APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1995, p. 1953–1958 Vol. 61, No.

5
0099-2240/95/$04.0010
Copyright q 1995, American Society for Microbiology

Purification and Characterization of the Bacillus subtilis

Levanase Produced in Escherichia coli
ERICH WANKER,1† ANTON HUBER,2 AND HELMUT SCHWAB1*
Institut für Biotechnologie, Arbeitsgruppe Genetik, Technische Universität,1 and Institut für
Physikalische Chemie, Karl-Franzens-Universität,2 A-8010 Graz, Austria
Received 11 August 1994/Accepted 2 March 1995

The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli
with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out
with ammonium sulfate and chromatography on DEAE–Sepharose CL-6B, S-Sepharose, and MonoQ-Sepha-

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rose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyac-
rylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase
was active on levan, inulin, and sucrose with Km values of 1.2 mM, 6.8 mM, and 65 mM, respectively. The pH
optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55&C. Levanase was rapidly
inactivated at 60&C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme
activity was completely inactivated by Ag1 and Hg21 ions, indicating that a sulfhydryl group is involved. A ratio
of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50
mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and
levan could be observed by thin-layer chromatography and size exclusion chromatography–low-angle laser
light scattering–interferometric differential refractive index techniques. This indicates that levanase is an
exoenzyme acting by the single-chain mode.

Enzymes involved in the hydrolysis of polyfructans are of
interest both for fundamental studies and for industrial appli-
cations. Especially inulin is of growing interest as a renewable
carbohydrate raw material for biotechnology. Two aspects are
of main importance: (i) production of pure fructose syrups,
so-called high-fructose inulin syrups, by enzymatic hydrolysis
of inulin (43) and (ii) direct fermentation of inulin by employ-
ing inulinase-producing microbes in order to synthesize various
products such as ethanol or aceton-butanol (20, 21).
b-D-Fructofuranosidases are usually classified upon their
ability to hydrolyze levan (levanases), inulin (inulinases), and
also the disaccharide sucrose (sucrases and invertases). How-
ever, many of these enzymes are capable of hydrolyzing more
than one type of these substrates. Inulinases (or inulases)
which are specific for inulin have been isolated only from
Jerusalem artichoke tubers (7, 12), whereas levanases which
are specific for levan have been isolated from bacteria only.
Examples are the levanases of Streptococcus salivarius KTA-19
(38) and Actinomyces viscosus ATCC 19246 (16). Conversely, a
variety of nonspecific b-D-fructofuranosidases have been found
in bacteria, yeasts, and fungi. For example, inulinases and
levanases which are capable of hydrolyzing inulin, levan, and
sucrose have been isolated from Bacillus subtilis (17), Actino-
myces viscosus ATCC 15987 (24), Streptococcus mutans (5),
Kluyveromyces fragilis (35), Chrysosporium pannorum (46, 47)
and Penicillium sp. strain (27). Enzymes active on inulin and
sucrose but not on levan have been found in filamentous fungi,
among them the b-fructofuranosidases (I to III) from Aspergil-
lus niger (42), the F2 inulinase from C. pannorum (47), and the
PII inulinase from Aspergillus niger (26). The inulinase II from
* Corresponding author. Mailing address: Institut für Biotechnolo-
gie, Arbeitsgruppe Genetik, Technische Universität, Petersgasse 12,
A-8010 Graz, Austria. Phone: (43) 316-873-8418. Fax: (43) 316-811-
050. Electronic mail address: genetik@fscm1.dnet.tu-graz.ac.at.
† Present address: Department of Biological Chemistry, UCLA
School of Medicine, Los Angeles, CA 90024-1737.
Arthrobacter ureafaciens (41) splits levan and inulin, but no
sucrose-hydrolyzing activity could be detected. However, un-
specific b-fructosidases which are active on levan and sucrose
but not on inulin have not been described yet.
Most inulinases and levanases from microorganisms which
were investigated in more detail have been found to be exoen-
zymes (11). They attack the inulin or levan molecules from the
fructose end and liberate fructose as the sole reaction product.
Exoinulinases or exolevanases are incapable of hydrolyzing
melezitose (3-O-a-D-glucopyranosyl-b-D-fructofuranosyl-a-D-
glucopyranoside), a trisaccharide with the same terminal con-
figuration as inulin (36). In melezitose the centrally located
fructose is protected from terminal hydrolysis by the second
glycosyl residue. Only endoinulinases are capable of hydrolyz-
ing melezitose. Most enzymes of this type have been isolated
and characterized from fungi, among them the endoinulinases
from Aspergillus ficuum (10) and the endoinulinase from C.
pannorum (47).
Most microbial levanases and inulinases which have been
purified and characterized in more detail were isolated from
yeasts (31, 32, 35, 36) and filamentous fungi (10, 11, 26, 27, 46,
47), whereas only a few bacterial enzymes have been purified
and characterized so far (5, 16).
The enzyme levanase from B. subtilis is a b-D-fructofurano-
sidase capable of hydrolyzing levan, inulin, and sucrose (17, 22,
23, 33, 34). B. subtilis levanase was first described by Kunst et
al. (17) and assigned as levanase because specific activity on
levan, inulin, sucrose, and raffinose was observed. However,
according to the Avigad and Bauer classification (2), it should
be assigned as a nonspecific b-fructofuranosidase to distin-
guish it from true levanases (specific 2 3 6 activity). Levanase
has been partially purified (22) and characterized with crude
protein extracts (17). Levanase expression in B. subtilis is
tightly regulated (23), and detectable amounts of enzyme are
found only with regulatory mutants (sacL mutants). The struc-
tural gene coding for levanase has been cloned in Escherichia
coli (13), sequenced, and characterized in detail (22, 33, 34).

1953
1954 WANKER ET AL.
The cloned gene has been overexpressed in E. coli, where a
levanase protein of about 75 kDa is found mainly intracellu-
larly, despite the presence of a secretion signal (44).
In this report we describe the purification and detailed char-
acterization, including the mechanism of action, of the B. sub-
tilis levanase overexpressed in E. coli, focusing on the inulin-
degrading activity of this enzyme.
MATERIALS AND METHODS
Bacterial strain and culture conditions. E. coli HB101 [ATCC 33649; F2
hsdS20(rB2 mB2) supE44 araA2 rpsL20(strR) xyl-5 mtl-I recA13] harboring
pESI7HE (44) was used in all experiments. The bacterial cells were routinely
grown at 378C in LB medium (1% Bacto Tryptone, 0.5% Bacto Yeast Extract,
0.5% NaCl) or M9 mineral salts medium (25), supplemented with essential
amino acids (20 mg/liter) and with thiamine (1 mg/liter), containing 0.5% glucose
or sucrose. For plasmid selection, ampicillin (100 mg/liter) was added. For
induction of the tac promoter, E. coli cultures were supplemented with 1 mM
isopropyl-b-D-thiogalactopyranoside (IPTG) at inoculation or in the middle of
the exponential phase. The cells were harvested by centrifugation after an incu-
bation of 16 to 24 h at a final optical density of about 0.5 (at 600 nm; Beckmann
DU 50 photometer) when grown in M9 mineral salts medium or of 1.5 to 2 when
grown in LB medium.
Protein determination and enzymatic assays. The protein concentration was
determined by the method of Bradford (4) by using a commercial reagent kit
(Bio-Rad, Richmond, Calif.) as recommended by the supplier. Bovine serum
albumin (Bio-Rad) was used as the standard.
Unless otherwise noted, all enzymatic assays were carried out in inulin reac-
tion buffer (0.2 M Na2HPO4, 0.2 M sodium acetate, pH 5.5) with dissolved (3 min
at 958C) or suspended (room temperature) inulin (Sigma, St. Louis, Mo.; average
molecular weight, 5,000), dissolved levan (Sigma; average molecular weight, 107),
or sucrose (Sigma) as a substrate.
For determination of inulinase activity, inulin was dissolved or suspended in
inulin reaction buffer to a final concentration of 50 mg/ml, and following addition
of enzyme (appropriate dilution) the mixture was incubated at 37 or 558C for 30
min. The levanase and sucrase activities were measured by using substrate con-
centrations of 25 and 50 mg/ml, respectively. Incubation was performed at 558C
over 30 min. With all three substrates the liberation of free fructose (and/or
glucose) was enzymatically determined (commercial test combination; Boehr-
inger GmbH, Mannheim, Germany) and was linear over this time period. One
unit of activity was defined as the amount of enzyme required to liberate 1 mmol
of fructose per min.
For determination of the pH optimum, inulinase activity was determined by
incubating purified levanase enzyme (0.625 mg/ml) with 50 mg of inulin per ml,
dissolved in inulin reaction buffer with pH values between 4 and 7, at 378C over
30 min. The temperature optimum was determined analogously with a constant
pH of 5.5 and different temperatures.
For determination of the thermostability, purified levanase enzyme (0.2 mg/
ml) was incubated in inulinase reaction buffer at 50, 55, and 608C. Samples were
taken after 30, 60, and 90 min, and retaining inulinase activity was measured with
50 mg of dissolved inulin per ml at 558C for 30 min.
The influence of additives on thermostability was investigated by incubating
purified levanase enzyme (0.2 mg/ml) at 608C in inulin reaction buffer (pH 5.5)
with fructose (30%, wt/vol) or glycerol (26.2%, wt/vol). Samples were taken after
10, 30, and 45 min, and inulinase activity (at 558C) was determined (as described
above).
For determination of the substrate specificity, purified levanase enzyme was
incubated with different substrates in inulin reaction buffer (pH 5.5) for 30 min
at 558C.
The influence of different ions and reagents on enzyme activity was investi-
gated by determining the inulinase activity of purified levanase in the presence of
1 mM ions or agents (558C).
Determination of enzyme parameters (Km and Vmax) was performed as fol-
lows: the concentrations of inulin (average molecular weight, 5,000), levan (av-
erage molecular weight, 107), and sucrose were varied between 2 and 14, 0.0001
and 0.011, and 10 and 150 mM, respectively. The substrates were dissolved in
inulin reaction buffer (pH 5.5) and incubated with purified levanase enzyme
(inulin and levan, 0.5 mg/ml; sucrose, 0.25 mg/ml) at 558C for 30 min. From the
amount of liberated fructose the initial velocity (v0) was determined. The enzyme
parameters Vmax and Km were calculated with a computer program based on the
evaluation methods of Wilkinson (45) and Eisenthal and Cornish-Bowden (9).
Atkins and Nimmo (1) tested different evaluation methods for the determination
of Km and Vmax and found that with these methods the best results were ob-
tained.
For the size exclusion chromatography–low-angle laser light scattering (SEC-
LALLS) and thin-layer chromatography (TLC) analyses inulin (50 and 100
mg/ml) and levan (30 mg/ml) were dissolved in inulin reaction buffer (pH 5.5)
and incubated with purified levanase (inulin, 5 mg/ml; levan, 4 mg/ml) at 558C.
Samples were taken in intervals over a longer period, and hydrolysis was stopped
APPL. ENVIRON. MICROBIOL.
FIG. 1. DEAE–Sepharose CL-6B chromatography of intracellular levanase
produced in E. coli HB101. Ammonium sulfate-precipitated protein was applied
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on a DEAE–Sepharose CL-6B column, and elution was performed with a 0 to 0.4
M NaCl gradient. The gradient was obtained by mixing a 25 mM Tris (pH 8)
buffer with a 1 M NaCl solution.
by heating samples to 958C. The fructose concentration in the samples was
determined enzymatically (test combination).
Purification of levanase. Recombinant E. coli cells were grown in shake flask
cultures (300 ml) with glucose M9 mineral salts medium containing ampicillin.
For induction of levanase expression, 1 mM IPTG was added at the time of
inoculation and cultures were incubated for 16 h at 378C (150 rpm). Cells were
harvested, washed with 0.9% NaCl, resuspended in 25 mM Tris-HCl buffer (pH
8), and disrupted by sonication (Braun Labsonic 2000) with four pulses of 30 s
each on ice. The cell debris was removed by centrifugation (65,500 3 g) for 1 h.
The supernatant was stored at 2208C until used for the following purification
steps.
(i) Ammonium sulfate precipitation. The crude lysate was first brought to 20%
saturation by addition of solid ammonium sulfate, and after incubation for 1 h
the precipitated protein was removed by centrifugation and discarded. To the
remaining supernatant solid ammonium sulfate was added stepwise to 40, 60, and
80% saturation, and the precipitated protein was harvested by centrifugation
after each step. The pellets obtained after each precipitation step were dissolved
in a small amount of 25 mM Tris-HCl buffer (pH 8), and the protein solution was
desalted by dialyzing overnight at 48C against the same buffer. The protein
concentration and inulinase activity were determined in the different protein
fractions.
(ii) DEAE–Sepharose CL-6B chromatography. Dialyzed protein solution (25
ml) was loaded on a Sepharose CL-6B anion-exchange column (HR 10/50)
preequilibrated with 25 mM Tris-HCl buffer (pH 8). The column was washed
with 50 ml of the same buffer, and proteins were eluted with a linear gradient of
NaCl from 0 to 0.4 M. Fractions (5 ml) were collected every 5 min.
(iii) S-Sepharose chromatography. Fractions with inulinase activity were
pooled, dialyzed overnight at 48C against 10 mM sodium acetate buffer (pH 5)
and loaded on an S-Sepharose cation-exchange column (HR 10/30) preequili-
brated with 10 mM sodium acetate buffer (pH 5). The columns were washed with
10 mM sodium acetate buffer (pH 5), and proteins were eluted with a linear
gradient of 0 to 1 M NaCl (1 ml/min).
(iv) MonoQ-Sepharose chromatography. Fractions with inulinase activity
eluted from the S-Sepharose column were pooled and dialyzed overnight at 48C
against 25 mM Tris-HCl–50 mM NaCl buffer (pH 8) and loaded on a MonoQ-
Sepharose column (HR 5/5) preequilibrated with the same buffer. Following
washing the column with 25 mM Tris-HCl–50 mM NaCl buffer (pH 8), proteins
were eluted with a linear NaCl gradient (0 to 0.5 M) at a flow rate of 0.5 ml/min.
SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) analysis was performed with the Mini-Protean II Dual Slab Cell System
(Bio-Rad). The stacking gel contained 5% (wt/vol) acrylamide, and the separat-
ing gel contained 7.5 or 12.5% acrylamide as described by Laemmli (18). The
samples were mixed in 1:1 ratio (vol/vol) with buffer containing 0.17 M Tris-HCl
(pH 6.8), 3% SDS, 30% glycerol, and 10% 2-mercaptoethanol and heated (5 min,
958C) before application. Approximate molecular masses were determined by
using commercially available marker proteins as standards (Pharmacia, Uppsala,
Sweden). The gels were stained for proteins with Coomassie brilliant blue R.
SEC-DRI-LALLS technique. For the determination of molecular weight av-
erages and molecular weight distributions of the dissolved polysaccharides, the
SEC-interferometric differential refractive index (DRI)-LALLS technique was
used. The experimental setup includes a chromatographic section with a series of
SEC columns containing TSK G5000 PW and TSK G4000 PW, each with a
length of 600 mm and an inner diameter of 7.5 mm, from Toyo Soda Manufac-
turing Co., Tokyo, Japan. For each characterization 200 ml of the sample solution
was injected at room temperature (208C), while the eluent (0.05 M aqueous
NaCl) was pumped through the SEC system with a constant flow rate of 0.8
VOL. 61, 1995 B. SUBTILIS LEVANASE PURIFICATION AND CHARACTERIZATION 1955

TABLE 1. Purification of B. subtilis levanase produced in E. coli

Purification
Step Protein (mg) Total activity (U)a Sp act (U/mg) Yield (%)
(fold)

Crude extract 1,940 4,100 2.1 100 1.0

Ammonium sulfate precipitation 1,170 3,760 3.2 91.5 1.5
DEAE–Sepharose CL-6B
Peak I 13.1 820 62.6 20 30
Peak II 12.7 1,140 89.4 27.7 43
S-Sepharoseb
Peak I 4.6 369 80.2 9 38
Peak II 2.4 206 84.3 5 40
MonoQ 1.3 122 97.4 3 46
a
Determined at 378C with 50 mg of suspended inulin per ml as the substrate in inulin reaction buffer, pH 5.0.
b
Proteins from the two peaks obtained by DEAE–Sepharose CL-6B chromatography were separately purified by S-Sepharose chromatography.

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ml/min by the high-pressure liquid chromatography pump (Constametric 3000; proved to be an advantageous step, as highly concentrated
LDC Analytical, Riviera Beach, Fla.). A 2-mm-pore-size metal filter block at the levanase protein was eluted as a single peak before increasing
reservoir end and a 0.45-mm-pore-size cellulose acetate in-line filter behind the
SEC columns purified the eluent and sample solution, respectively. In the de- the salt concentration, having a specific inulinase activity of 97
tection section of this experimental arrangement, a LALLS detector (KMX-6 U/mg at 378C. The levanase enzyme in this fraction was finally
from LDC Analytical) provides absolute information about molecular weight purified 46-fold with a 3% yield. Table 1 summarizes the re-
and an interferometric DRI detector (Optilab 903 with filter for l 5 630 nm sults of a typical purification experiment.
from Wyatt Technology, Santa Barbara, Calif.) provides information about the
actual concentrations of the separated sample components. Data acquisition and Substrate specificity. The substrate specificity of levanase
data processing were supported by the software packages PCLALLS and for poly- and oligosaccharides was examined by measuring the
PCGPC from LDC Analytical. liberation of fructose and glucose (Table 2). The purified
TLC of hydrolysis products. TLC of reaction products after enzymatic hydro- levanase enzyme mainly hydrolyzed levan, inulin, sucrose, and
lysis of inulin and levan was done on Kieselgel 60 plates (Merck, Darmstadt,
Germany) with 40:50:10 (vol/vol/vol) methanol-acetone-water as a solvent. The to a small extent raffinose, whereas melezitose, stachylose,
carbohydrates on TLC plates were visualized by heating the plates up to 958C cellobiose, maltose, and lactose were not hydrolyzed. Interest-
after spraying them with vanillin-sulfuric acid. ingly, the relative activity found with suspended inulin was only
about 50% lower than that found with dissolved inulin. This
RESULTS indicates that the B. subtilis levanase is well suited for hydro-
lyzing native inulin present as storage material in plants.
Culture conditions for levanase expression. Various culture Effects of pH, ions, and agents. The influence of pH on the
conditions were tested for optimal enzyme expression. Al- inulinase activity of levanase was examined as described in
though LB medium yielded the best growth after an incubation Materials and Methods. A broad pH optimum between pH 5
of 16 h, with a final optical density of 1.7 at 500 nm, compared and 6.5 was observed in assays with inulin reaction buffer. The
with an optical density of 0.5 obtained with glucose M9 mineral activity drops down drastically at pH values below 4.5 and
salts medium, cultures grown in the M9 medium yielded the above 7.
highest inulinase activities of about 30 to 40 U/ml in cell ex- The effects of various ions and agents on inulinase activity
tracts, compared with 3 to 4 U/ml for cultures grown in LB were investigated at a concentration of 1 mM. Hg21 and Ag1
medium. The use of glucose or sucrose as a substrate in M9 ions, which are known to affect thiol groups, totally inactivated
medium had no significant influence on the resulting inulinase inulinase activity. The addition of Fe21 (can be oxidized to
activity. An incubation time of 16 to 20 h was necessary for Fe31 in the presence of air) decreased the activity of levanase
efficient levanase production. Within the growth cycle, the to 16%, whereas Cs1, Na1, Li1, NH41, Cu21, Ca21, Zn21,
highest inulinase activities were reached at the end of the EDTA, dithiothreitol, and mercaptoethanol had no significant
exponential growth phase when induction was performed with influence on inulin-hydrolyzing activity.
1 mM IPTG at inoculation. When 1 mM IPTG was added in Temperature optimum and thermostability. The tempera-
the middle of the logarithmic phase, low levels of inulinase ture dependence of the inulinase activity of levanase was ex-
activity (3 to 4 U/ml) in M9 minimal medium were measured in
E. coli cell extracts. Despite the presence of a signal sequence,
almost the entire inulinase activity was always present intra-
cellularly (44). TABLE 2. Substrate specificity of levanase
Purification of levanase. The purification steps are described
in detail in Materials and Methods. During chromatography on Concn Relative activity (%)b
Substratea
a DEAE–Sepharose CL-6B column, inulinase activity was (mg/ml) Fructose Glucose
found in two peaks eluting at NaCl concentrations of 0.25 and
0.3 M (Fig. 1). However, during S-Sepharose chromatography, Sucrose 50 100.0 100.0
the levanase proteins of both fractions behaved identically and Sucrose 10 26.3 ND
could be eluted as uniform peaks at 0.71 M NaCl. In addition, Inulin, dissolved 50 81.4 0.0
Inulin, suspended 50 41.6 ND
when fractions of these two peaks were analyzed by SDS- Levan 25 52.9 2.2
PAGE, no differences in the molecular weights of the levanase Raffinose 50 3.6 1.4
proteins could be observed (data not shown). The S-Sepharose
a
chromatography step resulted in an apparently pure prepara- All substrates were dissolved or suspended in inulin reaction buffer, pH 5.5.
b
Sucrose (50 mg/ml) was used as the internal standard, and the resulting
tion of levanase protein. Although no impurities were detect- activities in terms of produced fructose and glucose were set at 100%. ND, not
able after analysis by SDS-PAGE and Coomassie staining, determined. No reaction was found with the sugars cellobiose, lactose, maltose,
subsequent chromatography on a MonoQ-Sepharose column and melezitose (50 mM each) and stachylose (10 mM).
1956 WANKER ET AL.
TABLE 3. Determination of enzyme parameters
Substrate Mol wt Km
Inulin 5 3 103a 6.7 mM
Levan 1 3 107a 1.2 mM
Sucrose 342.3 63.6 mM
a
Average molecular weight data provided by the manufacturer were used for calculations. The size distribution for inulin can be seen in Fig. 3C.
amined in the range between 20 and 708C. The enzyme was
shown to be most active between 47 and 558C in standard
30-min reactions. In addition, long-term temperature treat-
ment of levanase revealed high stability at 50 and 558C. No
significant loss of activity could be seen within 2 h at these
temperatures, whereas at 608C the enzyme was inactivated
rapidly and practically no activity was retained after 30 min.
However, adding fructose or glycerol to the enzyme prepara-
tion left enzyme activity almost unaffected after incubation of
40 min at 608C. A similar stabilization effect was obtained
when the enzyme was incubated with polyclonal levanase an-
tibodies.
Enzyme kinetics. The kinetic properties of levanase were
studied with the MonoQ-purified enzyme by measuring the
initial velocity of the reaction at 558C. The Km values of inulin,
levan, and sucrose were 6.7 mM, 1.2 mM, and 63.6 mM, re-
spectively. The maximum initial velocities for the conversion of
these substrates to fructose were 2.17 (inulin), 0.96 (levan),
and 0.91 (sucrose) nmol s21. From these data the turnover
numbers (kcat) and specificity constants (kcat/Km) for each sub-
strate were calculated with the equation Vmax 5 kcat 3 [E]0,
where [E]0 is the initial enzyme concentration. The determined
kinetic parameters are summarized in Table 3. All parameters
were calculated with enzyme concentrations which had been
determined with the micromethod of Bio-Rad (4) and the
molecular mass of 75 kDa determined for levanase.
Products and mechanism of hydrolysis. To determine
whether the levanase enzyme attacks inulin randomly or at
defined positions within the polymer (endoattack) or starts
hydrolysis from the fructose or glucose end (exoattack), the
purified enzyme was incubated with inulin and the liberation of
free fructose and glucose was determined enzymatically (Fig.
2). Samples taken at different time intervals were separated on
Kieselgel plates (TLC), and fructose was found to be the first
and only detectable monosaccharide which was liberated dur-
ing hydrolysis. It was further observed that over the entire
hydrolysis period the position of the oligosaccharide spot (in-
ulin) did not move to lower-molecular-weight positions; only
the intensity decreased with increasing incubation time. Glu-
cose liberation was below the detection limit on Kieselgel
plates. The absence of detectable amounts of lower-molecular-
weight oligosaccharides at any stage of hydrolysis encourages
the assumption of levanase being an exoacting enzyme.
For an actual screening of molecular weight distribution, the
intermediates of inulin at increasing stages of hydrolysis due to
levanase catalysis were investigated by means of the SEC-DRI-
LALLS technique, which has been proved over years to be a
powerful tool for the determination of absolute molecular
weights of plastics and biopolymers (3, 14) and recently was
successfully applied to determine changes in the molecular
weight distribution of inulin due to exoinulinase attack (15).
Stages of increasing hydrolysis shown in Fig. 2 were analyzed,
and the results of selected stages (a to d) are shown in Fig. 3.
Within experimental error the composition of the initially ap-
plied inulin substrate (G-Fn, where G is the glucose residue
and Fn is n fructose residues) remained constant till the end of
APPL. ENVIRON. MICROBIOL.
Vmax (nmol s21) kcat (s21) kcat/Km (M21 s21)
2.17 827 1.2 3 105
0.96 370 3.0 3 108
0.91 686 1.1 3 104
hydrolysis. No obvious shift toward midrange n values could be
observed, an endohydrolysis action of levanase could be ex-
cluded, and thus, the already expected exohydrolysis action of
levanase was supported. The product liberated from the very
beginning of the degradation process catalyzed by levanase was
again identified to be fructose. No significant diminishing of
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the highest-molecular-weight components of inulin could be
detected during the degradation process. This fact indicates a
single-chain mechanism of levanase action.
DISCUSSION
A standard method to discriminate between specificity for
high-molecular-weight versus low-molecular-weight substrates
of b(2 3 1) hydrolyzing b-fructofuranosidases (nonspecific or
inulinases) is the determination of ratios of sucrase to inulinase
activity (S/I values). S/I values close to 1 are found for only very
few inulinases from filamentous fungi, whereas typically S/I
values between 20 and 45 are reported for most inulinases
from yeast. Depending on the substrate concentration, S/I val-
ues between 0.32 and 1.23 were found for the investigated B.
subtilis levanase, indicating that this enzyme is more selective
for inulin than are enzymes from many other sources.
The homogeneous levanase fraction which was obtained af-
ter MonoQ chromatography showed a specific inulin-degrad-
ing activity of 175 U/mg at 378C with 50 mg of dissolved inulin
per ml as the substrate. No data on inulinase activity based on
solid (suspended) inulin could be found in the literature. How-
ever, the value of 97 U/mg measured with the purified levanase
protein (50 mg of suspended inulin per ml at 378C) indicates
FIG. 2. Preparative hydrolysis of inulin with purified levanase enzyme for
SEC-DRI-LALLS and TLC analysis. One milliliter of inulin solution (50 mg/ml)
was incubated with 5 mg of purified levanase per ml in a 1.5-ml Eppendorf vial
at 558C. Samples of 100 ml were taken at various times over a 300-min period,
and hydrolysis was stopped by heating samples to 958C for 5 min. The fructose
and glucose concentrations in the samples were determined enzymatically with a
commercial test combination (Boehringer GmbH). The indicated hydrolysis
stages a to d are illustrated in Fig. 3.
VOL. 61, 1995 B. SUBTILIS LEVANASE PURIFICATION AND CHARACTERIZATION
that this enzyme efficiently hydrolyzes the undissolved solid
substrate, a property of high value for industrial applications.
In comparison, for inulinase from Kluyveromyces marxianus (20
mg of dissolved inulin per ml at 508C) and for inulinases F2,
F3, and F4 from C. pannorum (5 mg of dissolved inulin per ml
at 308C) specific inulin-degrading activities of 119, 41.5, 10.5,
and 106 U/mg, respectively, have been determined (30, 46, 47).
The specific levan-degrading activity of 194 U/mg (30 mg of
dissolved levan per ml at 558C) found with the purified levan-
ase protein is lower but in the same order of magnitude as
reported for other microbial levanases. In comparison, levan-
ases from S. salivarius KTA-19 (38) and Actinomyces viscosus
ATCC 19246 (16) and the nonspecific exo-b-fructosidase from
S. salivarius KTA-19 (39) yielded levan-degrading activities of
about 850, 851, and 433 U/mg at 378C with 1 mg of dissolved
levan per ml.
The determined optimal pH and temperature ranges are in
good agreement with reported values for inulinases and levan-
ases from bacteria (8, 24), yeasts (28, 36), and filamentous
fungi (6, 43, 46, 47). In general, nonspecific b-fructofuranosi-
dases from yeasts and filamentous fungi are supposed to have
lower pH optima than bacterial enzymes, but comparison has
to be done cautiously, considering differences in substrate con-
centrations and assay conditions.
The inhibition patterns of the inulin-degrading activity by
different metal ions and agents maintain the similarity between
B. subtilis levanase and almost all inulinases, levanases, and
b-fructofuranosidases for which such studies are reported (5,
17, 27, 39, 47, 48). Because total inactivation can be achieved
by 1 mM Ag1 and Hg21 ions, there is strong evidence for a
situation in which SH groups are essential for the catalytic
activity. In the case of B. subtilis levanase, only one cysteine,
1957
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FIG. 3. Analysis of inulin hydrolysis with purified levanase by SEC-DRI-
LALLS technique. (A) Normalized mass chromatograms of inulin and fructose;
(B and C) normalized mass chromatograms (B) and normalized molecular
weight distributions (C) of inulin hydrolysis. Samples a to d were taken at the
times indicated in Fig. 2. Normalization: total peak areas were set to 1.0; V(ret),
retention volume; log(M), decadic logarithm of molecular weight; diff. wt., dif-
ferential weight.
which could be affected by these cations, is located (22, 33) in
the N-terminal region, at amino acid position 223. Potential
N-terminally located active centers have been proposed for the
invertase from Saccharomyces cerevisiae (40), the intracellular
invertase from Zymomonas mobilis (48), and the inulinase
from K. marxianus (19), strongly supporting the assumption of
a similar N-terminally located active center for all b-fructosi-
dases. The previously found fact, that deletion of 114 amino
acids from the C-terminal part of the levanase did not affect
inulinase activity (34), supports this suggestion.
Kinetic parameters of B. subtilis levanase were found to be in
the same range as those determined for other microbial
b-fructofuranosidases. However, one has to be aware of the
fact that the molecular weights of the polymeric substrates
inulin and levan are not clearly defined, as only average mo-
lecular weights are calculated. For sucrose, Km values between
2 and 65 mM have been found (11, 29, 31, 36), indicating a
broad range of substrate affinity. The lowest Km values have
been found with the invertases from Fusarium oxysporum, be-
tween 2.2 and 4.4 mM (29). With Km values between 61 and 65
mM, B. subtilis levanase is in the upper range.
For inulin, Km values between 0.0012 and 33 mM were
determined (8, 11, 26, 27). Compared with inulinases from
yeasts, inulinases from filamentous fungi usually have lower Km
values, indicating a higher substrate affinity for these enzymes.
Nevertheless, the lowest Km value (1.2 mM) was reported for a
bacterial inulinase from Clostridium acetobutylicum (8). An
expected high affinity of B. subtilis levanase for levan was ver-
ified with the determined low Km value of 1.2 mM. For the
hydrolysis of inulin with purified levanase, a kcat value of about
827 s21 was determined. This is a high value compared with the
known data of inulinases from Aspergillus ficuum (11) with kcat
values between 0.06 and 0.46 s21. In contrast, the Km values of
B. subtilis levanase and the inulinases from Aspergillus ficuum
range in the same order of magnitude (11). When comparing
the kcat/Km of these enzymes (1.2 3 105 for B. subtilis levanase
and 1 to 60 for the Aspergillus ficuum inulinases) it can be seen
that B. subtilis levanase is well suited for inulin hydrolysis. The
kcat/Km value of 108 for levan hydrolysis by B. subtilis levanase
already comes into the range of values found for enzymes that
have attained kinetic perfection (37).
1958 WANKER ET AL.
By means of analyzing reaction products of inulin hydrolysis
by TLC and SEC-DRI-LALLS techniques, the mode of attack
of the purified levanase was identified as an exohydrolase ac-
tion. As reported above, melezitose could not be hydrolyzed
(Table 2). This trisaccharide has a configuration identical to
that of inulin, but the medial fructose is protected from termi-
nal hydrolysis by glucosyl residues. Therefore, this fact pro-
vides additional evidence that levanase degrades inulin-like
molecules from the fructose end. Two possible mechanisms for
an exohydrolase can be distinguished: the multiple-chain and
single-chain modes. A multiple-chain mechanism would lead
to a situation in which the enzyme molecules randomly attack
oligosaccharide chains and thus because of the cut-and-go ac-
tion a shift in the molecular weight distribution towards
midrange- and low-n-value intermediates has to be expected.
With a single-chain mechanism an enzyme molecule attacks an
oligosaccharide chain and is bound to it until complete hydro-
lysis. In this case, and assuming that no significant preference
for any specific n-value components of the different G-Fn
chains exists, no detectable amounts of midrange- and low-n-
value oligosaccharides are expected. The finding that the initial
molecular weight distribution of inulin remains constant within
experimental error during the enzymatically catalyzed hydrolysis
yielding fructose as the only product favors the assumption of a
single-chain mechanism for the action of levanase on inulin.
ACKNOWLEDGMENTS
This work was supported by grants from the Bundesministerium für
Land- und Forstwirtschaft, the Bundesministerium für Wissenschaft
und Forschung, and the Land Steiermark.
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