231 (Oyo Toshitsu Kagaku, Vo1..42, No.3, p.231-236 (1995)) Purification and Characterization of Trehalase from Bacillus sp. T3 Tetsuya NAKADA,* Shoji IKEGAMI,* Tomoyuki NIsHIMOTO, * Hiroto CHAEN,* Toshiyuki SUGIMOTO* and Masashi KURIMOTO* * Hayashibara Biochemical Laboratories , Inc. (7- 7, Amase- minamimachi, Okayama 700, Japan) A trehalase was highly purified from Bacillus sp. T3 newly isolated from soil at 50•BC . The molecular weight of the trehalase was estimated to be 58,000 Da by SDS-polyacrylamide gel electrophoresis and 52,000 Da by gel filtration. The pI of the enzyme was 4.8. Its optimum pH and temperature were around 7.8 and 50•BC, respectively. The enzyme was stable between pH 6.0-9.5, and below 55•BC . The Km value for trehalose was 3.0 mM. The enzyme was inactivated by Cu2+ and Hg2+. Trehalases (ƒ¿, ƒ¿-Trehalase; ƒ¿, ƒ¿-Trehalose prepared by the method reported by YAMAUCHI glucohydrolase [EC 3.2.1.28]) which hydrolyze and Aso.10) All other chemicals used were of trehalose to glucose are distributed in micro- the highest grade available. organisms, plants, insects and mammalian Microorganism. The microorganism, strain tissues.l) There are several reports of purifi- T3, used in this study was isolated from soil at cation and characterization of trehalases from 50t and was identified according to "Bergey's fungi,2,3) yeasts,4) bacteria,5) algae6) and insects.7) Manual of Systematic Bacteriology."11) Some enzymes have been considered to play an Cultivation. The bacterium was cultivated important role in spore germination or in aerobically in 30 l fermenter (B. E. Marubishi degrading ingested trehalose.1 Recently, the Co.) containing 20 l of the liquid medium; 0.5% trehalase from Labosphaera sp. has been report- trehalose, 0.5% yeast extract, 1.0% polype- ed to also catalyze the condensation of glu ptone, 0.1% K2HPO4 and 0.05% MgSO4.7H2O cose.8) (pH 7.0) at 50•Ž for 28 hr. The culture super- In our laboratory, a strain producing tre- natant was obtained by continuous centrifuga- halase was isolated from soil and was identified tion at 15,000 x g, and was used for purification as a Bacillus species. In bacilli, B, cereus has of the trehalase. been reported to produce trehalase,9) although Enzyme assay. The reaction mixture consist- no detail on the enzyme has been given. In this ed of 4 ml of 1% trehalose in 20 mM sodium paper, the purification and properties of tre- phosphate buffer (pH 7.5) and 0.2 ml of enzyme halase from a strain designated Bacillus sp. T3 solution was incubated at 50•Ž for 20 min. The are described. reducing sugar increase was measured by the SOMOGYI-NELSON method12,13) using glucose as a MATERIALS AND METHODS standard. One unit of enzyme activity is de- fined as the amount of enzyme which catalyzed Materials. Trehalose, cellobiose and sucrose the liberation of 2,amol glucose per min. were obtained from Wako Pure Chemical Ltd. Protein assay. Protein was measured by the Nigerose was purchased from Sigma Chemical method of LOWRY et al.14) using bovine serum Co. Maltose, isomaltose and neotrehalose were albumin as a standard. Absorbance at 280 nm prepared in our laboratory. Kojibiose was was used for monitoring protein in column 232 Oyo Toshitsu Kagaku, Vol. 42, No. 3 (1995) eluate. buffer. The active fractions were combined Electrophoresis. Polyacrylamide gel electro- and concentrated by ultrafiltration (SIP-0013, phoresis (PAGE) was done by the method of Asahi Chemical Industry). DAVIS.15) Estimation of molecular weight of the Step 4. Ultrogel AcA44 gel filtration. The enzyme was done by SDS-PAGE by the methods concentrated enzyme solution was applied on an of LAEMMLI.16) After electrophoresis, protein Ultrogel AcA44 column (20 •~ 970 mm) equilib- was stained with Coomassie Brilliant Blue R- rated with 10 mM sodium phosphate buffer (pH 250 (Merck). Molecular weight markers from 7.0) containing 0.3 M NaCI, and eluted with the Bio-Rad Laboratories were used as standard same buffer at a flow rate of 13 ml/hr. The proteins. Estimation of isoelectric point of the active fractions were combined and concen- enzyme was done by gel isoelectric focusing by trated with ultrafiltration using the above mem- using Ampholine carrier ampholyte (Pharma- brane module. cia).•@•@ Step 5. DEAF- Toyopearl column chromatog- Gel filtration. The molecular weight of the raphy. The enzyme solution was dialyzed enzyme was also estimated by gel filtration on against 10 mM sodium phosphate buffer (pH TSKge1 G3000 SWxL (7.8 •~ 300 mm, Tosoh Co.) 7.0), and applied on a DEAE-Toyopearl 650S using a high-performance liquid chromatogra- column (11 x 100 mm) equilibrated with the phy system (Waters Ltd.) at a flow rate of 0.9 same buffer. The enzyme was eluted with a ml/min. Molecular markers from Bio-Rad linear gradient of NaCI from 0 to 0.4 M in the Laboratories were used as standard proteins. same buffer at flow rate of 0.5 ml/min. The Purification of the trehalase. active fractions were combined and dialyzed Step 1. Ammonium sulfate precipitation. against the same buffer. The culture supernatant was concentrated using Step 6. Mono Q column chromatography. an ultrafiltration membrane module AIP-2013 The dialyzed enzyme solution was applied on a (Asahi Chemical Industry) by about 20-fold. Mono Q HR5/5 column (5 •~ 50 mm) equilib- The concentrated enzyme solution was added rated with 10 mM sodium phosphate buffer (pH with ammonium sulfate to 70% saturation. 7.0), and the enzyme was eluted with a linear The resultant precipitates were dissolved in 10 gradient of NaCI from 0 to 0.4 M in the same mM sodium phosphate buffer (pH 7.0) and buffer at a flow rate of 0.45 ml/min. The active dialyzed against the same buffer. fractions were combined and ammonium sulfate Step 2. Sepabeads FP-DA13 column chro- was added to 80% saturation. The resultant matography. The dialyzed enzyme solution was precipitates were dissolved in 10 mM of sodium applied to Sepabeads FP-DA13 (Mitsubishi phosphate buffer (pH 7.0), and dialyzed against Chemical Industries) column (85 •~ 340 mm) the same buffer. The dialyzed enzyme solution equilibrated with 10 mM sodium phosphate was used as the purified enzyme preparation. buffer (pH 7.0), and the enzyme was eluted with Thin-layer chromatography. Thin-layer chro- a linear gradient of NaCI from 0 to 0.5 M in the matography (TLC) was done on Kieselgel 60 same buffer. The eluate was fractionated with plates' (Merck), using 1-butanol : pyridine 20 ml portions, and the active fractions were water (7: 31, v/v) as a solvent. Plates were combined. sprayed with 20 v/v% sulfuric acid in methanol, Step 3. Butyl- Toyopearl column chromatogra- and heated at 110 t for about 10 min to detect phy. Ammonium sulfate was added to the sugars. enzyme solution to a concentration of 1.2 M. Gas liquid chromatography. The preparation The solution was applied on a Butyl-Toyopearl of trimethylsilyl derivatives was performed by 650M column (26 •~ 830 mm) equilibrated with the method of SWEELEY et al.17) Gas liquid 10 mM sodium phosphate buffer (pH 7.0) con- chromatography (GLC) was done on a gas liq- taining 1.2 M ammonium sulfate. The adsorbed uid chromatograph, model GC-16A (Shimadzu enzyme was eluted with a linear gradient of Co.) equipped with a flame-ionization detector. ammonium sulfate from 1.2 to 0.6 M in the same A stainless steel column (0.3 x 200 cm) packed Purification and Characterization of Trehalase 233 with 2% Silicone OV-17 Chromosorb W (80-100 SWxL, a gel filtration column, and the molecu- mesh) was employed. Nitrogen was used at a lar weight of the enzyme was estimated to be flow rate of 40 ml/min. about 52,000 Da (Fig. 2). These results indicat- ed that the enzyme might be a monomeric RESULTS AND DISCUSSION Identification of microorganism. The mor- Table 1. Taxonomical properties'of strain T3 phological and physiological characteristics of producing trehalase. strain T3 are summarized in Table 1. The bacterium is a Gram-positive, aerobic rod with several peritrichous flagella, and it forms an endospore, indicating that it belongs to the genus Bacillus. Most characteristics of the strain are closely related to B, circurans or B. stearothermophilus, but the growth temperature does not agree with those two species. The strain was designated as Bacillus sp. T3. Formation of enzyme. Trehalase was pro- duced when the strain, Bacillus sp. T3, was cultivated on a medium containing trehalose as a sole carbon source. The enzyme activity of the culture supernatant was increased with lysis of cells, so the trehalase of Bacillus sp. T3 was considered to be intracellular. The enzyme was purified from the culture supernatant (0.14 units/ml) in this study. Purification of the enzyme. The results of the purification are summarized in Table 2. The enzyme was purified 425-fold over that of the culture supernatant, and had a specific activity of 93.5 units/mg protein. The preparation showed a single protein band in SDS-PAGE (Fig. 1). Molecular weight and isoelectric point. The molecular weight of the enzyme was estimated to be 58,000 Da by SDS-PAGE. The enzyme eluted as a single peak from TSKgel G3000 Table 2. Purification procedures of trehalase from Bacillus sp. T3. 234 Qyo Toslaitsau Kagahu, Vol. 42, No. 3 (1995) Fig. 2. Estimation of molecular weight of purified tre- halase by gel filtration. Gel filtration on a TSKgel G3000 SWxi, column. Molecular weight markers: a, gamma globulin (158 kl)a); b, ovalbumin (44 kDa); c, myoglobin (17 kDa).