrolase Family 26, a family containing mannanases and endoglucanases. Analysis of the substrate specificity of the mannanase showed that the enzyme hydrolysed mannan and galactomannan, but displayed little activity towards other polysaccharides located in the plant cell wall. The enzyme had a pH optimum of approx. 7.0, was resistant to proteolysis and had an M(r) of 46,000 when expressed by Escherichia coli.
Braithwaite
K L
KL
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, U.K.
Black
G W
GW
Hazlewood
G P
GP
Ali
B R
BR
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D008351:Mannans / Q000378:metabolism; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017385:Sequence Homology; D013379:Substrate Specificity
D004269:DNA, Bacterial; D008351:Mannans; D011993:Recombinant Fusion Proteins; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase