A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in pUC18 and Escherichia coli recombinants expressing 4-methylumbelliferyl beta-D-cellobioside-hydrolysing activity (MUCase) were isolated. Enzyme produced by MUCase-positive clones did not hydrolyse either cellobiose or cellotriose but converted cellotetraose into cellobiose and cleaved cellopentaose and cellohexaose, producing a mixture of cellobiose and cellotriose. There was no activity against CM-cellulose, insoluble cellulose or xylan. On this basis, the enzyme is identified as an endo-acting cellodextrinase and is designated cellodextrinase C (CELC). Nucleotide sequencing of the gene (celC) which directs the synthesis of CELC revealed an open reading frame of 2153 bp, encoding a protein of Mr 80,189. The deduced primary sequence of CELC was confirmed by the Mr of purified CELC (77,000) and by the experimentally determined N-terminus of the enzyme which was identical with residues 38-47 of the translated sequence. The N-terminal region of CELC showed strong homology with endoglucanase, xylanases and an arabinofuranosidase of Ps. fluorescens subsp. cellulosa; homologous sequences included highly conserved serine-rich regions. Full-length CELC bound tightly to crystalline cellulose. Truncated forms of celC from which the DNA sequence encoding the conserved domain had been deleted, directed the synthesis of a functional cellodextrinase that did not bind to crystalline cellulose. This is consistent with the N-terminal region of CELC comprising a non-catalytic cellulose-binding domain which is distinct from the catalytic domain. The role of the cellulose-binding region is discussed.
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November 1991
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Research Article|
November 01 1991
The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains
L M A Ferreira
;
L M A Ferreira
*Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
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G P Hazlewood
;
G P Hazlewood
†Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.
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P J Barker
;
P J Barker
†Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.
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H J Gilbert
H J Gilbert
*Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K.
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Biochem J (1991) 279 (3): 793–799.
Citation
L M A Ferreira, G P Hazlewood, P J Barker, H J Gilbert; The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains. Biochem J 1 November 1991; 279 (3): 793–799. doi: https://doi.org/10.1042/bj2790793
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