Purification and Characterization of the Bacillus Subtilis Levanase

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1995, p. 1953–1958 Vol. 61, No. 5 0099-2240/95/$04.00ϩ0 Copyright ᭧ 1995, American Society for Microbiology

Puriﬁcation and Characterization of the Bacillus subtilis Levanase Produced in Escherichia coli

1 2 1 ERICH WANKER, † ANTON HUBER, AND HELMUT SCHWAB * Institut fu¨r Biotechnologie, Arbeitsgruppe Genetik, Technische Universita¨t,1 and Institut fu¨r Physikalische Chemie, Karl-Franzens-Universita¨t,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- rose. The puriﬁed protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide 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 ␮M, 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 Ag؉ and Hg2؉ 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 Arthrobacter ureafaciens (41) splits levan and inulin, but no interest both for fundamental studies and for industrial appli- sucrose-hydrolyzing activity could be detected. However, un- cations. Especially inulin is of growing interest as a renewable specific ␤-fructosidases which are active on levan and sucrose carbohydrate raw material for biotechnology. Two aspects are but not on inulin have not been described yet. of main importance: (i) production of pure fructose syrups, Most inulinases and levanases from microorganisms which so-called high-fructose inulin syrups, by enzymatic hydrolysis were investigated in more detail have been found to be exoen- of inulin (43) and (ii) direct fermentation of inulin by employ- zymes (11). They attack the inulin or levan molecules from the ing inulinase-producing microbes in order to synthesize various fructose end and liberate fructose as the sole reaction product. products such as ethanol or aceton-butanol (20, 21). Exoinulinases or exolevanases are incapable of hydrolyzing ␤-D-Fructofuranosidases are usually classified upon their melezitose (3-O-␣-D-glucopyranosyl-␤-D-fructofuranosyl-␣-D- ability to hydrolyze levan (levanases), inulin (inulinases), and glucopyranoside), a trisaccharide with the same terminal con- also the disaccharide sucrose (sucrases and invertases). How- figuration as inulin (36). In melezitose the centrally located ever, many of these enzymes are capable of hydrolyzing more fructose is protected from terminal hydrolysis by the second than one type of these substrates. Inulinases (or inulases) glycosyl residue. Only endoinulinases are capable of hydrolyz- which are specific for inulin have been isolated only from ing melezitose. Most enzymes of this type have been isolated Jerusalem artichoke tubers (7, 12), whereas levanases which and characterized from fungi, among them the endoinulinases are specific for levan have been isolated from bacteria only. from Aspergillus ficuum (10) and the endoinulinase from C. Examples are the levanases of Streptococcus salivarius KTA-19 pannorum (47). (38) and Actinomyces viscosus ATCC 19246 (16). Conversely, a Most microbial levanases and inulinases which have been variety of nonspecific -D-fructofuranosidases have been found ␤ purified and characterized in more detail were isolated from in bacteria, yeasts, and fungi. For example, inulinases and yeasts (31, 32, 35, 36) and filamentous fungi (10, 11, 26, 27, 46, levanases which are capable of hydrolyzing inulin, levan, and 47), whereas only a few bacterial enzymes have been purified sucrose have been isolated from Bacillus subtilis (17), Actino- and characterized so far (5, 16). myces viscosus ATCC 15987 (24), Streptococcus mutans (5), The enzyme levanase from B. subtilis is a ␤-D-fructofurano- Kluyveromyces fragilis (35), Chrysosporium pannorum (46, 47) sidase capable of hydrolyzing levan, inulin, and sucrose (17, 22, and Penicillium sp. strain (27). Enzymes active on inulin and 23, 33, 34). B. subtilis levanase was first described by Kunst et sucrose but not on levan have been found in filamentous fungi, al. (17) and assigned as levanase because specific activity on among them the -fructofuranosidases (I to III) from Aspergil- ␤ levan, inulin, sucrose, and raffinose was observed. However, lus niger (42), the F2 inulinase from C. pannorum (47), and the according to the Avigad and Bauer classification (2), it should PII inulinase from Aspergillus niger (26). The inulinase II from be assigned as a nonspecific ␤-fructofuranosidase to distin- guish it from true levanases (specific 2 3 6 activity). Levanase has been partially purified (22) and characterized with crude * Corresponding author. Mailing address: Institut fu¨r Biotechnolo- gie, Arbeitsgruppe Genetik, Technische Universita¨t, Petersgasse 12, protein extracts (17). Levanase expression in B. subtilis is A-8010 Graz, Austria. Phone: (43) 316-873-8418. Fax: (43) 316-811- tightly regulated (23), and detectable amounts of enzyme are 050. Electronic mail address: [email protected]. found only with regulatory mutants (sacL mutants). The struc- † Present address: Department of Biological Chemistry, UCLA tural gene coding for levanase has been cloned in Escherichia School of Medicine, Los Angeles, CA 90024-1737. coli (13), sequenced, and characterized in detail (22, 33, 34).

1953 1954 WANKER ET AL. APPL.ENVIRON.MICROBIOL.

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 characterization, including the mechanism of action, of the B. subtilis 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; FϪ Ϫ Ϫ hsdS20(rB mB ) supE44 araA2 rpsL20(strR) xyl-5 mtl-I recA13] harboring pESI7HE (44) was used in all experiments. The bacterial cells were routinely grown at 37ЊC in LB medium (1% Bacto Tryptone, 0.5% Bacto Yeast Extract, 0.5% NaCl) or M9 mineral salts medium (25), supplemented with essential FIG. 1. DEAE–Sepharose CL-6B chromatography of intracellular levanase amino acids (20 mg/liter) and with thiamine (1 mg/liter), containing 0.5% glucose produced in E. coli HB101. Ammonium sulfate-precipitated protein was applied or sucrose. For plasmid selection, ampicillin (100 mg/liter) was added. For on a DEAE–Sepharose CL-6B column, and elution was performed witha0to0.4 induction of the tac promoter, E. coli cultures were supplemented with 1 mM M NaCl gradient. The gradient was obtained by mixing a 25 mM Tris (pH 8) isopropyl-␤-D-thiogalactopyranoside (IPTG) at inoculation or in the middle of bufferwitha1MNaCl solution. the exponential phase. The cells were harvested by centrifugation after an incubation 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 by heating samples to 95ЊC. The fructose concentration in the samples was determined by the method of Bradford (4) by using a commercial reagent kit determined enzymatically (test combination). (Bio-Rad, Richmond, Calif.) as recommended by the supplier. Bovine serum Purification of levanase. Recombinant E. coli cells were grown in shake flask albumin (Bio-Rad) was used as the standard. cultures (300 ml) with glucose M9 mineral salts medium containing ampicillin. Unless otherwise noted, all enzymatic assays were carried out in inulin reac- For induction of levanase expression, 1 mM IPTG was added at the time of tion buffer (0.2 M Na2HPO4, 0.2 M sodium acetate, pH 5.5) with dissolved (3 min inoculation and cultures were incubated for 16 h at 37ЊC (150 rpm). Cells were at 95ЊC) or suspended (room temperature) inulin (Sigma, St. Louis, Mo.; average harvested, washed with 0.9% NaCl, resuspended in 25 mM Tris-HCl buffer (pH molecular weight, 5,000), dissolved levan (Sigma; average molecular weight, 107), 8), and disrupted by sonication (Braun Labsonic 2000) with four pulses of 30 s or sucrose (Sigma) as a substrate. each on ice. The cell debris was removed by centrifugation (65,500 ϫ g)for1h. For determination of inulinase activity, inulin was dissolved or suspended in The supernatant was stored at Ϫ20ЊC until used for the following purification inulin reaction buffer to a final concentration of 50 mg/ml, and following addition steps. of enzyme (appropriate dilution) the mixture was incubated at 37 or 55ЊC for 30 (i) Ammonium sulfate precipitation. The crude lysate was first brought to 20% min. The levanase and sucrase activities were measured by using substrate con- saturation by addition of solid ammonium sulfate, and after incubation for 1 h centrations of 25 and 50 mg/ml, respectively. Incubation was performed at 55ЊC the precipitated protein was removed by centrifugation and discarded. To the over 30 min. With all three substrates the liberation of free fructose (and/or remaining supernatant solid ammonium sulfate was added stepwise to 40, 60, and glucose) was enzymatically determined (commercial test combination; Boehr- 80% saturation, and the precipitated protein was harvested by centrifugation inger GmbH, Mannheim, Germany) and was linear over this time period. One after each step. The pellets obtained after each precipitation step were dissolved unit of activity was defined as the amount of enzyme required to liberate 1 ␮mol in a small amount of 25 mM Tris-HCl buffer (pH 8), and the protein solution was of fructose per min. desalted by dialyzing overnight at 4ЊC against the same buffer. The protein For determination of the pH optimum, inulinase activity was determined by concentration and inulinase activity were determined in the different protein incubating purified levanase enzyme (0.625 ␮g/ml) with 50 mg of inulin per ml, fractions. dissolved in inulin reaction buffer with pH values between 4 and 7, at 37ЊC over (ii) DEAE–Sepharose CL-6B chromatography. Dialyzed protein solution (25 30 min. The temperature optimum was determined analogously with a constant ml) was loaded on a Sepharose CL-6B anion-exchange column (HR 10/50) pH of 5.5 and different temperatures. preequilibrated with 25 mM Tris-HCl buffer (pH 8). The column was washed For determination of the thermostability, purified levanase enzyme (0.2 mg/ with 50 ml of the same buffer, and proteins were eluted with a linear gradient of ml) was incubated in inulinase reaction buffer at 50, 55, and 60ЊC. Samples were NaCl from 0 to 0.4 M. Fractions (5 ml) were collected every 5 min. taken after 30, 60, and 90 min, and retaining inulinase activity was measured with (iii) S-Sepharose chromatography. Fractions with inulinase activity were 50 mg of dissolved inulin per ml at 55ЊC for 30 min. pooled, dialyzed overnight at 4ЊC against 10 mM sodium acetate buffer (pH 5) The influence of additives on thermostability was investigated by incubating and loaded on an S-Sepharose cation-exchange column (HR 10/30) preequili- purified levanase enzyme (0.2 mg/ml) at 60ЊC in inulin reaction buffer (pH 5.5) brated with 10 mM sodium acetate buffer (pH 5). The columns were washed with with fructose (30%, wt/vol) or glycerol (26.2%, wt/vol). Samples were taken after 10 mM sodium acetate buffer (pH 5), and proteins were eluted with a linear 10, 30, and 45 min, and inulinase activity (at 55ЊC) was determined (as described gradient of 0 to 1 M NaCl (1 ml/min). above). (iv) MonoQ-Sepharose chromatography. Fractions with inulinase activity For determination of the substrate specificity, purified levanase enzyme was eluted from the S-Sepharose column were pooled and dialyzed overnight at 4ЊC incubated with different substrates in inulin reaction buffer (pH 5.5) for 30 min against 25 mM Tris-HCl–50 mM NaCl buffer (pH 8) and loaded on a MonoQ- at 55ЊC. Sepharose column (HR 5/5) preequilibrated with the same buffer. Following The influence of different ions and reagents on enzyme activity was investi- washing the column with 25 mM Tris-HCl–50 mM NaCl buffer (pH 8), proteins gated by determining the inulinase activity of purified levanase in the presence of were eluted with a linear NaCl gradient (0 to 0.5 M) at a flow rate of 0.5 ml/min. 1 mM ions or agents (55ЊC). SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- Determination of enzyme parameters (Km and Vmax) was performed as fol- PAGE) analysis was performed with the Mini-Protean II Dual Slab Cell System lows: the concentrations of inulin (average molecular weight, 5,000), levan (av- (Bio-Rad). The stacking gel contained 5% (wt/vol) acrylamide, and the separat- erage molecular weight, 107), and sucrose were varied between 2 and 14, 0.0001 ing gel contained 7.5 or 12.5% acrylamide as described by Laemmli (18). The and 0.011, and 10 and 150 mM, respectively. The substrates were dissolved in samples were mixed in 1:1 ratio (vol/vol) with buffer containing 0.17 M Tris-HCl inulin reaction buffer (pH 5.5) and incubated with purified levanase enzyme (pH 6.8), 3% SDS, 30% glycerol, and 10% 2-mercaptoethanol and heated (5 min, (inulin and levan, 0.5 ␮g/ml; sucrose, 0.25 ␮g/ml) at 55ЊC for 30 min. From the 95ЊC) before application. Approximate molecular masses were determined by amount of liberated fructose the initial velocity (v0) was determined. The enzyme using commercially available marker proteins as standards (Pharmacia, Uppsala, parameters Vmax and Km were calculated with a computer program based on the Sweden). The gels were stained for proteins with Coomassie brilliant blue R. evaluation methods of Wilkinson (45) and Eisenthal and Cornish-Bowden (9). SEC-DRI-LALLS technique. For the determination of molecular weight av- Atkins and Nimmo (1) tested different evaluation methods for the determination erages and molecular weight distributions of the dissolved polysaccharides, the of Km and Vmax and found that with these methods the best results were ob- SEC-interferometric differential refractive index (DRI)-LALLS technique was tained. used. The experimental setup includes a chromatographic section with a series of For the size exclusion chromatography–low-angle laser light scattering (SEC- SEC columns containing TSK G5000 PW and TSK G4000 PW, each with a LALLS) and thin-layer chromatography (TLC) analyses inulin (50 and 100 length of 600 mm and an inner diameter of 7.5 mm, from Toyo Soda Manufac- mg/ml) and levan (30 mg/ml) were dissolved in inulin reaction buffer (pH 5.5) turing Co., Tokyo, Japan. For each characterization 200 ␮l of the sample solution and incubated with purified levanase (inulin, 5 ␮g/ml; levan, 4 ␮g/ml) at 55ЊC. was injected at room temperature (20ЊC), while the eluent (0.05 M aqueous Samples were taken in intervals over a longer period, and hydrolysis was stopped 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 37ЊC 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. 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-␮m-pore-size metal filter block at the levanase protein was eluted as a single peak before increasing reservoir end and a 0.45-␮m-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 37ЊC. 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 ␭ϭ630 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 95ЊC 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 hydrolyzing 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. Hg2ϩ and Agϩ 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 Fe2ϩ (can be oxidized to activity. An incubation time of 16 to 20 h was necessary for Fe3ϩ in the presence of air) decreased the activity of levanase efficient levanase production. Within the growth cycle, the ϩ ϩ ϩ ϩ 2ϩ 2ϩ 2ϩ to 16%, whereas Cs ,Na ,Li ,NH4 ,Cu ,Ca ,Zn , 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 b in detail in Materials and Methods. During chromatography on Concn Relative activity (%) 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 All substrates were dissolved or suspended in inulin reaction buffer, pH 5.5. chromatography step resulted in an apparently pure prepara- b tion of levanase protein. Although no impurities were detect- Sucrose (50 mg/ml) was used as the internal standard, and the resulting 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. APPL.ENVIRON.MICROBIOL.

TABLE 3. Determination of enzyme parameters

Ϫ1 Ϫ1 Ϫ1 Ϫ1 Substrate Mol wt Km Vmax (nmol s ) kcat (s ) kcat/Km (M s ) Inulin 5 ϫ 103a 6.7 mM 2.17 827 1.2 ϫ 105 Levan 1 ϫ 107a 1.2 ␮M 0.96 370 3.0 ϫ 108 Sucrose 342.3 63.6 mM 0.91 686 1.1 ϫ 104

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 70ЊC. The enzyme was hydrolysis. No obvious shift toward midrange n values could be shown to be most active between 47 and 55ЊC in standard observed, an endohydrolysis action of levanase could be ex- 30-min reactions. In addition, long-term temperature treat- cluded, and thus, the already expected exohydrolysis action of ment of levanase revealed high stability at 50 and 55ЊC. No levanase was supported. The product liberated from the very significant loss of activity could be seen within2hatthese beginning of the degradation process catalyzed by levanase was temperatures, whereas at 60ЊC the enzyme was inactivated again identified to be fructose. No significant diminishing of rapidly and practically no activity was retained after 30 min. the highest-molecular-weight components of inulin could be However, adding fructose or glycerol to the enzyme prepara- detected during the degradation process. This fact indicates a tion left enzyme activity almost unaffected after incubation of single-chain mechanism of levanase action. 40 min at 60ЊC. A similar stabilization effect was obtained when the enzyme was incubated with polyclonal levanase an- DISCUSSION tibodies. Enzyme kinetics. The kinetic properties of levanase were A standard method to discriminate between specificity for studied with the MonoQ-purified enzyme by measuring the high-molecular-weight versus low-molecular-weight substrates initial velocity of the reaction at 55ЊC. The Km values of inulin, of ␤(2 3 1) hydrolyzing ␤-fructofuranosidases (nonspecific or levan, and sucrose were 6.7 mM, 1.2 ␮M, and 63.6 mM, re- inulinases) is the determination of ratios of sucrase to inulinase spectively. The maximum initial velocities for the conversion of activity (S/I values). S/I values close to 1 are found for only very these substrates to fructose were 2.17 (inulin), 0.96 (levan), few inulinases from filamentous fungi, whereas typically S/I and 0.91 (sucrose) nmol sϪ1. From these data the turnover values between 20 and 45 are reported for most inulinases numbers (kcat) and specificity constants (kcat/Km) for each sub- from yeast. Depending on the substrate concentration, S/I val- strate were calculated with the equation Vmax ϭ kcat ϫ [E]0, ues between 0.32 and 1.23 were found for the investigated B. where [E]0 is the initial enzyme concentration. The determined subtilis levanase, indicating that this enzyme is more selective kinetic parameters are summarized in Table 3. All parameters for inulin than are enzymes from many other sources. were calculated with enzyme concentrations which had been The homogeneous levanase fraction which was obtained af- determined with the micromethod of Bio-Rad (4) and the ter MonoQ chromatography showed a specific inulin-degrad- molecular mass of 75 kDa determined for levanase. ing activity of 175 U/mg at 37ЊC with 50 mg of dissolved inulin Products and mechanism of hydrolysis. To determine per ml as the substrate. No data on inulinase activity based on whether the levanase enzyme attacks inulin randomly or at solid (suspended) inulin could be found in the literature. How- defined positions within the polymer (endoattack) or starts ever, the value of 97 U/mg measured with the purified levanase hydrolysis from the fructose or glucose end (exoattack), the protein (50 mg of suspended inulin per ml at 37ЊC) indicates 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 during hydrolysis. It was further observed that over the entire hydrolysis period the position of the oligosaccharide spot (inulin) 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 FIG. 2. Preparative hydrolysis of inulin with purified levanase enzyme for weight distribution of inulin due to exoinulinase attack (15). SEC-DRI-LALLS and TLC analysis. One milliliter of inulin solution (50 mg/ml) Stages of increasing hydrolysis shown in Fig. 2 were analyzed, was incubated with 5 ␮g of purified levanase per ml in a 1.5-ml Eppendorf vial and the results of selected stages (a to d) are shown in Fig. 3. at 55ЊC. Samples of 100 ␮l were taken at various times over a 300-min period, Within experimental error the composition of the initially ap- and hydrolysis was stopped by heating samples to 95ЊC for 5 min. The fructose and glucose concentrations in the samples were determined enzymatically with a plied inulin substrate (G-Fn, where G is the glucose residue commercial test combination (Boehringer GmbH). The indicated hydrolysis and Fn is n fructose residues) remained constant till the end of stages a to d are illustrated in Fig. 3. VOL. 61, 1995 B. SUBTILIS LEVANASE PURIFICATION AND CHARACTERIZATION 1957

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., differential 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 ␤-fructosidases. The previously found fact, that deletion of 114 amino acids from the C-terminal part of the levanase did not affect that this enzyme efficiently hydrolyzes the undissolved solid inulinase activity (34), supports this suggestion. substrate, a property of high value for industrial applications. Kinetic parameters of B. subtilis levanase were found to be in In comparison, for inulinase from Kluyveromyces marxianus (20 the same range as those determined for other microbial mg of dissolved inulin per ml at 50ЊC) and for inulinases F2, ␤-fructofuranosidases. However, one has to be aware of the F3, and F4 from C. pannorum (5 mg of dissolved inulin per ml fact that the molecular weights of the polymeric substrates at 30ЊC) specific inulin-degrading activities of 119, 41.5, 10.5, inulin and levan are not clearly defined, as only average mo- and 106 U/mg, respectively, have been determined (30, 46, 47). lecular weights are calculated. For sucrose, Km values between The specific levan-degrading activity of 194 U/mg (30 mg of 2 and 65 mM have been found (11, 29, 31, 36), indicating a dissolved levan per ml at 55ЊC) found with the purified levan- broad range of substrate affinity. The lowest Km values have ase protein is lower but in the same order of magnitude as been found with the invertases from Fusarium oxysporum, be- reported for other microbial levanases. In comparison, levan- tween 2.2 and 4.4 mM (29). With Km values between 61 and 65 ases from S. salivarius KTA-19 (38) and Actinomyces viscosus mM, B. subtilis levanase is in the upper range.

ATCC 19246 (16) and the nonspecific exo-␤-fructosidase from For inulin, Km values between 0.0012 and 33 mM were S. salivarius KTA-19 (39) yielded levan-degrading activities of determined (8, 11, 26, 27). Compared with inulinases from about 850, 851, and 433 U/mg at 37ЊC with 1 mg of dissolved yeasts, inulinases from filamentous fungi usually have lower Km levan per ml. values, indicating a higher substrate affinity for these enzymes.

The determined optimal pH and temperature ranges are in Nevertheless, the lowest Km value (1.2 ␮M) was reported for a good agreement with reported values for inulinases and levan- bacterial inulinase from Clostridium acetobutylicum (8). An ases from bacteria (8, 24), yeasts (28, 36), and filamentous expected high affinity of B. subtilis levanase for levan was ver- fungi (6, 43, 46, 47). In general, nonspecific ␤-fructofuranosi- ified with the determined low Km value of 1.2 ␮M. For the dases from yeasts and filamentous fungi are supposed to have hydrolysis of inulin with purified levanase, a kcat value of about lower pH optima than bacterial enzymes, but comparison has 827 sϪ1 was determined. This is a high value compared with the to be done cautiously, considering differences in substrate con- known data of inulinases from Aspergillus ficuum (11) with kcat Ϫ1 centrations and assay conditions. values between 0.06 and 0.46 s . In contrast, the Km values of The inhibition patterns of the inulin-degrading activity by B. subtilis levanase and the inulinases from Aspergillus ficuum different metal ions and agents maintain the similarity between range in the same order of magnitude (11). When comparing 5 B. subtilis levanase and almost all inulinases, levanases, and the kcat/Km of these enzymes (1.2 ϫ 10 for B. subtilis levanase ␤-fructofuranosidases for which such studies are reported (5, and 1 to 60 for the Aspergillus ficuum inulinases) it can be seen 17, 27, 39, 47, 48). Because total inactivation can be achieved that B. subtilis levanase is well suited for inulin hydrolysis. The ϩ 2ϩ 8 by1mMAg and Hg ions, there is strong evidence for a kcat/Km value of 10 for levan hydrolysis by B. subtilis levanase situation in which SH groups are essential for the catalytic already comes into the range of values found for enzymes that activity. In the case of B. subtilis levanase, only one cysteine, have attained kinetic perfection (37). 1958 WANKER ET AL. APPL.ENVIRON.MICROBIOL.

By means of analyzing reaction products of inulin hydrolysis 17. Kunst, F., M. Steinmetz, J. A. Lepesant, and R. Dedonder. 1977. Presence of by TLC and SEC-DRI-LALLS techniques, the mode of attack a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168. Biochimie 59:287–292. of the purified levanase was identified as an exohydrolase ac- 18. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of tion. As reported above, melezitose could not be hydrolyzed the head of bacteriophage T4. Nature (London) 227:680–685. (Table 2). This trisaccharide has a configuration identical to 19. Laloux, O., J.-P. Cassart, J. Delcour, J. van Beeumen, and J. Vandenhaute. that of inulin, but the medial fructose is protected from termi- 1991. Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var. marxianus ATCC 12424. FEBS Lett. 289:64–68. nal hydrolysis by glucosyl residues. Therefore, this fact pro- 20. Marchal, R., D. Blanchet, and J. P. Vandecasteele. 1985. Industrial optimi- vides additional evidence that levanase degrades inulin-like zation of acetone-butanol fermentation: a study of the utilization of Jerusa- molecules from the fructose end. Two possible mechanisms for lem artichokes. Appl. Microbiol. Biotechnol. 23:92–98. an exohydrolase can be distinguished: the multiple-chain and 21. Margaritis, A., and P. Bajpai. 1982. Ethanol production from Jerusalem artichoke tubers (Helianthus tuberosus) using Kluyveromyces marxianus and single-chain modes. A multiple-chain mechanism would lead Saccharomyces rosei. Biotechnol. Bioeng. 24:941–953. to a situation in which the enzyme molecules randomly attack 22. Martin, I., M. Debarbouille, E. Ferrari, A. Klier, and G. Rapoport. 1987. oligosaccharide chains and thus because of the cut-and-go ac- Characterization of the levanase gene of Bacillus subtilis which shows ho- tion a shift in the molecular weight distribution towards mology to yeast invertase. Mol. Gen. Genet. 208:177–184. 23. Martin, I., M. Debarbouille, A. Klier, and G. Rapoport. 1989. Induction and midrange- and low-n-value intermediates has to be expected. metabolite regulation of levanase synthesis in Bacillus subtilis. J. Bacteriol. With a single-chain mechanism an enzyme molecule attacks an 171:1885–1892. oligosaccharide chain and is bound to it until complete hydro- 24. Miller, C. H., and P. J. B. Somers. 1978. Degradation of levan by Actino- lysis. In this case, and assuming that no significant preference myces viscosus. Infect. Immun. 22:266–274. 25. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor for any specific n-value components of the different G-Fn Laboratory, Cold Spring Harbor, N.Y. chains exists, no detectable amounts of midrange- and low-n- 26. Nakamura, T., S. Hoashi, and S. Nakatsu. 1978. Culture conditions for value oligosaccharides are expected. The finding that the initial inulase production by Aspergillus. Nippon Nogeikagaku Kaishi 52:105–110. molecular weight distribution of inulin remains constant within 27. Nakamura, T., and S. Nakatsu. 1977. General properties of extracellular inulase from Penicillium. Nippon Nogeikagaku Kaishi 51:681–689. experimental error during the enzymatically catalyzed hydrolysis 28. Negoro, H. 1978. Inulase from Kluyveromyces fragilis. J. Ferment. Technol. yielding fructose as the only product favors the assumption of a 56:102–107. single-chain mechanism for the action of levanase on inulin. 29. Nishizawa, M., Y. Maruyama, and M. Nakamura. 1980. Purification and characterization of invertase isozymes from Fusarium oxysporum. Agric. Biol. Chem. 44:489–498. ACKNOWLEDGMENTS 30. Rouwenhorst, R. J. 1992. Production and localization of inulinases in Kluyveromyces yeasts. Ph.D. thesis. Technical University of Delft, Delft, The This work was supported by grants from the Bundesministerium fu¨r Netherlands. Land- und Forstwirtschaft, the Bundesministerium fu¨r Wissenschaft 31. Rouwenhorst, R. J., M. Hensing, J. Verbakel, W. A. Scheffers, and J. P. van und Forschung, and the Land Steiermark. Dijken. 1990. Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556. Appl. Environ. Microbiol. 56:3337–3345. REFERENCES 32. Rouwenhorst, R. J., W. S. Ritmeester, W. A. Scheffers, and J. P. van Dijken. 1. Atkins, G., and I. A. Nimmo. 1975. A comparison of seven methods for fitting 1990. Localization of inulinase and invertase in Kluyveromyces species. Appl. the Michaelis-Menten equation. Biochem. J. 149:775–777. Environ. Microbiol. 56:3329–3336. 2. Avigad, G., and S. Bauer. 1966. Fructan hydrolase. Methods Enzymol. 8:621–628. 33. Scho¨rgendorfer, K., H. Schwab, and R. M. Lafferty. 1987. Nucleotide se- 3. Barth, H. G., and B. E. Boys. 1992. Size exclusion chromatography. Anal. quence of a cloned 2.5 kb PstI-EcoRI Bacillus subtilis DNA fragment coding Chem. 64:428–442. for levanase. Nucleic Acids Res. 22:9606. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of 34. Scho¨rgendorfer, K., H. Schwab, and R. M. Lafferty. 1988. Molecular char- microgram quantities of protein utilizing the principle of protein dye-bind- acterization of Bacillus subtilis levanase and a C-terminal deleted derivative. ing. Anal. Biochem. 72:248–254. J. Biotechnol. 7:247–258. 5. Burne, R. A., K. Schilling, W. Bowen, and R. E. Yasbin. 1987. Expression, 35. Snyder, H. E., and H. J. Phaff. 1960. Studies on a beta-fructosidase (inulinase) produced by Saccharomyces fragilis. Antonie van Leeuwenhoek 26: purification, and characterization of an exo-␤-D-fructosidase of Streptococcus mutans. J. Bacteriol. 169:4507–4517. 433–452. 6. Derycke, D. G., and E. J. Vandamme. 1984. Production and properties of 36. Snyder, H. E., and H. J. Phaff. 1962. The pattern of action of inulinase from Aspergillus niger inulinase. J. Chem. Technol. Biotechnol. 34:45–51. Saccharomyces fragilis on inulin. J. Biol. Chem. 237:2438–2441. 7. Edelmann, J., and T. G. Jefford. 1964. The metabolism of fructose polymers 37. Stryer, L. 1988. Biochemistry. W. H. Freeman & Co., New York. in plants. 4. ␤-Fructofuranosidases of tubers of Helianthus tuberosus. Bio- 38. Takahashi, N., F. Mizuno, and K. Takamori. 1983. Isolation and properties chem. J. 93:148–161. of levanase from Streptococcus salivarius KTA-19. Infect. Immun. 42:231– 8. Efstathiou, I., G. Reysset, and N. Truffaut. 1986. A study of inulinase activity 236. in the Clostridium acetobutylicum strain ABKn8. Appl. Microbiol Biotechnol. 39. Takahashi, N., F. Mizuno, and K. Takamori. 1985. Purification and prelim- 25:143–149. inary characterization of exo-␤-D-fructosidase in Streptococcus salivarius 9. Eisenthal, R., and A. Cornish-Bowden. 1974. The direct linear plot: a new KTA-19. Infect. Immun. 47:271–276. graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 40. Taussig, R., and M. Carlson. 1983. Nucleotide sequence of the yeast SUC2 139:715–720. gene for invertase. Nucleic Acids Res. 11:1943–1954. 10. Ettalibi, M., and J. C. Baratti. 1987. Purification, properties and comparison 41. Uchiyama, T. 1975. Action of Arthrobacter ureafaciens inulinase II on several of invertase, exoinulinases and endoinulinases of Aspergillus ficuum. Appl. oligofructans and bacterial levans. Biochim. Biophys. Acta 397:153–163. Microbiol. Biotechnol. 26:13–20. 42. Uhm, T., D. Jeon, S. Byun, J. Hong, and J. W. D. GrootWassink. 1987. 11. Ettalibi, M., and J. C. Baratti. 1990. Molecular and kinetic properties of Purification and properties of ␤-fructofuranosidase from Aspergillus niger. Aspergillus ficuum inulinases. Agric. Biol. Chem. 54:61–66. Biochim. Biophys. Acta 926:119–126. 12. Flood, A. E., P. P. Rutherford, and E. W. Weston. 1967. Effects of 2:4- 43. Vandamme, E. J., and D. G. Derycke. 1983. Microbial inulinases: fermenta- dichlorophenoxyacetic acid on enzyme systems in Jerusalem artichoke tubers tion process, properties and applications. Adv. Appl. Microbiol. 29:139–176. and chicory roots. Nature (London) 214:1049–1050. 44. Wanker, E., K. Scho¨rgendorfer, and H. Schwab. 1991. Expression of the 13. Friehs, K., K. Scho¨rgendorfer, and H. Schwab. 1986. Cloning and pheno- Bacillus subtilis levanase gene in Escherichia coli and Saccharomyces cerevi- typic expression in Escherichia coli of a Bacillus subtilis gene fragment coding siae. J. Biotechnol. 18:243–254. for sucrose hydrolysis. J. Biotechnol. 3:333–341. 45. Wilkinson, G. N. 1961. Statistical estimations in enzyme kinetics. Biochem. J. 14. Harding, S. E., D. B. Sattelle, and V. A. Bloomfield (ed.). 1992. Laser light 80:324. scattering in biochemistry. RSC, Cambridge. 46. Xiao, R., M. Tanida, and S. Takao. 1989. Purification and characteristics of 15. Huber, A., W. Praznik, T. Spies, S. Kammerer, and C. Knabl. 1993. Prepa- two exoinulinases from Chrysosporium pannorum. J. Ferment. Bioeng. 67: ration and characterisation of aqueous inulin/exo-inulinase systems, p. 231– 331–334. 240. In A. Fuchs (ed.), Inulin and inulin-containing crops—studies in plant 47. Xiao, R., M. Tanida, and S. Takao. 1989. Purification and some properties of science. Elsevier, Amsterdam. endoinulinase from Chrysosporium pannorum. J. Ferment. Bioeng. 67:244–248. 16. Igarashi, T., M. Takahashi, A. Yamamoto, Y. Etoh, and K. Takamori. 1987. 48. Yanase, H., H. Fukushi, N. Ueda, Y. Maeda, A. Toyoda, and K. Tonomura. Purification and characterization of levanase from Actinomyces viscosus 1991. Cloning, sequencing, and characterization of the intracellular invertase ATCC 19246. Infect. Immun. 55:3001–3005. gene from Zymomonas mobilis. Agric. Biol. Chem. 55:1383–1390.