APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

June 1999, p. 2598–2605 Vol. 65, No. 6

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Mannan-Degrading Enzymes from Cellulomonas fimiDOMINIK STOLL, HENRIK STÅLBRAND,† AND R. ANTONY J. WARREN*

Department of Microbiology and Immunology and The Protein Engineering Network of Centres of Excellence,The University of British Columbia, Vancouver, British Columbia, Canada

Received 15 December 1998/Accepted 22 February 1999

The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and b-mannosidase2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising acatalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknownfunction. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments ofthe enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth onmannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of theMan26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalyticmodule in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalianb-mannosidases.

Members of the genus Cellulomonas are common in envi-ronments rich in decaying plant material. They degrade cellu-lose, xylans, glucans, mannans, chitin, and starch. Extracellularenzymes hydrolyzing some of these polysaccharides have beendescribed from a number of strains. As with other polysaccha-ride-degrading microorganisms, purification of an enzyme iscomplicated by the production of complex systems of enzymeswith similar activities. Consequently, cloning of the genes en-coding the enzymes and expression of the genes in heterolo-gous hosts are used extensively to obtain individual enzymes(4). Several cellulases and xylanases from Cellulomonas fimiwere characterized in this manner (7, 10, 12, 39, 40, 46, 54, 64),but mannan-degrading enzymes have not been described.

Mannans are linear polymers of mannose, or of mannoseand glucose, and are linked b-1,4. The principal hemicellulosesin soft woods, accounting for up to 25% of the dry weight, areO-acetylgalactoglucomannans in which the backbone com-prises mannose and glucose in the ratio 3:1; the glucose resi-dues may be distributed randomly (49, 61). Galactose mono-mers are linked a-1,6 to some of the mannose residues (61);some 2- and 3-hydroxyls of the mannose residues and, to alesser extent, of the glucose residues, are acetylated (49). Insome green algae, the crystalline structural component of thecell wall is mannan, not cellulose. Mannans function as storagecarbohydrates in the bulbs and endosperm of some plants:ivory nut mannan (INM), from Phytelphus macrocarpa, is aninsoluble crystalline material comprising only mannose, with abackbone conformation very similar to that of cellulose; thebackbone of locust bean gum (LBG), from Ceratonia siliqua,also comprises only mannose, with galactose monomers linkedto it randomly by a-1,6 bonds. The hydrolysis of O-acetylga-lactomannan into its monomeric components requires endo-b-mannanase, b-mannosidase, b-glucosidase, a-galactosidase,and acetyl esterase (49). The hydrolysis of INM requires onlyendo-b-mannanase and b-mannosidase; hydrolysis of LBG re-quires a-galactosidase in addition to these two enzymes.

b-Mannanases (mannan endo-1,4-b-mannosidase; EC3.2.1.78) are produced by plants, fungi, and bacteria. Theycatalyze the random hydrolysis of b-1,4 mannosidic linkageswithin the backbones of mannans, galactomannans, and gluco-mannans. Well-characterized enzymes include Man1 fromTrichoderma reesei (25, 56), ManA from Streptomyces lividans(2), ManA from Pseudomonas fluorescens subsp. cellulosa (8,9), and an enzyme from Aspergillus niger (38). The amino acidsequences of several enzymes, deduced from the sequences ofthe genes encoding them (2, 9, 41, 42, 55), place them infamilies 5 and 26 of the glycosyl hydrolases (26–28), both ofwhich are in clan GH-A of retaining enzymes (8, 29). Thecrystal structure of a mannanase from family 5 has been de-termined (31). A particular enzyme may comprise one or moremodules (9, 21, 42, 55).

b-Mannosidases (b-1,4-mannoside mannohydrolase; EC3.2.1.25) catalyze the removal of b-D-mannose residues fromthe nonreducing ends of oligosaccharides. Some eucaryoticb-mannosidases, in family 2 of glycosyl hydrolases, process theN-linked oligosaccharides of glycoproteins (1, 11, 15, 35). A.niger, Aspergillus awamori, and T. reesei fungi secrete b-man-nosidases that act preferentially on shorter manno-oligosac-charides (43). An enzyme from the archaeon Pyrococcus furio-sus, the only procaryotic b-mannosidase sequenced to date, isin family 1 of the glycosyl hydrolases (3).

a-Galactosidases (melibiase or a-D-galactoside galactohy-drolase; EC 3.2.1.22) remove a-1,6-linked galactose residuesfrom galactomannan polymers. They are in families 27 and 36of the glycosyl hydrolases (28). Bacterial enzymes are intracel-lular (20, 65) or extracellular (37, 38, 58).

This study describes the cloning and sequencing of two genesfrom C. fimi that encode a b-mannanase and a b-mannosidase,their expression in Escherichia coli, and the preliminary char-acterization of the encoded enzymes. The enzymes belong tofamilies 26 and 2, respectively, of the glycosyl hydrolases. Theb-mannanase has an unusual modular structure.

MATERIALS AND METHODS

Materials. Restriction endonucleases were from New England Biolabs (Mis-sissauga, Ontario, Canada), Gibco-BRL (Burlington, Ontario, Canada), orBoehringer Mannheim (Laval, Quebec, Canada). T4 DNA ligase was fromGibco-BRL. Vent DNA polymerase was from New England Biolabs. PWO andHiFi DNA polymerases were from Boehringer Mannheim. Isopropyl-b-D-thio-galactopyranoside (IPTG), 4-methylumbelliferyl-b-D-mannoside (MUbMan),

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of British Columbia, 300-6174 Uni-versity Blvd., Vancouver, BC, Canada V6T 1Z3. Phone: (604) 822-2376. Fax: (604) 822-6041. E-mail: rajw@unixg.ubc.ca.

† Present address: Department of Biochemistry, University of Lund,Lund, Sweden.

2598

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

4-methylumbelliferyl-a-D-galactoside (MUaGal), 5-bromo-4-chloro-3-indolyl-b-D-glucoside (X-Gluc), p-nitrophenyl glycosides (other than that of manno-biose), LBG (purity not specified), and carboxymethyl cellulose (CM-cellulose;sodium salt, low viscosity; nominal degree of polymerization, 400; nominal de-gree of substitution, 0.7) were from Sigma (St. Louis, Mo.). INM (purity, .98%)and azo-carob galactomannan (ACG; low viscosity, purity, .98%) were fromMegazyme (North Rocks, New South Wales, Australia). p-Nitrophenyl-b-man-nobioside (PNPM2) was synthesized by enzymatic transglycosylation (unpub-lished data). Birchwood xylan was from Carl Roth KG (Karlsruhe, Germany).Zeocin was from Invitrogen (San Diego, Calif.). Mouse anti-His6 serum was fromDianova (Hamburg, Germany). Rabbit anti-mouse immunoglobulin G serumconjugated to horseradish peroxidase was from Dako Diagnostics (Carpinteria,Calif.).

Bacterial strains, plasmids, and bacteriophages. The E. coli strains used wereJM101 (51), DH5a (51), BL21(DE3) (24), and XLOLR (Stratagene). The C.fimi strain used was ATCC 484. The plasmids used were pZErO (Invitrogen),pBluescript SK and KS (Stratagene) and pET27b and pET28a (Novagen). Thebacteriophages used were lZAPII and ExAssist (Stratagene). Bacterial stockswere maintained at 270°C in Luria-Bertani (LB) medium containing 15% glyc-erol. Plasmid DNA was stored in water at 220°C. Bacteriophage lambda wasstored at 4°C or 270°C in TYP medium.

Media and growth conditions. E. coli strains were grown routinely in LBmedium (51) at 37°C or in TYP medium (51) at room temperature for proteinproduction. Media were supplemented with 50 mg of kanamycin or 100 mg ofampicillin ml21, depending on the plasmid-encoded resistance. Strains carryingpZErO and derivatives thereof were grown in low-salt LB (51) supplementedwith 50 mg of Zeocin ml21. C. fimi was grown at 30°C in basal salts medium (57)supplemented with 0.2% (wt/vol) carbon source and 50 mg of kanamycin ml21.Liquid cultures were shaken at 220 rpm. Solid media contained 1.5% agar(Difco).

DNA manipulations. All oligodeoxynucleotide primers were synthesized bythe Nucleic Acid and Protein Service (NAPS) Unit of the University of BritishColumbia by using an Applied Biosystems model 380A DNA synthesizer andwere purified by extraction with n-butanol (53). DNA was sequenced by theNAPS Unit by using the AmpliTaq dye termination cycle sequencing protocolwith the addition of 5% dimethyl sulfoxide (DMSO) and an Applied Biosystemsmodel 377 sequencer. C. fimi DNA was amplified by PCR. Reaction mixturescontained 10 to 100 ng of template, 40 pmol of each primer, the recommendedpolymerase buffer, 10% DMSO, 0.2 mM 29-deoxynucleoside 59-triphosphates,and 1 U of Vent, PWO, or HiFi polymerase in a final volume of 100 ml.Incubation was for 30 cycles of 94°C for 30 s, 64 to 67°C for 30 s, and 72°C for45 to 90 s, depending on the reaction. DNA was manipulated routinely asdescribed previously (51). Restriction endonucleases were used as recommendedby the suppliers. DNA fragments were separated by agarose gel electrophoresis(51); they were then recovered from the gels and purified by using the Qiaex IIGel Extraction Kit (Qiagen, Santa Clarita, Calif.). Ligation reaction mixturescontained 100 to 500 ng of total DNA at an insert-to-vector molar ratio of 10, 1U of T4 DNA ligase, and the recommended buffer in a final volume of 100 ml.Mixtures were incubated at 23°C for 2 h and then desalted by butanol precipi-tation (62). E. coli cells were transformed either by electroporation (GenePulser;Bio-Rad) or by heat shock of cells prepared by the quick chemical competent cellprotocol in TSS buffer (51).

Lambda library screening. The agar in petri dishes (150 mm in diameter)containing 30 ml of NZY medium (51) was overlaid with 10 ml of NZY mediumcontaining 0.4% ACG and 1.5% agar. After solidification, the plates were driedfor several hours before they were overlaid with a mixture of E. coli cells and anappropriate dilution of a C. fimi l ZAPII library (39, 40) in 3.5 ml of NZYmedium with 0.1 to 0.5 mM IPTG and 0.7% agar. Mannanase-positive clonesproduced haloes of hydrolysis after incubation at 37°C for 16 to 24 h (9). Aftersecondary and tertiary screening, mannanase-positive phagemids (pBluescriptSK) containing genomic DNA inserts were excised in vivo from lambda DNA,recircularized, and transduced into E. coli XLOLR cells (54). The excised formof the entire genomic l ZAPII library was screened for E. coli clones producingb-mannosidase by plating appropriate dilutions on NZY plates and then, afterincubation at 37°C overnight, replicating the colonies onto NZY plates contain-ing 0.5 mM MUbMan and 0.3 mM IPTG. Positive clones fluoresced under UVlight (63).

Detection of enzyme activity. Mannanase-positive E. coli clones were detectedby plating on LB containing 0.2% ACG (9). Zones of hydrolysis were visualizedafter incubation at 37°C for 12 to 16 h. b-Mannanase or a-galactosidase activitywas detected on plates containing 0.1 to 0.5 mM MUbMan or MUaGal, respec-tively. After incubation, the hydrolysis product was detected under UV light.Mannanases in culture supernatants or cell extracts were screened by zymogramsby using nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gels incor-porating 0.5% ACG (9). b-Mannosidase, a-galactosidase, and b-glucosidase inextracts were screened by incubating nonreducing SDS-polyacrylamide gels afterelectrophoresis in 1 mM MUbMan, 1 mM MUaGal, or 0.5 mM X-Gluc, respec-tively (33, 63). Mannanase activity was screened during purification by spotting10- to 20-ml samples onto plates containing 2.0% agar and 0.5% LBG in 50 mMphosphate buffer (pH 7.0) (56); after incubation for 12 to 16 h at 37°C, hydrolysiswas detected by staining the plates with Congo red (60).

Location of mannanase activity in C. fimi. A liquid culture of C. fimi was grownfor 6 days in minimal medium containing 0.2% LBG. One hour before harvest-ing, chloramphenicol was added (40 mg ml21) to stop further protein synthesis.Mannanase activity was measured in samples of the whole culture, the culturesupernatant, and the cells. The cells were recovered by centrifugation, washedthree times with 50 mM phosphate buffer (pH 7.0), and resuspended to thestarting volume in the same buffer. Activities were determined by incubating210-ml samples of each preparation with 75 ml of 2% ACG, 5 ml of 10% sodiumazide, and 6 ml of chloramphenicol (20 mg ml21) for 16 h at 37°C. Reactions werestopped by adding 750 ml of 95% ethyl alcohol. The precipitated ACG wasremoved by centrifugation for 2 min at 13,000 rpm. The release of solubleazo-manno-oligosaccharides was determined from the A590 of the supernatant.The results were normalized to the activity in the whole culture being 100%.

Production and purification of enzymes. Cloned genes were expressed frompET vectors in E. coli BL21(DE3) cells. Cultures, shaken at room temperature,were induced with 0.2 to 0.4 mM IPTG in mid-exponential phase and thenincubated for a further 24 to 36 h. The cultures were centrifuged at 5,000 rpmand 4°C for 10 min. Mannanase or mannosidase was purified from the cells asfollows. The cells were resuspended in binding buffer (5 mM imidazole, 500 mMNaCl, 20 mM Tris-HCl; pH 7.9) and ruptured by passage three times through aFrench pressure cell. Debris and unbroken cells were removed by centrifugationat 15,000 rpm and 4°C for 30 min. The supernatant was passed through a 5- to10-ml His-Bind metal chelating affinity column at a flow rate of 0.5 ml min21.Mannanase was also purified from the supernatant of the E. coli culture. Thesupernatant was concentrated to 50 ml and exchanged into binding buffer byusing an Ultrasette tangential flow concentrator with a 1-kDa cutoff (Filtron).The concentrated solution was passed through a 15-ml His-Bind column at a flowrate of 0.5 ml min21. In all cases, the His-Bind columns were washed with 3 to6 volumes of binding buffer at a flow rate of 1 ml min21, after which adsorbedproteins were eluted by stepwise increases in the concentration of imidazole inthe buffer from 5 to 500 mM. The protein content was screened by measuring theA280. Fractions of 5 ml were collected. Protein-containing fractions werescreened for enzyme activity. The enzymes were eluted, usually with 50 to 120mM imidazole. Enzyme-containing fractions were pooled and then concentrated,and the buffer was exchanged by diafiltration through an Amicon PM10 mem-brane. Purity was evaluated by SDS-polyacrylamide gel electrophoresis (PAGE)(34). b-Mannosidase was partially purified from cells of C. fimi as follows. C. fimiwas grown overnight at 30°C in 2 liters of basal salts medium with 0.2% glucose.The cells were recovered by centrifugation for 10 min at 5,000 rpm and 4°C,resuspended in 50 ml of buffer, and ruptured by four passages through a Frenchpressure cell. Debris and unbroken cells were removed by centrifugation at 4,000rpm and 4°C for 1 h. The buffer in the supernatant was changed to 20 mMTris-HCl (pH 5.8) by diafiltration. The extract was passed through an EconoQ(Bio-Rad) column. Adsorbed proteins were eluted with 20 column volumes of alinear gradient of 0 to 700 mM NaCl in the same buffer. Fractions of 5 ml werecollected and screened for activity. The fractions with the highest activity werepooled and concentrated as described above.

Analysis of proteins. The concentrations of proteins were determined bymeasuring the A280 (47). N-terminal amino acid sequences of proteins or frag-ments thereof were determined after separation by SDS-PAGE, electroblottingto polyvinylidene membranes, and excision of the appropriate bands after visu-alization by staining with Coomassie blue. Automated Edman sequencing wasdone by the NAPS Unit at the University of British Columbia or at the ProteinMicrochemistry Facility, University of Victoria, British Columbia, Canada, byusing Applied Biosystems 476A gas-phase sequenators. The molecular weights ofproteins were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Protein samples (0.5 to 3.0 mg ml21) were desalted bydepositing 5- to 10-ml drops onto dialysis membranes (Millipore MF; 0.025-mmpore size) floating on distilled water. After 12 to 16 h, 1 ml of dialyzed sample wastransferred to the sample holder and dried for 5 min. The sample was overlaidwith 1 ml of matrix (supersaturated sinapinic acid solution in 70% acetonitrileand 0.1% trifluoroacetate) and dried for 5 min (32). Spectra were obtained witha Mass Phoresis instrument (Ciphergen, Inc.).

RESULTS

Galactomannan-degrading enzymes produced by C. fimi.After growth of C. fimi for 6 days in minimal medium withLBG as a carbon source, most of the mannanase activity was inthe culture supernatant; there was a low level of activity in acell extract. Supernatants from cultures grown with differentcarbon sources were screened for mannanases by zymogramswith ACG as substrate. There were three proteins with endo-mannanase activity when LBG was the carbon source: two majorproteins of 75 and 30 kDa and a minor protein of 100 kDa (Fig.1). The N-terminal sequence of the mannanase of 75 kDa wasAPADET. The production of all three proteins was reducedwhen glucose and LBG were added together as carbon sources.

VOL. 65, 1999 MANNAN-DEGRADING ENZYMES FROM C. FIMI 2599

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

Only the 30-kDa protein was produced with CM-cellulose as acarbon source. Mannanases were not produced with mannose,galactose, xylan, or glycerol as a carbon source.

b-Mannosidase and a-galactosidase activities were presentonly in the cell extract; zymograms revealed single proteins of60 and 120 kDa, respectively. The mannosidase comigratedwith a b-glucosidase (63), so it was partially purified. Its N-terminal amino acid sequence was MITQDLYD. Zymogramsof cell extracts showed that b-mannosidase was produced onlyin the presence of mannan or mannose.

Screening of a genomic library of C. fimi DNA for mannan-ase genes and sequencing of a mannanase gene. A library of C.fimi genomic DNA was prepared previously in l ZAPII (39).The library was screened for mannanase-positive clones onACG plates with IPTG. Eight positive plaques were obtained.Phagemids (pBluescript with C. fimi DNA inserts) were ex-cised from the clones and transferred to E. coli XLOLR. Thestrains were designated CMan1 to CMan8; all of them accu-mulated in the culture medium at least two proteins with man-nanase activity and of similar sizes, the largest being approxi-mately 100 kDa. The clones producing the most activity,CMan2 and CMan4, were analyzed further. They carried plas-mids, pCMan2 and pCMan4, with inserts of 4.3 and 6.3 kbp,respectively. Restriction mapping and partial sequencingshowed that the inserts had a DNA sequence in common.

Compared with that in pCMan2, the insert in pCMan4 had anextra 65 bp at the 59 end, after which the sequences were thesame. As it appeared that the two inserts encoded the samemannanase, the insert in pCMan2 was sequenced in its en-tirety.

A putative translational start codon was not present in thesequence of the insert in pCMan2. The similarity of the man-nanase profiles in strains CMan1 to CMan8 led to the librarybeing screened for further mannanase-positive clones. Fivefurther clones were treated as described above and screened byzymograms. CMan30 produced two mannanases similar in sizeto those produced by C. fimi. It carried a plasmid with an insertof 3.0 kbp, the restriction pattern for which showed that it alsocontained the sequence common to pCMan2 and pCMan4.Sequencing of the 59 end of the insert in pCMan30 revealed anATG start codon 540 bp downstream of its 59 end and 6 and 72bp, respectively, upstream of the 59 ends of the inserts inpCMan4 and pCMan2, thus completing the sequence of aputative mannanase gene (GenBank accession numberAF126471).

The open reading frame covered by the inserts in pCMan2,pCMan4, and pCMan30 was 3,033 bp long, encoding apolypeptide of 1,011 amino acids. The molecular weight of thepolypeptide, calculated from the deduced amino acid se-quence, was 107,033. The N-terminal amino acid sequence wascharacteristic of a signal peptide, with the cleavage site pre-dicted to be between Ala40 and Ala41 in the sequence37PAPA2APV43 (44). This finding agreed with the consensusleader peptide cleavage sequence A/VXA2A derived fromother extracellular enzymes from C. fimi (12, 39, 40, 46, 54, 64).Cleavage at this point would give a mature polypeptide ofabout 103 kDa, similar to that of the largest polypeptide withmannanase activity (;100 kDa) detected in supernatants fromC. fimi cultures. Amino acids 50 to 55 in the deduced sequencewere APADET, matching the N-terminal sequence of themannanase of 75 kDa in the supernatants. Ala50 in theMan26A sequence is designated residue 1 in the maturepolypeptide, which is 961 amino acids long.

Analysis of the deduced amino acid sequence of the man-nanase. Alignment of the deduced amino acid sequence of themannanase with those of proteins in the database revealed amodular structure. The N-terminal part of the polypeptideshared sequence identity with the catalytic modules of enzymesin family 26 of the retaining glycosyl hydrolases, which containsmannanases and glucanases. Therefore, in keeping with therecently proposed nomenclature for enzymes hydrolyzing thepolysaccharides in the cell walls of plants (30), the enzymefrom C. fimi is designated Man26A. Man26A shares the great-est identity with mannanase ManA from P. fluorescens subsp.cellulosa, a single module polypeptide (9): amino acids 1 to 398of Man26A are 40% identical to amino acids 3 to 383 ofmature ManA (Fig. 2). Thus, the catalytic module of Man26Acomprises at least residues 1 to 398, based on the alignmentwith ManA. Glu174 and Glu282 in mature ManA are thecatalytic carboxylic amino acids (8). These residues are strictlyconserved in the enzymes of family 26; the corresponding res-idues in mature Man26A are Glu176 and Glu283 (Fig. 2).

Amino acids 637 to 830 of mature Man26A share 23 to 30%sequence identity with S-layer homology (SLH) domains,which typically comprise three repeats of about 60 amino acidseach (45). There are three repeats of about 60 amino acidseach that are 20% identical in this part of Man26A, coveringresidues 647 to 826 (Fig. 3). None of the extracellular proteinsfrom C. fimi described previously contain SLH modules. Suchmodules usually anchor the proteins containing them to thebacterial cell wall. They are present in some xylanases and

FIG. 1. Mannanases from C. fimi analyzed by nonreducing SDS-PAGE usingzymograms of culture supernatants. The cultures were grown in minimal mediumwith no addition (lane 1) or supplemented (0.2%) with LBG (lane 2), mannose(lane 3), glycerol (lane 4), glucose and LBG (lane 5), xylan (lane 6), CM-cellulose(lane 7), or galactose (lane 8). Prestained molecular weight standards are shownin lane M. The cultures were incubated for 2 days (A), 6 days (B), and 11 days(C).

2600 STOLL ET AL. APPL. ENVIRON. MICROBIOL.

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

pullulanases and in cellulosome-anchoring proteins (19, 36,50).

Amino acids 450 to 636 and 831 to 961 of Man26A do notshare sequence identity with any proteins in the database. Theymay comprise functional modules.

Such a multimodule structure is common in the extracellularenzymes produced by C. fimi (10).

Production of Man26A in E. coli. The man26A gene wassubcloned into the expression vector pET27b as a fusion en-coding Man26A with a C-terminal H6 tag. The plasmid,pET27Man26A, was transformed into E. coli BL21(DE3). Thisstrain yielded about 70 mg of active Man26A liter21 afterpurification (see Materials and Methods), with ca. 10 mg re-covered from the culture supernatant and ca. 60 mg from thecell extract.

Properties of Man26A. The Man26A produced by E. coli was;100 kDa (Fig. 4); its N-terminal amino acid sequence wasAPAPAAP, corresponding to residues 214 to 28 relative tothe N terminus of the enzyme produced by C. fimi. This meanseither that E. coli and C. fimi use different leader peptideprocessing sites in proMan26A or that they use the same siteand C. fimi removes further amino acids from the N terminus.Man26A hydrolyzed PNPM2 very slowly; prolonged incubation

was required to detect activity. The hydrolysis of LBG wasassayed at concentrations from 0.01 to 4.5 mg ml21. Higherconcentrations were not tested because of the viscosity. Anapparent kcat/Km of 1,150 ml mg21 min21 was calculated fromthe initial slope of a plot of v versus [S]. The pH and temper-ature optima were 5.5 and 42°C, respectively. The enzyme wasstable for .2 h at this temperature.

Proteolysis of Man26A. C. fimi secretes a protease(s) thatreleases discrete fragments, corresponding to individual mod-ules, from other polysaccharide hydrolases secreted by theorganism (22, 23, 52). When Man26A produced in E. coli wasdigested with the protease (23), several discrete fragmentswere released, two of which were resistant to further hydrolysis(Fig. 4). The largest stable fragment, which arose by hydrolysisof a fragment of 55 to 60 kDa, had the N-terminal amino acidsequence AGALP and a mass of 50 kDa, making it about 460amino acids long. Therefore, it comprised the catalytic moduleplus 50 to 60 amino acids at the C terminus. The sequenceAGALP starts nine residues after the N terminus of Man26Aproduced by E. coli and six residues before the N terminus of

FIG. 4. Digestion of Man26A, produced in E. coli, with protease from C.fimi. Man26A was digested with protease (1 mg of protease and 60 mg ofMan26A) at 37°C. Samples were removed at intervals and boiled in loadingbuffer for 2 min to stop digestion. Digestion times were 0, 15, 30, 45, 60, and 90min (lanes 1 to 6) and 2, 3, 5, 7, 16, and 24 h (lanes 7 to 12). Molecular weightstandards are in lanes M1 and M2. The N termini of the polypeptides markedwith an asterisk were sequenced.

FIG. 2. Alignment of the first 398 amino acids from C. fimi Man26A (Cf Man26A; GenBank AF126471) with the entire sequence of ManA from P. fluorescens subsp.cellulosa (Pf ManA; GenBank X82179). Asterisks indicate identical residues. The catalytic glutamates are in boldface.

FIG. 3. Alignment of the SLH domain repeats of Man26A with the SLHrepeats from bacterial proteins. The numbers indicate the positions of the firstamino acid residue of each sequence in the different proteins. Cf, C. fimiMan26A; Ta, S-layer protein from Thermus aquaticus (GenBank A47024); Ba,S-layer protein from Bacillus anthracis (GenBank X99724); Ct, cellulosome-anchoring protein from Clostridium thermocellum (GenBank X67506); Tm, outermembrane protein from Thermotoga maritima (GenBank X68276). Asterisksindicate residues that are identical or conserved in all sequences.

VOL. 65, 1999 MANNAN-DEGRADING ENZYMES FROM C. FIMI 2601

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

the mature protein produced by C. fimi. The other stablefragment had the N-terminal amino acid sequence AHPGVE,corresponding to residues 637 to 642 of the mature protein,just before the first SLH repeat starting at residue 647; its masswas 21 kDa, making it about 190 amino acids long. This stablefragment arose by trimming of the N and C termini of afragment of about 28 kDa with the N-terminal amino acidsequence VNSAE, corresponding to residues 618 to 622 of themature protein.

The nature of these fragments supported the modular struc-ture proposed for Man26A. As with other polysaccharide hy-drolases produced by C. fimi, the initial sites of proteolysiswere between the modules. Besides cutting Man26A from E.coli between the modules, the protease from C. fimi alsotrimmed the N terminus of the catalytic module.

When supernatants from cultures of C. fimi grown withINM, LBG, CM-cellulose, or LBG and CM-cellulose werescreened by zymogram after nondenaturing gel electrophore-sis, the patterns of active bands were identical except for thatfor the culture grown with CM-cellulose. Furthermore, thesame patterns were obtained for Man26A produced in E. coliand digested either with C. fimi protease or with the superna-tant from the culture of C. fimi grown with LBG (Fig. 5). Thissuggests that Man26A is the only mannanase produced by C.fimi and that all of the multiple bands seen on the zymograms(Fig. 1) arise by proteolysis of this 100-kDa protein in thecultures.

Location of mannanase activity. After growth of a culture ofC. fimi for 6 days in the presence of LBG, the relative levels ofmannanase activity in the whole culture, associated with thecells, and in the culture supernatant were 100, 80, and 55%,respectively. Thus, a significant fraction of the activity ap-peared to be cell associated, possibly through binding to thesurface by the SLH domain. The discrepancy between theactivity in the culture and the sum of the activities associatedwith the cells and in the culture supernatant could have beencaused by residual LBG in the culture interfering with hydro-lysis of the test substrate, ACG. Any residual LBG would havebeen separated from the cells by centrifugation. Man26A pro-duced in E. coli did not bind to a peptidoglycan fraction pre-pared from C. fimi.

Isolation and sequencing of a mannosidase gene. Mannosi-dase-positive plaques were not obtained by direct screening ofthe l ZAPII library with MUbMan. Therefore, the library wasrescreened in its excised form as E. coli colonies carrying

pBluescript phagemids with inserts of C. fimi DNA. Two pos-itive clones were obtained. The plasmids they carried weredesignated pCManI and pCManII and contained fragments ofC. fimi DNA of 2.6 and 6.5 kbp, respectively. Restriction map-ping and partial sequencing showed them to have a DNAfragment in common. The 59 end of the insert in pCManI was150 bp downstream of the 59 end of that in pCManII. There-fore, the insert in pCManI was sequenced. It contained anopen reading frame of 2,526 bases (GenBank accession num-ber AF126472), encoding a protein of 842 amino acids with acalculated molecular weight of 94,960.

Analysis of the deduced amino acid sequence of the manno-sidase. The N-terminal amino acid sequence deduced for themannosidase matched that for the enzyme partially purifiedfrom C. fimi. Signature consensus sequences are defined foreach family of glycosyl hydrolases to reduce the ambiguity inthe classification of newly reported enzymes. The mammalianmannosidases are in family 2 of the glycosyl hydrolases, buttheir alignments with the consensus sequences for this familysuggest that they form a subfamily within it. Alignment of itsamino acid sequence placed the enzyme from C. fimi in thissubfamily (Fig. 6); it was designated Man2A (30). At present,it is the only procaryotic enzyme in the subfamily. The con-served catalytic carboxyls in family 2 are glutamates. InMan2A, Glu429 and Glu519 are predicted to be the acid-basecatalyst and the nucleophile, respectively (Fig. 6). UnlikeMan26A, Man2A comprises only a catalytic module.

Production of Man2A in E. coli. The man2A gene was sub-cloned into the expression vector pET28A(1), yielding theplasmid pET28Man. The protein encoded by this plasmid car-ried a C-terminal H6 tag for purification. Cell extracts from E.coli BL21(D3) carrying pET28Man yielded up to 300 mg ofpurified Man2A liter21 of culture. The purity was estimated tobe .95% by SDS-PAGE. The N-terminal amino acid se-quence of the enzyme was MITQDLYDG, matching that ofthe enzyme produced in C. fimi.

Properties of Man2A. Man2A hydrolyzed p-nitrophenyl-b-mannoside (PNPM) readily. It had low activity on p-nitrophe-nyl-b-galactoside. It did not hydrolyze p-nitrophenyl derivatives ofa-mannose, b-N-acetylglucosamine, b-glucose, b-xylose, b-cel-lobiose, or b-gentiobiose. The optimum pH for the hydrolysisof PNPM was 7.0. Although the rate of hydrolysis of PNPMwas fastest at 55°C, the enzyme was unstable at this tempera-ture. The half-life of the enzyme was 27 h at 37°C. Man2A wasinhibited by PNPM at concentrations .400 mM (data notshown), so the values of 167 s21 for kcat, 0.3 mM for Km, and500 s21 z mM21 for kcat/Km were only estimates.

Size exclusion chromatography of Man2A gave a mass of103 kDa, indicating that the native enzyme was a monomer.

DISCUSSION

All of the extracellular glycosyl hydrolases produced by C.fimi, including Man26A, are modular proteins, comprising acatalytic module and at least one other module. Man26A is theonly one with an SLH module. It appears to contain two othermodules, comprising amino acids 450 to 636 and 831 to 961,that are unrelated to modules in the database and are ofunknown function. The mannanases produced by other micro-organisms vary in complexity; they range in length from 306 (2)to 1,332 (42) amino acids. Mannanases from S. lividans and P.fluorescens subsp. cellulosa comprise catalytic modules only,from families 5 and 26, respectively, of the glycosyl hydrolases(2, 9). A mannanase from Agaricus bispora comprises a cata-lytic module from family 5 connected to an N-terminal cellu-lose-binding module from family II by a proline-rich linker

FIG. 5. Origin of the multiple mannanases from C. fimi analyzed by usingnonreducing SDS-PAGE zymograms. Cultures of C. fimi were incubated for 11days at 30°C in minimal medium supplemented (0.2%) with INM (lane 1), LBG(lane 2), CM-cellulose (lane 3), or LBG and CM-cellulose (lane 4). Samples ofthe culture supernatants were analyzed. Man26A (0.05 mg) produced in E. coliwas analyzed without pretreatment (lane 5), after digestion (1 h, 37°C) withprotease from C. fimi (lane 6), or with the supernatant from a culture of C. fimigrown with LBG (lane 7).

2602 STOLL ET AL. APPL. ENVIRON. MICROBIOL.

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

FIG. 6. Alignment of the b-mannosidase subfamily in family 2 of the glycosyl hydrolases. Goat Man, Capra hicus b-mannosidase (GenBank U46067); Bovine Man,Bos taurus b-mannosidase (GenBank U17432); Human Man, Homo sapiens b-mannosidase (GenBank U60337); C. fimi Man, C. fimi b-mannosidase (GenBankAF126472). Asterisks indicate identical residues. The putative catalytic glutamates are in boldface.

VOL. 65, 1999 MANNAN-DEGRADING ENZYMES FROM C. FIMI 2603

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

(66). Thermophilic Caldocellulosiruptor spp. produce a numberof complex enzymes with two catalytic modules, at the N and Ctermini, connected by one or two cellulose-binding modulesfrom family III; in some of them, one of the catalytic modulesis a mannanase from family 5 (42) or family 26 (21). Theactivity of Man26A against LBG is comparable to those ofother mannanases (2, 8, 14, 56), but exact comparisons aredifficult because of the viscosity of the substrate.

SLH modules are present in other polysaccharidases, includ-ing xylanases, pullulanases, lichenases, and endoglucanases(6), where they may be involved in attaching the enzymes tothe cell surfaces. In growing cells of Clostridium thermocellum,cellulosomes are attached to the cell envelope through SLHmodules (5). Although some SLH modules bind to peptidogly-can, others appear to bind to secondary cell wall polysaccha-rides rather than to peptidoglycan (16, 50). It is possible, giventheir diverse sequences, that not all SLH modules recognizeand interact with the same components of the cell surface;some interact with other SLH modules (36). Man26A pro-duced by C. fimi appears to be transiently cell associated butnot to bind to peptidoglycan. Further analysis is required todefine the exact role(s) of the SLH module in the functioningand location of the enzyme.

Man26A appears to be processed further by C. fimi afterremoval of the leader peptide. The N terminus of the matureenzyme from C. fimi is 13 amino acids downstream of the siteprocessed by E. coli. The N-terminal amino acid sequences ofsome enzymes secreted by C. fimi are the same when theenzymes are produced by E. coli or C. fimi (7, 12, 39, 40, 46, 64),but, like Man26A, cellobiohydrolase Cel48A (formerly CbhB;see reference 30) also appears to be processed further by C.fimi after removal of the leader peptide (54). The relevantsequences are as follows: Cel48A, PAIA21AAGAGQPATVTVPAASPVRA22AVDGE; Man26A, PAQS21APAPAAPVAGALPT22APADE; 21 and 22 indicate the sites of hy-drolysis producing the N termini of the enzymes produced byE. coli and C. fimi, respectively. T. reesei removes a furthereight amino acids from the N terminus of Man1 after theprocessing of the leader peptide (55).

Family 2 of the glycosyl hydrolases includes enzymes fromeubacteria and eucaryotes, but Man2A is the first eubacterialb-mannosidase to be assigned to the family. An interestingdifference between Man2A and the mammalian enzymes in thesame subfamily is the cysteine residues: the mammalian en-zymes contain 13 conserved cysteines; Man2A contains only 3of these conserved cysteines plus 2 others (Fig. 6). Unlike someother glycosidases, Man2A appears to be a monomer. Its ac-tivity is comparable to that reported for a mannosidase fromThermotoga neopolitana (14). Like Man2A, some other glyco-sidases, including a b-glucosidase (17) and b-galactosidases(48), are inhibited by substrate.

Detailed characterization of Man26A and Man2A, includingtheir combined actions on manno-oligosaccharides and man-nans, is in progress.

ACKNOWLEDGMENTS

We thank N. R. Gilkes, D. G. Kilburn, P. Tomme, and S. G. Withersfor helpful discussions and Brad McLean for help with the preparationof the figures and tables.

This research was supported by the Natural Sciences and Engineer-ing Research Council of Canada and the Protein Engineering Networkof Centers of Excellence.

REFERENCES

1. Alkhayat, A. H., S. A. Kraemer, J. R. Leipprandt, M. Macek, W. J. Kleijer,and K. H. Friderici. 1998. Human b-mannosidase cDNA characterization

and first identification of a mutation associated with human b-mannosidosis.Hum. Mol. Genet. 7:75–83.

2. Arcand, N., D. Kluepfel, F. W. Paradis, R. Morosoli, and F. Sharek. 1993.b-Mannanase of Streptomyces lividans 66: cloning and DNA sequencing ofthe manA gene and characterization of the enzyme. Biochem. J. 290:857–863.

3. Bauer, M. W., E. J. Bylina, R. V. Swanson, and R. M. Kelly. 1996. Compar-ison of a b-glucosidase and a b-mannosidase from the hyperthermophilicarchaeon Pyrococcus furiosus. J. Biol. Chem. 271:23749–23755.

4. Beguin, P., N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., G. P. O’Neill, andR. A. J. Warren. 1987. Cloning of cellulase genes. Crit. Rev. Biotechnol.6:129–162.

5. Beguin, P., and M. Lemaire. 1996. The cellulosome: an exocellular, multi-protein complex specialized in cellulose degradation. Crit. Rev. Biochem.Mol. Biol. 3:201–236.

6. Beveridge, T. J., P. H. Pouwels, M. Sars, A. Kotiranta, K. Lounatma, K.Kari, E. Keruso, M. Haapasolo, E. M. Egelseer, I. Scocher, U. B. Sleytr, L.Morelli, M.-L. Callegari, J. F. Nomellini, W. H. Bingle, J. Smit, E. Leibovitz,M. Lemaire, I. Miras, S. Salamitou, P. Beguin, H. Ohayon, P. Gounon, M.Matuschek, K. Sahm, H. Bahl, R. Grogono-Thomas, J. Dworkin, M. J.Blaser, R. M. Woodland, D. G. Newell, M. Kessel, and S. F. Koval. 1997.Function of S-layers. FEMS Microbiol. Rev. 20:99–149.

7. Black, G. W., G. P. Hazlewood, S. J. Millward-Sadler, J. I. Laurie, and H. J.Gilbert. 1995. A modular xylanase containing a novel non-catalytic xylan-specific binding domain. Biochem. J. 307:191–195.

8. Bolam, D. N., N. Hughes, R. Virden, J. H. Lakey, G. P. Hazlewood, B.Henrissat, K. L. Braithwaite, and H. J. Gilbert. 1996. Mannanase A fromPseudomonas fluorescens subsp. cellulosa is a retaining glycosyl hydrolase inwhich E12 and E320 are the putative catalytic residues. Biochemistry 35:16195–16204.

9. Braithwaite, K. L., G. W. Black, G. P. Hazlewood, B. R. Ali, and H. J. Gilbert.A non-modular endo-b-mannanase from Pseudomonas fluorescens subsp.cellulosa. Biochem. J. 305:1005–1010.

10. Bray, M. R., A. L. Creagh, H. G. Damude, N. R. Gilkes, C. A. Haynes, E.Jervis, D. G. Kilburn, A. M. MacLeod, A. Meinke, R. C. Miller, Jr., D. R.Rose, H. Shen, P. Tomme, D. Tull, A. White, S. G. Withers, and R. A. J.Warren. 1996. b-1,4-Glycanases of Cellulomonas fimi: families, mechanisms,and kinetics. ACS (Am. Chem. Soc.) Symp. Ser. 655:64–84.

11. Chen, H., J. R. Leipprandt, C. E. Travis, B. L. Sopher, M. Z. Jones, K. T.Cavanagh, and K. H. Friderici. 1995. Molecular cloning and characterizationof bovine b-mannosidase. J. Biol. Chem. 270:3841–3848.

12. Clarke, J. H., K. Davidson, H. J. Gilbert, C. M. G. A. Fontes, and G. P.Hazlewood. 1996. A modular xylanase from mesophilic Cellulomonas fimicontains the same cellulose-binding and thermostabilising domains as xyla-nases from thermophilic bacteria. FEMS Microbiol. Lett. 139:27–35.

13. Clarke, J. H., J. I. Laurie, H. J. Gilbert, and G. P. Hazlewood. 1991. Multiplexylanases of Cellulomonas fimi are encoded by distinct genes. FEMS Micro-biol. Lett. 83:305–310.

14. Duffaud, G. D., C. M. McCutchen, P. Leduc, K. N. Parker, and R. M. Kelly.1997. Purification and characterization of extremely thermostable b-man-nanase, b-mannosidase, and a-galactosidase from the hyperthermophiliceubacterium Thermotoga neopolitana 5068. Appl. Environ. Microbiol. 63:169–177.

15. Duran, P., P. Lehn, I. Callebaut, S. Fabrega, B. Henrissat, and J.-P. Mor-non. 1997. Active-site motifs of lysosomal acid hydrolases: invariant featuresof clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis.Glycobiology 7:277–284.

16. Egelseer, E. M., K. Leitner, M. Jarosch, C. Hotzy, S. Zayni, U. B. Sleytr, andM. Sara. 1998. The S-layer proteins of two Bacillus stearothermophilus wild-type strains are bound via their N-terminal region to a secondary cell wallpolymer of identical composition. J. Bacteriol. 180:1488–1495.

17. Estrada, P., I. Mata, J. M. Dominguez, M. P. Castillon, and C. Acebal. 1990.Kinetic mechanism of b-glucosidase from Trichoderma reesei QM 9414. Bio-chim. Biophys. Acta 1033:298–304.

18. Fanutti, C., T. Ponyi, G. W. Black, G. P. Hazlewood, and H. J. Gilbert. 1995.The conserved non-catalytic 40-residue sequence in cellulases and hemicel-lulases from anaerobic fungi functions as a protein docking domain. J. Biol.Chem. 270:29314–29322.

19. Fujino, T., P. Beguin, and J.-P. Aubert. 1993. Organization of a Clostridiumthermocellum gene cluster encoding the cellulosomal scaffolding proteinCipA and a protein possibly involved in the attachment of the cellulosome tothe cell surface. J. Bacteriol. 175:1891–1899.

20. Gherardini, F., M. Babcock, and A. Salyers. 1985. Purification and charac-terization of two a-galactosidases associated with the catabolism of guar gumand other b-galactosides by Bacteroides ovatus. J. Bacteriol. 161:500–506.

21. Gibbs, M. D., A. U. Elinder, R. A. Reeves, and P. L. Bergquist. 1996.Sequencing, cloning and expression of a b-1,4-mannanase gene, manA, fromthe extremely thermophilic anaerobic bacterium, CaldicellulosiruptorRT8B.4. FEMS Microbiol. Lett. 141:37–43.

22. Gilkes, N. R., D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren. 1989.Structural and functional analysis of a bacterial cellulase by proteolysis.J. Biol. Chem. 264:17802–17808.

2604 STOLL ET AL. APPL. ENVIRON. MICROBIOL.

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

23. Gilkes, N. R., R. A. J. Warren, R. C. Miller, Jr., and D. G. Kilburn. 1988.Precise excision of the cellulose-binding domain from two Cellulomonas fimicellulases by a homologous protease and the effects on catalysis. J. Biol.Chem. 263:10401–10407.

24. Grodber, J., and J. J. Dunn. 1988. ompT encodes the Escherichia coli outermembrane protease that cleaves T7 RNA polymerase during purification. J.Bacteriol. 170:1245–1253.

25. Harjunpaa, V., A. Teleman, M. Siika-Aho, and T. Drakenberg. 1995. Kineticand stereochemical studies of mannooligosaccharide hydrolysis catalyzed byb-mannanases from Trichoderma reesei. Eur. J. Biochem. 234:278–283.

26. Henrissat, B. 1990. A classification of glycosyl hydrolases based on aminoacid sequence similarities. Biochem. J. 280:304–316.

27. Henrissat, B., and A. Bairoch. 1993. New families in the classification ofglycosyl hydrolases based on amino acid sequence similarities. Biochem. J.293:781–788.

28. Henrissat, B., and A. Bairoch. 1996. Updating the sequence based classifi-cation of glycosyl hydrolases. Biochem. J. 316:695–696.

29. Henrissat, B., I. Callebaut, S. Fabrega, P. Lehn, J.-P. Mornon, and G.Davies. 1995. Conserved catalytic machinery and the prediction of a commonfold for several families of glycosyl hydrolases. Proc. Natl. Acad. Sci. USA92:7090–7094.

30. Henrissat, B., T. T. Teeri, and R. A. J. Warren. 1998. A scheme for desig-nating enzymes that hydrolase the polysaccharides in the cell walls of plants.FEBS Lett. 425:352–354.

31. Hilge, M., S. M. Gloor, W. Rypniewski, O. Sauer, T. D. Heightman, W.Zimmerman, K. Winterhalter, and K. Piontek. 1998. High-resolution nativeand complex structures of thermostable b-mannanase from Thermomonos-pora fusca—substrate specificity in glycosyl hydrolase family 5. Structure6:1433–1444.

32. Kallweit, U., K. O. Boernsen, G. M. Kresbach, and H. M. Widmer. 1996.Matrix compatible buffers for analysis of proteins with matrix-assisted laserdesorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom.10:845–849.

33. Kohchi, C., and A. Toh-e. 1986. Cloning of a Candida pelliculosa b-glucosi-dase gene and its expression in Saccharomyces cerevisiae. Mol. Gen. Genet.203:89–94.

34. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

35. Leipprandt, J. R., S. A. Kraemer, B. E. Haithcock, H. Chen, J. L. Dyme,K. H. Friderici, and M. Z. Jones. Caprine b-mannosidase: sequencing andcharacterization of the cDNA and identification of the molecular defect ofcaprine b-mannosidosis. Genomics 37:51–56.

36. Lemaire, M., H. Ohayon, P. Gounon, T. Fujino, and P. Beguin. 1995. OlpB,a new outer layer protein of Clostridium thermocellum, and binding of itsS-layer-like domains to components of the cell envelope. J. Bacteriol. 177:2451–2459.

37. Margolles-Clark, E., M. Tenkanen, E. Luonteri, and M. Penttila. 1996.Three a-galactosidase genes of Trichoderma reesei cloned by expression inyeast. Eur. J. Biochem. 240:104–111.

38. McLeary, B. V., F. R. Taravel, and J.-P. Joseleau. 1983. Characterisation ofthe oligosaccharides produced on hydrolysis of galactomannan with b-D-mannanase. Carbohydr. Res. 118:91–109.

39. Meinke, A., N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J.Warren. 1993. Cellulose-binding polypeptides from Cellulomonas fimi: en-doglucanase D (CenD), a family A b-1,4-glucanase. J. Bacteriol. 175:1910–1918.

40. Meinke, A., N. R. Gilkes, E. Kwan, D. G. Kilburn, R. C. Miller, Jr., andR. A. J. Warren. 1994. Cellobiohydrolase A (CbhA) from the cellulolyticbacterium Cellulomonas fimi is a b-1,4-exocellobiohydrolase analogous toTrichoderma reesei CBHII. Mol. Microbiol. 12:413–442.

41. Millward-Sadler, S. J., J. Hall, G. W. Black, G. P. Hazlewood, and H. J.Gilbert. 1996. Evidence that the Piromyces gene family encoding endo-1,4-mannanases arose through gene duplication. FEMS Microbiol. Lett. 141:183–188.

42. Morris, D. D., R. A. Reeves, M. D. Gibbs, D. J. Saul, and P. Bergquist. 1995.Correction of the the b-mannanase domain of the celC pseudogene fromCaldocellulosiruptor saccharolyticus and activity of the gene product on kraftpulp. Appl. Environ. Microbiol. 61:2262–2269.

43. Neustroev, K. N., A. S. Krylov, L. M. Firsov, O. N. Abroskina, and A. Y.Khorlin. 1991. Isolation and properties of b-mannosidase from Aspergillusawamori. Biokhimiya 56:1406–1412.

44. Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identifica-tion of prokaryotic and eukaryotic signal peptides and prediction of theircleavage sites. Prot. Eng. 10:1–6.

45. Olabarria, G., J. Carrascosa, M. A. Pedro, and J. Berenguer. 1996. Aconserved motif in S-layer proteins is involved in peptidoglycan binding inThermus thermophilus. J. Bacteriol. 178:4765–4772.

46. O’Neill, G., S. H. Goh, R. A. J. Warren, D. G. Kilburn, and R. C. Miller, Jr.1986. Structure of the gene encoding the exoglucanase of Cellulomonas fimi.Gene 44:325–330.

47. Pace, N. C., F. Vajdos, L. Fee, G. Grimsley, and T. Gray. 1995. How tomeasure and predict the molar absorption coefficient of a protein. ProteinSci. 4:2411–2423.

48. Posci, I., S. A. Taylor, A. C. Richardson, B. V. Smith, and R. G. Price. 1993.Comparison of several new chromogenic galactosides as substrates for var-ious b-D-galactosidases. Biochim. Biophys. Acta 1163:54–60.

49. Puls, J., and J. Schuseil. 1996. Chemistry of hemicelluloses: relationshipbetween hemicellulose structure and enzymes required for hydrolysis, p.1–28. In M. P. Coughlan and G. P. Hazlewood (ed.), Hemicelluloses andhemicellulases. Portland Press, London, England.

50. Ries, W., C. Hotzy, I. Schocher, U. B. Sleytr, and M. Sagra. 1997. Evidencethat the N-terminal part of the S-layer protein from Bacillus stearothermophi-lus PV72/p2 recognizes a secondary cell wall polymer. J. Bacteriol. 179:3892–3898.

51. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

52. Sandercock, L., A. Meinke, and R. A. J. Warren. 1996. Degradation ofcellulases in cultures of Cellulomonas fimi. FEMS Microbiol. Lett. 143:7–12.

53. Sawadago, M., and M. W. van Dyke. 1991. A rapid method for the purifi-cation of deprotected oligodeoxynucleotides. Nucleic Acids Res. 19:674.

54. Shen, H., N. R. Gilkes, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren.1995. Cellobiohydrolase B, a second exocellobiohydrolase from the cellulo-lytic bacterium Cellulomonas fimi. Biochem. J. 311:67–74.

55. Stålbrand, H., A. Saloheimo, J. Vehmaanpera, B. Henrissat, and M. Pent-tila. 1995. Cloning and expression in Saccharomyces cerevisiae of a Tricho-derma reesei b-mannanase gene containing a cellulose-binding domain. Appl.Environ. Microbiol. 61:1090–1097.

56. Stålbrand, H., M. Siika-Aho, M. Tenkanen, and L. Viikari. 1993. Purificationand characterisation of two b-mannanases from Trichoderma reesei. J. Bio-technol. 29:229–242.

57. Stewart, B. J., and J. M. Leatherwood. 1976. Derepressed synthesis of cel-lulase by Cellulomonas. J. Bacteriol. 128:609–615.

58. Talbot, G., and J. Sygusch. 1990. Purification and characterization of ther-mostable b-mannanase and a-galactosidase from Bacillus stearothermophi-lus. Appl. Environ. Microbiol. 56:3505–3510.

59. Tamaru, Y., T. Araki, H. Amagoi, H. Mori, and T. Morishita. 1995. Purifi-cation and characterization of an extracellular b-1,4-mannanase from a ma-rine bacterium, Vibrio sp. strain MA-138. Appl. Environ. Microbiol. 61:4454–4458.

60. Teather, R. M., and P. J. Wood. 1982. Use of Congo red-polysaccharideinteractions in enumeration and characterization of cellulolytic bacteria fromthe bovine rumen. Appl. Environ. Microbiol. 43:777–780.

61. Tenkanen, M., M. Makkonen, M. Perttula, L. Viikari, and A. Teleman. 1997.Action of Trichoderma reesei on galactoglucomannan in pine kraft pulp.J. Biotechnol. 57:191–204.

62. Thomas, M. G. 1994. A procedure for second-round differential screening ofcDNA libraries. BioTechniques 16:988–989.

63. Wakarchuk, W. W., D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren.1984. b-Glucosidases of Cellulomonas fimi. J. Gen. Microbiol. 130:1385–1389.

64. Wong, W. K. R., B. Gerhard, Z. M. Guo, D. G. Kilburn, R. A. J. Warren, andR. C. Miller, Jr. 1986. Characterisation and structure of an endoglucanasegene cenA of Cellulomonas fimi. Gene 44:315–324.

65. Xiuzhu, D., P. J. Y. M. J. Schyns, and A. J. M. Stams. 1991. Degradation ofgalactomannan by a Clostridium butyricum strain. Antonie Leewenhoek 60:109–114.

66. Yague, E., M. Mehak-Zunic, L. Morgan, D. A. Wood, and C. F. Thurston.1997. Expression of CEL2 and CEL4, two proteins from Agaricus bisporuswith similarity to fungal cellobiohydrolase I and b-mannanase, respectively,is regulated by carbon source. Microbiology 143:239–244.

VOL. 65, 1999 MANNAN-DEGRADING ENZYMES FROM C. FIMI 2605

on Decem

ber 5, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from