scholarly journals A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain

1993 ◽  
Vol 294 (2) ◽  
pp. 349-355 ◽  
Author(s):  
L M Ferreira ◽  
T M Wood ◽  
G Williamson ◽  
C Faulds ◽  
G P Hazlewood ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Methoxycinnamic Acid ◽  
Cellulose Binding ◽  
Derivatives Of

The 5′ regions of genes xynB and xynC, coding for a xylanase and arabinofuranosidase respectively, are identical and are reiterated four times within the Pseudomonas fluorescens subsp. cellulosa genome. To isolate further copies of the reiterated xynB/C 5′ region, a genomic library of Ps. fluorescens subsp. cellulosa DNA was screened with a probe constructed from the conserved region of xynB. DNA from one phage which hybridized to the probe, but not to sequences upstream or downstream of the reiterated xynB/C locus, was subcloned into pMTL22p to construct pFG1. The recombinant plasmid expressed a protein in Escherichia coli, designated esterase XYLD, of M(r) 58,500 which bound to cellulose but not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from destarched wheat bran. The nucleotide sequence of the XYLD-encoding gene, xynD, revealed an open reading frame of 1752 bp which directed the synthesis of a protein of M(r) 60,589. The 5′ 817 bp of xynD and the amino acid sequence between residues 37 and 311 of XYLD were almost identical with the corresponding regions of xynB and xynC and their encoded proteins XYLB and XYLC. Truncated derivatives of XYLD lacking the N-terminal conserved sequence retained the capacity to hydrolyse ester linkages, but did not bind cellulose. Expression of truncated derivatives of xynD, comprising the 5′ 817 bp sequence, encoded a non-catalytic polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domain and a C-terminal catalytic domain.

scholarly journals The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains

1991 ◽  
Vol 279 (3) ◽  
pp. 793-799 ◽  
Author(s):  
L M A Ferreira ◽  
G P Hazlewood ◽  
P J Barker ◽  
H J Gilbert
Keyword(s):  
Pseudomonas Fluorescens ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Terminal Region ◽  
Crystalline Cellulose ◽  
Nucleotide Sequencing ◽  
Reading Frame ◽  
Strong Homology ◽  
Cellulose Binding

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.

scholarly journals Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes

1990 ◽  
Vol 272 (2) ◽  
pp. 369-376 ◽  
Author(s):  
L E Kellett ◽  
D M Poole ◽  
L M Ferreira ◽  
A J Durrant ◽  
G P Hazlewood ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Pseudomonas Fluorescens ◽  
Crystalline Cellulose ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Binding Domains ◽  
Cellulose Binding ◽  
Derivatives Of

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5′ end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.

scholarly journals Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action

1997 ◽  
Vol 323 (2) ◽  
pp. 547-555 ◽  
Author(s):  
Vincent A. McKIE ◽  
Gary W. BLACK ◽  
Sarah J. MILLWARD-SADLER ◽  
Geoffrey P. HAZLEWOOD ◽  
Judith I. LAURIE ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Mode Of Action ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Single Copy ◽  
Terminal Sequence ◽  
Reading Frame ◽  
Significant Release

Pseudomonas fluorescens subsp. cellulosa expressed arabinanase activity when grown on media supplemented with arabinan or arabinose. Arabinanase activity was not induced by the inclusion of other plant structural polysaccharides, and was repressed by the addition of glucose. The majority of the Pseudomonas arabinanase activity was extracellular. Screening of a genomic library of P. fluorescens subsp. cellulosa DNA constructed in Lambda ZAPII, for recombinants that hydrolysed Red-dyed arabinan, identified five arabinan-degrading plaques. Each of the phage contained the same Pseudomonas arabinanase gene, designated arbA, which was present as a single copy in the Pseudomonas genome. The nucleotide sequence of arbA revealed an open reading frame of 1041 bp encoding a protein, designated arabinanase A (ArbA), of Mr 39438. The N-terminal sequence of ArbA exhibited features typical of a prokaryotic signal peptide. Analysis of the primary structure of ArbA indicated that, unlike most Pseudomonas plant cell wall hydrolases, it did not contain linker sequences or have a modular structure, but consisted of a single catalytic domain. Sequence comparison between the Pseudomonas arabinanase and proteins in the SWISS-PROT database showed that ArbA exhibits greatest sequence identity with arabinanase A from Aspergillus niger, placing the enzyme in glycosyl hydrolase Family 43. The significance of the differing substrate specificities of enzymes in Family 43 is discussed. ArbA purifed from a recombinant strain of Escherichia coli had an Mr of 34000 and an N-terminal sequence identical to residues 32–51 of the deduced sequence of ArbA, and hydrolysed linear arabinan, carboxymethylarabinan and arabino-oligosaccharides. The enzyme displayed no activity against other plant structural polysaccharides, including branched sugar beet arabinan. ArbA produced almost exclusively arabinotriose from linear arabinan and appeared to hydrolyse arabino-oligosaccharides by successively releasing arabinotriose. ArbA and the Aspergillus arabinanase mediated a decrease in the viscosity of linear arabinan that was associated with a significant release of reducing sugar. We propose that ArbA is an arabinanase that exhibits both an endo- and an exo- mode of action.

scholarly journals The non-catalytic C-terminal region of endoglucanase E from Clostridium thermocellum contains a cellulose-binding domain

1991 ◽  
Vol 273 (2) ◽  
pp. 289-293 ◽  
Author(s):  
A J Durrant ◽  
J Hall ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Amino Acids ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Binding Domain ◽  
Full Length ◽  
Crystalline Cellulose ◽  
Cellulose Binding Domain ◽  
Beta Glucan ◽  
Cellulose Binding

Mature endoglucanase E (EGE) from Clostridium thermocellum consists of 780 amino acid residues and has an Mr of 84,016. The N-terminal 334 amino acids comprise a functional catalytic domain. Full-length EGE bound to crystalline cellulose (Avicel) but not to xylan. Bound enzyme could be eluted with distilled water. The capacity of truncated derivatives of the enzyme to bind cellulose was investigated. EGE lacking 109 C-terminal residues (EGEd) or a derivative in which residues 367-432 of the mature form of the enzyme had been deleted (EGEb), bound to Avicel, whereas EGEa and EGEc, which lack 416 and 246 C-terminal residues respectively, did not. The specific activity of EGEa, consisting of the N-terminal 364 amino acids, was 4-fold higher than that of the full-length enzyme. The truncated derivative also exhibited lower affinity for the substrate beta-glucan than the full-length enzyme. It is concluded that EGE contains a cellulose-binding domain, located between residues 432 and 671, that is distinct from the active site. The role of this substrate-binding domain is discussed.

scholarly journals A non-modular endo-β-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa

1995 ◽  
Vol 305 (3) ◽  
pp. 1005-1010 ◽  
Author(s):  
K L Braithwaite ◽  
G W Black ◽  
G P Hazlewood ◽  
B R S Ali ◽  
H J Gilbert
Keyword(s):  
Cell Wall ◽  
Pseudomonas Fluorescens ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Ph Optimum ◽  
Reading Frame ◽  
Protein Motifs

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.

scholarly journals The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel

1995 ◽  
Vol 309 (3) ◽  
pp. 749-756 ◽  
Author(s):  
J Hall ◽  
G W Black ◽  
L M A Ferreira ◽  
S J Millward-Sadler ◽  
B R S Ali ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Primary Structure ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Cellulase Activity ◽  
Full Length ◽  
Type I ◽  
Catalytic Domains ◽  
Cellulose Binding

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA, constructed in lambda ZAPII, was screened for carboxymethyl-cellulase activity. The pseudomonad insert from a recombinant phage which displayed elevated cellulase activity in comparison with other cellulase-positive clones present in the library, was excised into pBluescript SK- to generate the plasmid pC48. The nucleotide sequence of the cellulase gene, designated celE, revealed a single open reading frame of 1710 bp that encoded a polypeptide, defined as endoglucanase E (CelE), of M(r) 59663. The deduced primary structure of CelE revealed an N-terminal signal peptide followed by a 300-amino-acid sequence that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase Family 5. Adjacent to the catalytic domain was a 40-residue region that exhibited strong sequence identity to non-catalytic domains located in two other endoglucanases and a xylanase from P. fluorescens. The C-terminal 100 residues of CelE were similar to Type-I cellulose-binding domains (CBDs). The three domains of the cellulase were joined by linker sequences rich in serine residues. Analysis of the biochemical properties of full-length and truncated derivatives of CelE confirmed that the enzyme comprised an N-terminal catalytic domain and a C-terminal CBD. Analysis of purified CelE revealed that the enzyme had an M(r) of 56000 and an experimentally determined N-terminal sequence identical to residues 40-54 of the deduced primary structure of full-length CelE. The enzyme exhibited an endo mode of action in hydrolysing a range of cellulosic substrates including Avicel and acid-swollen cellulose, but did not attack xylan or any other hemicelluloses. A truncated form of the enzyme, which lacked the C-terminal CBD, displayed the same activity as full-length CelE against soluble cellulose and acid-swollen cellulose, but exhibited substantially lower activity than the full-length cellulase against Avicel. The significance of these data in relation to the role of the CBD is discussed.

scholarly journals Isolation of a cDNA Encoding a β-1,4-endoglucanase in the Root-Knot Nematode Meloidogyne incognita and Expression Analysis During Plant Parasitism

1999 ◽  
Vol 12 (7) ◽  
pp. 585-591 ◽  
Author(s):  
Marie-Noëlle Rosso ◽  
Bruno Favery ◽  
Christine Piotte ◽  
Laury Arthaud ◽  
Jan M. De Boer ◽  
...  
Keyword(s):  
Meloidogyne Incognita ◽  
Catalytic Domain ◽  
Cellulase Activity ◽  
Root Surface ◽  
Binding Domain ◽  
Cellulose Binding Domain ◽  
Root Knot Nematode ◽  
Cyst Nematodes ◽  
Adult Females ◽  
Cellulose Binding

A β-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of β-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode β-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a β-1,4-endoglucanase devoid of a cellulose-binding domain.

Characterization of aNeocallimastixpatriciarumxylanase gene and its product

1999 ◽  
Vol 45 (11) ◽  
pp. 970-974 ◽  
Author(s):  
Jin-Hao Liu ◽  
Brent L Selinger ◽  
Cheng-Fang Tsai ◽  
Kuo-Jaon Cheng
Keyword(s):  
Catalytic Domain ◽  
Xylanase Activity ◽  
Binding Domain ◽  
Glycosyl Hydrolases ◽  
Cellulose Binding Domain ◽  
Xylanase Gene ◽  
Neocallimastix Patriciarum ◽  
Cellulose Binding ◽  
Linker Domain

A xylanase gene (xynC) isolated from the anaerobic ruminal fungus Neocallimastix patriciarum was characterized. The gene consists of an N-terminal catalytic domain that exhibited homology to family 11 of glycosyl hydrolases, a C-terminal cellulose binding domain (CBD) and a putative dockerin domain in between. Each domain was linked by a short linker domain rich in proline and alanine. Deletion analysis demonstrated that the CBD was essential for optimal xylanase activity of the enzyme, while the putative dockerin domain may not be required for enzyme function.Key words: xylanase, cellulose binding domain, Neocallimastix patriciarum.

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