A non-modular endo-β-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.

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

Biochemical Journal ◽

10.1042/bj3230547 ◽

1997 ◽

Vol 323 (2) ◽

pp. 547-555 ◽

Cited By ~ 55

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.

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Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria

Biochemical Journal ◽

10.1042/bj3070151 ◽

1995 ◽

Vol 307 (1) ◽

pp. 151-158 ◽

Cited By ~ 102

Author(s):

C M G A Fontes ◽

G P Hazlewood ◽

E Morag ◽

J Hall ◽

B H Hirst ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Genomic Library ◽

Thermophilic Bacteria ◽

Cross Hybridization ◽

General Role ◽

Sequence Identity ◽

Cell Wall Hydrolases

A genomic library of Clostridium thermocellum DNA constructed in lambda ZAPII was screened for xylanase-expressing clones. Cross-hybridization experiments revealed a new xylanase gene isolated from the gene library, which was designated xyn Y. The encoded enzyme, xylanase Y (XYLY), displayed features characteristic of an endo-beta1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and was active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates. The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinant XYLY, expressed by Escherichia coli had a maximum Mr of 116000. The nucleotide sequence of xyn Y contained an open reading frame of 3228 bp encoding a protein of predicted Mr 120 105. The encoded enzyme contained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity. Downstream of domain A was a 351-residue xylanase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28% sequence identity with a thermostable domain of Thermoanaerobacterium saccharolyticum xylanase A. The C-terminal portion of XYLY comprised the 23-residue duplicated docking sequence found in all other C. thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, followed by a 286-residue domain which exhibited 32% sequence identity with the N-terminal region of C. thermocellum xylanase Z. The enzyme did not contain linker sequences found in other C. thermocellum plant cell wall hydrolases. Analysis of truncated forms of XYLY and hybrid proteins, comprising segments of XYLY fused to the E. coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and an adjacent thermostable domain. The C-terminal domain did not bind to cellulose or xylan. Western blot analysis using antiserum raised against XYLY showed that the xylanase was located in the cellulosome and did not appear to be extensively glycosylated. The non-catalytic domains of XYLY are discussed in relation to the general stability of thermophilic xylanases.

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Characterization of the gene celD and its encoded product 1,4-β-d-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa

Biochemical Journal ◽

10.1042/bj2850947 ◽

1992 ◽

Vol 285 (3) ◽

pp. 947-955 ◽

Cited By ~ 30

Author(s):

J E Rixon ◽

L M A Ferreira ◽

A J Durrant ◽

J I Laurie ◽

G P Hazlewood ◽

...

Keyword(s):

Pseudomonas Fluorescens ◽

Plant Cell Wall ◽

Genomic Library ◽

Cell Envelope ◽

Sequence Similarity ◽

Significant Proportion ◽

Structural Similarity ◽

Reading Frame ◽

E Coli ◽

Beta Glucosidase

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA constructed in pUC18 and expressed in Escherichia coli was screened for recombinants expressing 4-methylumbelliferyl beta-D-glucoside hydrolysing activity (MUGase). A single MUGase-positive clone was isolated. The MUGase hydrolysed cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose to glucose, by sequentially cleaving glucose residues from the non-reducing end of the cello-oligosaccharides. The Km values for cellobiose and cellohexaose hydrolysis were 1.2 mM and 28 microM respectively. The enzyme exhibited no activity against soluble or insoluble cellulose, xylan and xylobiose. Thus the MUGase is classified as a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) and is designated 1,4-beta-D-glucan glucohydrolase D (CELD). When expressed by E. coli, CELD was located in the cell-envelope fraction; a significant proportion of the native enzyme was also associated with the cell envelope when synthesized by its endogenous host. The nucleotide sequence of the gene, celD, which encodes CELD, revealed an open reading frame of 2607 bp, encoding a protein of M(r) 92,000. The deduced primary structure of CELD was confirmed by the M(r) of CELD (85,000) expressed by E. coli and P. fluorescens subsp. cellulosa, and by the experimentally determined N-terminus of the enzyme purified from E. coli, which showed identity with residues 52-67 of the celD translated sequence. The structure of the N-terminal region of full-length CELD was similar to the signal peptides of P. fluorescens subsp. cellulosa plant-cell-wall hydrolases. Deletion of the N-terminal 47 residues of CELD solubilized MUGase activity in E. coli. CELD exhibited sequence similarity with beta-glucosidase B of Clostridium thermocellum, particularly in the vicinity of the active-site aspartate residue, but did not display structural similarity with the mature forms of cellulases and xylanases expressed by P. fluorescens subsp. cellulosa.

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

Biochemical Journal ◽

10.1042/bj2790793 ◽

1991 ◽

Vol 279 (3) ◽

pp. 793-799 ◽

Cited By ~ 18

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.

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Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals

Biochemical Journal ◽

10.1042/bj3320507 ◽

1998 ◽

Vol 332 (2) ◽

pp. 507-515 ◽

Cited By ~ 353

Author(s):

Stephen C. FRY

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Hydroxyl Radicals ◽

Plant Cell Wall ◽

Oh Radicals ◽

Radical Scavengers ◽

Cell Wall Polysaccharides ◽

Ph Optimum ◽

Living Plant

Scission of plant cell wall polysaccharides in vivo has generally been assumed to be enzymic. However, in the presence of l-ascorbate, such polysaccharides are shown to undergo non-enzymic scission under physiologically relevant conditions. Scission of xyloglucan by 1 mM ascorbate had a pH optimum of 4.5, and the maximum scission rate was reached after a 10–25-min delay. Catalase prevented the scission, whereas added H2O2 (0.1–10 mM) increased the scission rate and shortened the delay. Ascorbate caused detectable xyloglucan scission above approx. 5 µM. Dehydroascorbate was much less effective. Added Cu2+ (> 0.3 µM) also increased the rate of ascorbate-induced scission; EDTA was inhibitory. The rate of scission in the absence of added metals appeared to be attributable to the traces of Cu (2.8 mg·kg-1) present in the xyloglucan. Ascorbate-induced scission of xyloglucan was inhibited by radical scavengers; their effectiveness was proportional to their rate constants for reaction with hydroxyl radicals (•OH). It is proposed that ascorbate non-enzymically reduces O2 to H2O2, and Cu2+ to Cu+, and that H2O2 and Cu+ react to form •OH, which causes oxidative scission of polysaccharide chains. Evidence is reviewed to suggest that, in the wall of a living plant cell, Cu+ and H2O2 are formed by reactions involving ascorbate and its products, dehydroascorbate and oxalate. Systems may thus be in place to produce apoplastic •OH radicals in vivo. Although •OH radicals are often regarded as detrimental, they are so short-lived that they could act as site-specific oxidants targeted to play a useful role in loosening the cell wall, e.g. during cell expansion, fruit ripening and organ abscission.

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Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from theRuminococcus flavefacienscellulosome

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14025576 ◽

2015 ◽

Vol 71 (1) ◽

pp. 45-48 ◽

Cited By ~ 1

Author(s):

Immacolata Venditto ◽

Arun Goyal ◽

Andrew Thompson ◽

Luis M. A. Ferreira ◽

Carlos M. G. A. Fontes ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Cellulolytic Bacterium ◽

Modular Architecture ◽

Carbohydrate Binding Modules ◽

Fundamental Biological Process

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connectedvialinker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium,Ruminococcus flavefaciensstrain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using theCCP4 program suite andSHELXC/D/E.

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

Biochemical Journal ◽

10.1042/bj2940349 ◽

1993 ◽

Vol 294 (2) ◽

pp. 349-355 ◽

Cited By ~ 101

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.

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Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins

Molecules ◽

10.3390/molecules26144335 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4335

Author(s):

Gerasimos Daras ◽

Dimitris Templalexis ◽

Fengoula Avgeri ◽

Dikran Tsitsekian ◽

Konstantina Karamanou ◽

...

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Economic Value ◽

Cellulose Synthase ◽

Protein Isoforms ◽

Research Progress ◽

Cellulose Synthesis ◽

Biotic Stresses

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.

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

Biochemical Journal ◽

10.1042/bj3090749 ◽

1995 ◽

Vol 309 (3) ◽

pp. 749-756 ◽

Cited By ~ 54

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.

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Characterization of Two Noncellulosomal Subunits, ArfA and BgaA, from Clostridium cellulovorans That Cooperate with the Cellulosome in Plant Cell Wall Degradation

Journal of Bacteriology ◽

10.1128/jb.184.24.6859-6865.2002 ◽

2002 ◽

Vol 184 (24) ◽

pp. 6859-6865 ◽

Cited By ~ 43

Author(s):

Akihiko Kosugi ◽

Koichiro Murashima ◽

Roy H. Doi

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Plant Cell Wall ◽

Genomic Library ◽

Amino Acid Sequences ◽

Glycosyl Hydrolases ◽

Cell Wall Degradation ◽

Clostridium Cellulovorans ◽

Molecular Weights ◽

Plant Cell Wall Degradation

ABSTRACT Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the α-l-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with α-l-arabinofuranosidase or α-l-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to α-l-arabinofuranosidase and β-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl α-l-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl α-l-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl α-l-arabinopyranoside but also p-nitrophenyl β-d-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl α-l-arabinopyranoside and p-nitrophenyl β-d-galactopyranoside were compared, the Km values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for α-l-arabinopyranose residues than for β-d-galactopyranoside residues and possessed a novel enzymatic property for a family 42 β-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with β-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.

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