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 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-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 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 b-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 b-D-Fructofuranosidases are usually classified upon their melezitose (3-O-a-D-glucopyranosyl-b-D-fructofuranosyl-a-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 b 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 b-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- b 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 b-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 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 2 2 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 378C 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-b-D-thiogalactopyranoside (IPTG) at inoculation or in the middle of bufferwitha1MNaCl solution. 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 by heating samples to 958C. 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 378C (150 rpm). Cells were at 958C) 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 3 g)for1h. For determination of inulinase activity, inulin was dissolved or suspended in The supernatant was stored at 2208C 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 558C 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 558C the precipitated protein was removed by centrifugation and discarded.