a-L-Fucosidase from
a
Soil Bacterium
K. MORTENSSON-EGNUND, R. SCHbYEN, C. HOWE, L. T. LEE, AND A. HARBOE
Virus Department B, National InstituteofPublicHealth, Oslo,Norway,andDepartmentofMicrobiology,
Columbia University, College of PhysiciansandSurgeons, New York,New York 10032
Received forpublication31December1968
Intracellular glycosidases were measured in cell-free extracts obtainedby
ultra-sonicdisruption of a gram-negative soil coccobacillus (Chase, 1938). From these extracts, a-L-fucosidase was purified about 120-fold by saltingout with
(NH4)2SO4,
ionexchange chromatography, and gel filtration. Theapproximate molecular weight of theenzyme was 50,000; its pH optimum was5. Theenzymewas inhibited by
L-fucose and split this sugar from a purified acid mucopolysaccharide from chicken
chorioallantoic fluid. The acidmucopolysaccharideis identical with a component
(hostantigen) of the hemagglutinin of influenza virus.Its antigenic reactivity is
altered by cell-tree extracts ofthe bacterium, in which the responsible enzyme is
thoughttobe ana-L-fucosidase.
A sulfated mucopolysaccharide derived from chicken chorioallantoic fluidhas been shown to be identical with aportion of the hemagglutinin
of egg-grown influenza virus (9-11). This
rela-tionship was demonstrated by the ability of the soluble mucopolysaccharide
[host
factor (HF)] to block the anti-hemagglutinin (antibody) forinfluenzavirus producedinrabbits in response to theinjection ofnormalchorioallantoicmembrane. Purified HFcontains sugar constituents charac-teristic of blood group substances, notably hexosamine, galactose, and fucose (10). It was
thought, therefore, that enzymes known to de-grade blood group substances might be useful in further elucidating the structure of HF. One sourceof such enzymes was an unclassified cocco-bacillus originally isolated from soil by Chase
(2) andshownbyhimtodecompose blood group substances. Subsequent studies with this
bac-terium, by Gilmore and Howe (8, 8a), showed that cell-freeextracts (CFE) degradedA, B, and
0(H) substances down to diffusible oligosac-charides and monosacoligosac-charides. CFE from the
Chase organism have recently been found also
to destroy the serological activity of HF on the intact virion and in purified water-soluble form. Evidence to be reported separately supports the hypothesis that the enzyme responsible is an a-L-fucosidase. The present paper concerns at-tempts at separation of this enzyme from other glycosidases present in the crude CFE of the soil bacterium.
MATERIALS AND METHODS
Chemicals. p-Nitrophenyl-a-L-fucopyranoside, not
commerciallyavailablewhen we started the
investiga-tions,wassynthesizedaccordingto amethod described
byConchie andLevvy (4). Later, this substrate was obtained fromKoch-Light Laboratories Ltd., along
withp-nitrophenylglycosides of thefollowingsugars:
,B-L-fucopyranose, 2-acetamido-2-deoxy-,f-D-galacto-pyranose,2-acetamido-2-deoxy-f-D-glucopyranose, a-and 3-D-galactopyranose. Methyl-a-L-fucopyranoside
and methyl-,j-L-fucopyranoside were synthesized by
standard methods(19).
L-Fuconolactoneswereobtainedbybromine
oxida-tion(12) of L-fucose.Twocrystallinereactionproducts
wereisolated and characterizedby meltingpoint and
byinfraredspectrophotometricanalysis.Theprincipal
product wasthe known -y-lactone (1), which had a
melting point of104 to 104Candacarbonyl
absorp-tion at 1,750 cm-'. The other product, which isnot
described in the literature, had a melting point of
145 to 148 C and a carbonyl absorption at 1,717 cm-';itwasthoughtpossiblytobe the 4-lactone.
Ammonium sulfate (special enzyme grade) and
streptomycin sulfate were obtained from Mann
Re-search Laboratories Inc.; sucrose was from Difco;
andp-(hydroxy)mercuribenzoate (sodium salt),
a-D-fucose, a-L-fucOse, alkaline phosphatase type III-S
(E.coli),andhorseradishperoxidase(type VIapprox.
R.Z. 3.2) were fromSigma Chemical Co.
Diethyla-minoethyl (DEAE) cellulose (DE-22) was obtained
fromWhatman, CM-Sephadex C-50fromPharmacia,
andBio-Gel P-200 from Bio-Rad Laboratories.
Mcllvaine citric acid-phosphate, consisting of 0.1
M citric acid-0.2 M Na2HPO4, was used in the pH
range 3.0 to 7.8. Buffer solution A was Mcllvaine
buffer, pH 7, diluted 1:10 with distilled water;
buffersolutionBwasMcllvainecitric acidbuffer, pH
7, diluted 1:50 with distilled water. Gomory Tris
buffer, pH7.2, contained 0.2 M
tris(hydroxymethyl)-aminomethane (Tris)-0.2 N HCl. Thesolutions were stored in the cold with toluene to avoid bacterial
contamination. Methylpentose was determined by the method of Dische and Shettles (5).
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,a-L-FUCOSIDASE FROM A SOIL BACTERIUM
Propagation oforganism. The Chase coccobacillus
(unclassified, accessionno. 13949) wasobtainedfrom
theAmerican TypeCultureCollectionand was prop-agated accordingto theprocedures described by Gil-more and Howe (8) in a 0.1% solution of hog gastric mucin in 0.1% (NH4)2SO4 and 0.2% K2HPO4 ad-justed to pH 7.2. For large-scale production slight
modifications wereintroduced.A 40-gamountof hog
gastric mucin (Koch-Light Laboratories Ltd.) was
dissolved in 12 liters of distilled water and dialyzed
against tap water for 48 hr and distilled water for 18 hr. Themucin solution was then clarified by
continuous-flowcentrifugation in aServall centrifuge (12,000 x
g). Volumes of 300 ml of mucin were made up to 1 liter with salt solution [0.1% (NH4)2SO4 and 0.2%
K2HPO4, pH 7.2]. Dry weight determinations
indi-catedthe finalconcentration of mucin to be 0.01%. The medium was autoclaved at 120 C for 30 min.
Theorganismwaspropagatedin 10-literbottles.Each
bottle received aseedinoculumof 150 ml of a
3-day-oldcultureand wasincubatedat 30Cfor 2 to 3 days,
during which time aeration was effected for periods
of 1 hr, spacedat6-hr intervals. The organismswere
harvestedbycentrifugationat roomtemperaturein a
continuous-flow centrifuge (27,000 X g), and the cells
were washed in cold 0.01 Mcitric acidbuffer, pH 7.
Thereafter, all procedures were carried out at 4 C.
Onevolume of sedimented bacteria wasresuspended
in sixvolumes of the same buffer and disrupted by ultrasonicvibrations with aBransonSonifier (model S-125, position5) for 5min.Thedisruptedcells were
centrifugedat 12,000 X gfor 45 min in aServall
high-speedcentrifuge. The sediment wasdiscardedand the
CFE wasfrozen.
Enzyme assays. The glycosidaseswere assayed by
theprocedureofLewyandMcAllan (13),withminor
modifications. Theincubation mixture contained the
following components: 0.45 ml of Mcllvaine citric
acid-phosphate buffer (pH 5), 0.45 ml ofsubstrate (2
mMp-nitrophenyl-a-L-fucopyranoside or 5 mm solu-tions of other p-nitrophenylglycopyranosides) and
0.10 mlof suitably diluted enzyme solution. After 1
hr at 30C, the reaction wasstopped by the addition of3.5 mlof 1 N NaOH.The resultingyellow color,
which wasstable inthe alkalinemedium, was
meas-uredat420 nminaBeckmanBmodel
spectrophotom-eter. The amount of liberated p-nitrophenol was
determined by referenceto acalibrationcurve.
Suita-ble enzyme and substrate controls were included. A
unit ofenzyme wasdefinedasthe amount thatwould
liberate 1 ,umole of p-nitrophenol from the
p-ni-trophenyl-glycopyranoside per min at 30 C. The
specific activity was expressed as the number of
enzyme units per milligram ofprotein. The protein
determinations were performed according to the
method of Lowry et al. (15) with crystalline serum albumin used as a standard. Alkaline phosphatase
was assayed in a mixture containing 0.45 ml of
p-nitrophenyl-phosphate (4mg/ml),0.45 mlof alkaline
glycinebuffer (pH10.5), and 0.10 ml ofenzyme
solu-tion.After incubationfor 15 min at 30C, thereaction
wasstoppedbyadding3.5ml of1NNaOH, and the
absorbance wasmeasured at420nm.Peroxidasewas
assayed accordingtoamethod developed by
Worth-ington Biochemical Corp., based upon the use of
o-anisidine as the hydrogen donor. The rate ofthe
color development was determined by measurement ofoptical densityat460nmat20 C.
RESULTS
Purification of the enzyme. The
purification
procedures
were carried out at 0 to 4 C. Thefrozen,
crude CFE was thawed and mixed with10% (w/v)
streptomycinsulfate, pH7 (20ml/100
ml of thawed CFE). After standing for about 30minwith occasionalshaking, the mixturewas
centrifugedandthesediment wasdiscarded. The supernatantfluidwasmade upto33%saturation with solid (NH4)2SO4 (196 g/1,000 ml) and allowed to stand for 14to 16 hr.After
centrifu-gation
at12,000
X gfor 1 hr,theprecipitatewasdiscarded and the supematant fluidwasmade up
to 60% saturation (1,777 g/1,000 ml). After
standingfor another 14to16hr,themixturewas
centrifuged and the supernatant fluid was dis-carded. The redissolved precipitate was exten-sively dialyzed against buffer B to remove the
(NH4)2SO4. This preparation was assayed for enzymeactivity. Specificactivities of the various enzymes in (NH4)2SO4 33 to 60% precipitate
were as follows (102 units per mg of
protein):
f3-L-fucosidase,
0.0; a-L-fucosidase, 2.8;13-D-galactosidase, 13.8; a-D-galactosidase,
14.2;
N-acetyl-,3-D-galactosaminidase, 57.9; andN-acetyl-(3-D-glucosaminidase, 118.5.
A column (2.6 by 20 cm) of DE-22 cellulose (Whatman) was equilibrated with buffer B. A 50-mlamountof thedialyzed preparationwas clarified by centrifugation at low speed and
ap-pliedtothe column. BufferBwasusedaseluant. The enzymescameoffjustafter the voidvolume,
and about30%of the activitieswerecollected in the first 100 ml of buffer. This fraction was freeze-dried at once and dissolved in distilled
water, giving a protein concentration of about 10mg/ml.
A column (1.3 by 35 cm) of CM-Sephadex
C-50 (Pharmacia)wasequilibrated with buffer A. A 5-ml sample of enzyme preparation from the DEAE cellulose columns was dialyzed against
buffer A andappliedtothecolumn. The column
wasfirst eluted with about 60 ml of the same
buffer to wash off unabsorbed protein, which represented about 60% of the applied material. Itwasthen eluted with the same buffer contain-ing 0.2 M NaCl. The peak of the enzymesemerged with 0.10 to 0.14 M NaCl in the effluent. The frac-tions were pooled, dialyzed against distilled water,freeze-dried,and dissolved in avery small volume of buffer A, resulting in a decrease of about30% in the total amount of thefucosidase activity.
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Acolumn (1.8 by 30 cm) of Bio-Gel (Bio-Rad Laboratories) P-200, 100 to 200mesh, was equi-librated with buffer A. The concentrated enzyme fractions pooled from the CM-Sephadex columns were applied in a volume of 1 ml containing 20
mg ofprotein. The column was eluted with the
same buffer and the flow rate was adjusted to about 1 ml/hr. Fractions (1.7 ml) werecollected and assayed for protein and enzyme activities. Atypical elution pattern is shown in Fig. 1.
Toobtain a better separation of a-L-fucosidase from the other enzymes, material was rechro-matographed on Bio-Gel P-200under conditions identical to those of the first Bio-Gel step. All fractions containing a-L-fucosidase (peak I in Fig. 1) were pooled, dialyzed, and concentrated by freeze-drying, resulting in some loss of
ac-tivity.Inthiswayit waspossible to obtain some fractions with a-L-fucosidase activity and no detectable a- or ,B-galactosidase activities. How-ever, small amounts of N-acetylhexosaminidase
activity were still present (Fig. 2). The averages of results in several experiments arepresented in Table 1. The total activity was increased after
streptomycin sulfate precipitation, possibly ow-ing toremoval of an inhibitor. The final product
represented a purification of specific a-L-fucosi-dase about 120 times over that of the original
crudeextract (Table1).
Properties of the a-L-fucosidase. Crude
prep-arations of the enzyme were stable at 4 C for several weeks. The enzyme in a purified concen-trated preparation did not show any significant
max 2.6. I ' 1.8 - 1.4 z wI o1.0 0.6 a. 16 20 24 28 32 36 40 44 FRACTION NUMBER
FIG. 1.ElutionpatternfromaBio-Gel P-200 column.
0, N-acetyl-8-D-glucosaminidase; 0,
ci-D-galactosid-ase; U, ,B-D-galactosidase; A, a-L-fucosidase. All
enzyme activities are expressedas optical density at
420nm. 6 z w 0 C4 -Z CL 0 0 x 16 20 24 28 32 36 40 FRACTION NUMBER
Fio. 2. Elution patternfrom rechromatography of peak I in Fig. I on a Bio-Gel P-200 column. 0, N-acetyl-j3-D-glucosaminidase; 0, ce-D-galactosidase; *,
,B-D-galactosidase; A, ca-L-fucosidase;X, ,g ofprotein
per ml. All enzyme activities areexpressedasoptical density at 420 nm.
TABLE 1. Steps inpurification of a-L-fucosidase Step Cell-free extract... Streptomycin sul-fate Supernatant fluid... Ammonium sulfate 33-60% Precipi-tate ... DE-22 cellulose Freeze-dried fractions... CM-Sephadex Fractions in 0.10-0.14M NaCl... Samefractions freeze-dried.... Bio-Gel P-200 Peak I inFig. 1.. Specific activitya 0.88 1.27 2.83 11.16 55.16 41.33 105.16 Vol ml
444
533 105 8 15 0.7 18.7 Total unitsb 28.3 31.1 27.5 8.3 7.0 5.2 4.8 Yield 100 110 97 29 25 18 17aExpressed as
10'
units of enzyme activity permilligram ofprotein.
bOne unit = amount liberating 1 ,umole of
p-nitrophenol fromsubstrate per minat30C.
decrease inactivity when tested after2 weeks of
storage at 4C, butdiluted solutions rapidlylost activity. Freezingand thawing causeda 30% loss ofactivity. The effect ofpH on the rate of hy-drolysis of
p-nitrophenyl-a-L-fucopyranoside
is shown in Fig. 3I Maximal enzyme activity was obtained at pH 5. On varying the enzymecon-centration against a constant amount of
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a-L-FUCOSIDASE FROM A SOIL BACTERIUM 1.3 1.1 E 0 C- a-0 0.9
[
0.7[ 0.5p 0.3 0.1 3 4 5 pH x0 O 30 0 VI o 20. 10 z I CL 0-jc
6 7 8FIG. 3. pH optimumofa-L-fucosidase. Assays were performed under standard conditions in citric acid-phosphate buffer; the enzyme activities are expressed
asopticaldensityat420 nm.
strate under standard conditions at pH 5, a
linear relation was demonstrated between the
amount ofchromogen released and theamount
of enzyme added. The effect of substrate
con-centration on the reaction rate at pH 5 was studied. The rate ofhydrolysis as a function of p-nitrophenyl-a-L-fucopyranoside concentration is shown in Fig. 4. The Lineweaver-Burk plot
(14) was found to be linear; the estimated Km
valuewas3.6 X 104M.
Theenzyme wastested forsusceptibilityto
in-hibition by monosaccharides and other
inhibi-tors. Theincubation mixtures had the following
composition: 0.45 ml of citric acid-phosphate
buffer (pH 5), 0.45mlof2 mm p-nitrophenyl-a-L-fucopyranoside, 0.10 ml of enzyme solution, and 0.10 ml ofinhibitor solution. All inhibitor solutions were freshly prepared just before use. After 1 hrat 30 C, the reactionwas stopped by adding 3.4 ml of 1 N NaOH. a-L-Fucose was
found to be a
competitive
inhibitor for thea-L-fucosidaseasdetermined bythe Lineweaver-Burkplots(Fig. 5).Theenzyme wasalsostrongly inhibited by p-hydroxymercuribenzoate. With p-nitrophenyl-a-L-fucopyranoside, the Ki values for theL-fucoseand the
p-hydroxymercuribenzo-atewereestimatedtobe 2.3 X 10 4Mand7.1 X 10-8 M, respectively. Assays with D-fucose,L-fucono-y-lactone,
L-fucono-6-lactone,
D-galac-tose, and D-galactono-y-lactone, in
concentra-tions from 5 to 100 mm, showed no
inhibitory
effect. Neither a-methyl- norf,-methyl-fucoside
affected the activity oftheenzyme.
The purified influenza virus host antigen, a
fucose-containing
acid mucopolysaccharide (10),o0.5 1 2
p-NITROPHENYL-Jl-L-FUCOPYRANOSIDE mM (S)
FIG.4. Effect of varying substrate concentration (S) onthe reaction rate (V) at pH 5. Enzyme activity is
ex-pressedas micromoles of p-nitrophenol liberatedper
hour times102. Innergraph (1/Vversus 1/S) is Line-weaver-Burk plot.
2 4 6 8 10 1 2
S [MM]
FIG. 5. Inhibition ofa-fucosidase activity by
L-fucose andp-hydroxymercuribenzoate assayed by the
method of Lineweaver and Burk (13). p-Nitrophenyl-a-L-fucopyranosidewasusedassubstrate.
was dissolved in citric acid-phosphate buffer (pH 5) at a concentration of 1 mg/ml. Enzyme
(from the first Bio-Gel chromatogram, peak I, Fig. 1) was added, and the mixture was incu-batedat30Cfor48 hr.Afterheatingto56 Cfor
10 min to inactivate the enzyme, the reaction
mixture was dialyzed against distilled water for
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18 hr. The methylpentose content of the
non-dialyzable residue was found to be
approxi-mately 20% of that of the untreated
mucopoly-saccharide, asshown by the cysteine-sulfuric acid
reaction (5). The dialysate was concentrated to
dryness and dissolved in about 100 ,uliters of
distilledwater.Thepresence of fucose was
identi-fied by paper chromatography in
ethylacetate-pyridine-water [12:5:4 (21)1 developed with
AgNO3.
Sucrose gradient centrifugation. A Christ
Omega II ultracentrifuge with the swinging bucket
(3 by 5 ml) rotor 9750 was used for sucrose
densitygradientanalyses (18). Samples (0.25 ml)
fromaDEAEcellulose column preparation
con-taining 0.5 to 1.0% protein in Tris buffer were
appliedto agradientof 5 to 20% sucrose (4.6 ml)
in0.05 MTris-hydrochloridebuffer (pH 7.2) and
centrifuged at 4 C for 18 hr at 100,000 X g.
Twenty fractions of approximately 0.25 ml were
collected from each of the tubes by introducing glycerol at constantvelocity from the bottom by
means ofa syringe. This procedure effected
par-tial separation of the different enzymes. Typical results are shown in Fig. 6. Peroxidase
[horse-radish type VI; molecular weight 40,000 (16)]
and alkaline phosphatase [E. coli type III-S;
molecular weight 80,000 (7)] were used as
ref-erence enzymes. The molecular weight of the
a-L-fucosidase calculated on the basis of the ultracentrifuge analyses was approximately
50,000.
DISCUSSION
The presence of a-L-fucosidase in the
cocco-bacillus described by Chase (2) had not
previ-(I) LA c - :3 N < z ._ A 0) 0 K : Vl 0 L. 0 L-A A. 5 10 15 -bot tom FRACTION NUMBER
FIG. 6. Ultracentrifugal analysis in sucrose density
gradient (S to20%). After 18 hrat 100,000 X g;20
fractionswere collected, numberIat thetop. Enzyme activitiesweremeasured accordingtomethods described in thisreportand plottedinarbitrary units.
ously been reported. In addition to the
a-L-fucosidase, this coccobacillus contained several
other glycosidases butwasfree of,B-L-fucosidase.
In crude preparations, thespecific a-L-fucosidase activity was lower than either galactosidase or
N-acetylhexosaminidase activities assayed with corresponding p-nitrophenyl-glycosides as
sub-strates. Among several methods tested for
puri-fication, that described earlier in this report
seemedto berelatively simple and reproducible. Conventional methodswereused,namely salting outwith (NH4)2SO4, ion exchange
chromatogra-phy, and gel filtration. The a-L-fucosidase
finally isolated was 120 times more active than
the crude CFE. The molecular weight was
esti-mated to be approximately 50,000. The
determi-nationof a-L-fucosidase activity as afunction of
pH indicated an optimum ofpH 5; there was
little activity below pH 3.4 or above pH 7.8. ThispH optimumiswithin the rangefound for fucosidases from other sources. Thus,
a-L-fucosidases in homogenates from various
mam-malian tissues were studied by Levvy and McAllan (13). Thehighestactivity wasfound in
rat epididymis and ox liver with pH optima of 6.1 and 5.6, respectively. The same authors
re-ported the visceral hump of the limpet, Patella vulgata, to be a better source of a-L-fucosidase than mammalian tissues. Marnay etal. (17) de-scribedtwo different fucosidases fromthe
diges-tive juice of Helix pomatia, an a-L-fucosidase with a pH optimum of 3.2 anda 3-D-fucosidase
with a pH optimum of5.5. Tanaka et al. (20)
reported the isolation and
purification
of a-L-fucosidase fromabalonelivers.Theirpurificationprocedure increased the specific activity about eightfold. Two types of a-L-fucosidase were re-ported, differing in pH optimum and substrate specificity. One had a pH optimum at about 5,
tested with
p-nitrophenyl-a-L-fucopyranoside,
but didnot act on thefucosidiclinkages of
por-cine submaxillary mucin. The other had a pH
optimum at about 2, tested with the synthetic
substrate as well as with
porcine submaxillary
mucin. Wefound only one pHoptimum (pH 5)
withenzyme preparation from the Chase
cocco-bacillus, tested with the
p-nitrophenyl-a-L-fucopyranoside. Our results, however, do not
exclude thepresence ofisoenzymes. Theactivity
of the presentpreparation on
p-nitrophenyl-a-L-fucopyranoside washighly sensitive to
hydroxy-mercuribenzoate, a finding which suggestedthat
sulfhydryl groups mightbe essential foractivity.
The curve shown in Fig. 4 is consistent with
simple Michaelis-Mentel kinetics. L-Fucose
com-petitively inhibited the reaction with p-nitro-phenyl-a-L-fucopyranoside. Surprisingly, the
re-action was not affected by fucono-y-lactone or
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a-L-FUCOSIDASE FROM A SOIL BACTERIUM byfucono-5-lactone, although the transformation
from y to 6 configuration has been reported to
increase the inhibitory power of lactones in certain instances (6). Likewise, methylfucosides were ineffective as inhibitors and were not sus-ceptible to the action of the enzyme. In these respects, our findings accorded with the results reported by Levvy and McAllan (13) regarding the characteristics of mammalian fucosidases.
Our preparations of fucosidase from the coccobacillus of Chase were active against the purified host antigen (HF) of influenza virus hemagglutinin, 80% of the fucose normally con-tained in the acid mucopolysaccharide being rendered dialyzable as the result of enzyme action. Concomitantly, the antigenic specificity of HF was altered. These latter results, to be reported in detail subsequently, suggested that fucose may beinvolved in the structural
configu-rations which determine the antigenic specificity
of the hostantigen. The presentdata, however, are not sufficient to exclude the presence in our preparations ofadditional enzymeswhich might
act in concert with the fucosidase on this and
othersubstrates.
ACKNOWLEDGMENTS
We areindebted to E. Jantzen, Methodology Department, National Institute of Public Health, Oslo, Norway, for the syn-thesis ofp-nitrophenyl-a-L-fucopyranoside.
This investigation was supported by Public Health Service grantAI-03168 from the National Instituteof Allergy and In-fectiousDiseases.
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