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).

Fig. 1. SDS-polyacrylamide gel electrophoresis of the ram. The K,,, of the enzyme for trehalose was trehalase. 3.0 mM. SDS-polyacrylamide gel (10%) electrophoresis of Effects of pH and temperature on activity purified trehalase (lane 1) and molecular weight and stability. The enzyme activity was mea- markers (lane 2). a, phosphorylase b (97.1 kDa); b, bovine serum albumin (66.2 kDa); c, ovalbumin (45 sured from pH 5 to 10. As shown in Fig. 3, kDa); d, carbonic anhydrase (31 kDa); e, soybean maximal activity was observed at pH around trypsin inhibitor (21.5 kDa); f, lysozyme (14.4 kDa). 7.8 under standard enzyme assay conditions. The enzyme was stable from pH 6.0 to 9.5 at 4•Ž protein. The molecular weight of the trehalase for 24 hr. The optimum pH of trehalase of of Bacillus sp. T3 was smallest of the microbial Bacillus sp. T3 was alkaline, in contrast with trehalases.2,3.5,6) The isoelectric point was p1I many other microbial trehalases" except that 4.8. of Mycobacterium smegmatis.19)

Substrate specificity. Substrate specificities The effect of temperature on the enzyme were examined using various oligosaccharides activity was measured from 30 to 70•Ž at pH as follows. The trehalase (0.1 units/ml) was 7.5 for 20 min. As shown in Fig. 4, maximal incubated with 1.0% of each substrate in 20 ms activity was observed at 50•Ž. The enzyme was sodium phosphate buffer (pH 7.5) at 45•Ž for 20 stable below 55°C under the condition of pH 7.5 min, and the amount of glucose liberated was for 1 hr. measured by the glucose-oxidase method.18) Effects of Chemicals. The enzyme was in- The enzyme hydrolyzed trehalose specifically, cubated with various metal ions and EDTA in but not neotrehalose, kojibiose, nigerose, malt- 20 mvi glycero-phosphate buffer (pH 7.0) at ose, isomaltose, cellobiose or sucrose. The 30•Ž for 1 hr, and the remaining activities were trehalase of Bacillus sp. T3 was highly specific measured under standard assay conditions for trehalose as well as those from other (Table 3). The enzyme was inactivated by microbes. The products of enzymatic hydroly- Cu2+ and Hg2+. No effect was observed by sis of trehalose were analyzed by TLC or GLC. EDTA.

Only glucose was detected on each chromatog- Condensation reaction. NAKANO et al. re- Purification and Characterization of Trehalase 235

Fig. 3. Effect of pH on activity and stability of the Fig. 4. Effect of temperature on activity and stability trehalase. of the trehalase. •@•@ Effect on activity (•›,•¢,• )LID: the reaction mixture Effect on activity (•›) : the reaction mixture consist- consisting of 0.2 ml of the enzyme solution and 4 ml of ing of 0.2 ml of the enzyme solution and 4 ml of 1 1% trehalose in 100 mM MCILVaiH buffer (pH 4 .5-8.0), trehalose in 20 mM sodium phosphate buffer (pH 7.5) 100 mM KOLTFIOFF buffer (pH 7.5-9.5), 10 .0 mM NaHC03- was incubated at various temperatures for 20 min. Na2C03 buffer (pH 9.0-10.5) were incubated at 50•Ž Effect on stability (•œ) : the enzyme was incubated at for 20 min. various temperatures for 1 hr in 20 mM sodium phos- Effect on stability (•œ,•£,•¡) : the enzyme solution phate buffer (pH 7.5), and the residual activities were (0.5 units/ml) was kept in 100 ms MCILVAINE buffer (pH assayed at 50t . 4.5-8.0), 100 mM KoLTxoFF buffer (pH 7.5-9.5), 100 mM NaHC03-Na2C03 buffer (pH 9.0-10.5) at 4t for 24 hr. Then these solutions were diluted 10-fold with 100 mM sodium phosphate buffer, pH 7.5, and the residual activ- ities were assayed. Table 3. Effect of various metal ions and EDTA on trehalase activity.

ported that the trehalase of Labosphaeya sp. also catalyzed the condensation of glucose.8) The trehalase of Bacillus sp. T3 was examined for the condensation of glucose. The enzyme (50 units/ml) was incubated with 50% glucose in 20 mM sodium phosphate buffer (pH 7.5) at 45•Ž for 72 hr. The reaction products were assayed by TLC or GLC. No product was detected on each chromatogram. The trehalase of Bacillus sp. T3, was distinct from the enzyme of Labos-

phaera, sp. in terms of its action on glucose. Tyehalose determination. The determination of trehalose using the trehalase of Bacillus sp. T3 was examined in combination by the glu- cose-oxidase method. The reaction mixture

(pH 7.0) containing trehalose (0-96 nmol) and trehalase (0.1 units) were incubated at 45t for 120 min. The glucose content in the reaction 236 Oyo Toshitsu Kagaku, Vol. 42, No. 3 (1995)

MOUMRIKOFF: Biochimie, 53, 853-857 (1971). 6) H. NAKANO,M. MORIWAKI,T. WASHING,T. KING, H. YOSHIZUMIand S. KITAHATA: Biosci. Biotech. Bio chem., 58, 1430-1434 (1994). 7) S. SAITO : J. Biochem., 48, 101-109 (1960). 8) H. NAKANO, M. MORIWAKI, T. WASHINO and S. KITAHATA: Biosci. Biotech. Biochem., 58, 1435-143 8 (1994). 9) L. H. CRABBE,A. E. S. GUSSIN and E. A. TYRRELL : Experientia, 25, 793 (1969). 10) F. YAMAUCHIand K. Aso : Nature, 189, 753 (1961). 11) D. CLAUSand R. C. W. BERKELEY: Bergey's Manual of Systematic Bacteriology, Vol. 2, P. H. A. SNEATH, N. S. MAIR, M. E. SHARP and J. G. HALT, eds.,

Fig. 5. Calibration curve for trehalose determination Williams and Wilkins Co., Baltimore, p. 1105-1139 using the trehalase and glucose-oxidase method. (1986).

The reaction mixture consisting of 0.1 ml of trehalase 12) M. S0M0GYI : J. Biol. Chem., 195, 19-23 (1952).

(0.1 units) in 100 mM sodium phosphate buffer (pH 7.5) 13) N. NELSON : J. Biol. Chem., 153, 375-380 (1944). and 0.4 ml of trehalose solution (0-96 nmol) was in- 14) 0. H. LOWRY,N. J. ROSEBROUGH,A. L. FARR and R. cubated at 45t for 120 min. To the reaction mixture J. RANDALL: J. Biol. Chem., 193, 265-275 (1951). 0.5 ml of glucose-oxidase reagent was added,18) and 15) B. J. DAVIS : Ann. N. Y. Acad. Sci., 121, 404-427 the mixture was incubated at 40t for 30 min. Then (1964). 2.5 ml of 5 N HCl was added to measure the absorbance 16) U. K. LAEMMLI: Nature, 227, 680-685 (1970). at 525 nm. Regression equation (trehalose 0-72 nmol) : 17) C. C. SWEELEY,R. BENTLEY,M. MAKITA and W. W. Y = 1.97(•}0.0052)X; Y, glucose measured (nmol) ; X, WELLS : J. Am. Chem. Soc., 85, 2497 (1963). trehalose (nmol). 18) J. B. LLOYDand W. J. WHELAN : Anal. Biochem., 30, 467-470 (1969). 19) B. W. PATTERSON,A. H. FERGUSON,M. MATULAand mixture was assayed by the glucose-oxidase A. D. ELBEIN : Methods Enzymol., 28, 996-1000 method. (1972). As shown in Fig. 5, a good linearity up to 70 nmol of trehalose was obtained in the relation (Received March 31, 1995; Accepted May 25, 1995) between the amount of trehalose and that of glucose measured, and the recovery was 98.5% Bacillussp.T3株 の 生 産 す る of the theoretical value. Good linearity and ト レ ハ ラー ゼ の 精 製 と 性 質 recovery were also obtained in the presence of 5

1umol of maltose (data not shown). The tre- 仲 田 哲 也*,池 上 庄 治*,西 本 友 之* halase of Bacillus sp. T3 is considered to be 茶 圓 博 人*,杉 本 利 行*,栗 本 雅 司* available for enzymatic assay of the amount of *林 原 生 物 化 学 研 究 所 trehalose in the mixture of oligosaccharides. (700岡 山 市 天 瀬 南 町7-7) REFERENCES 土 壌 か ら分 離 し たBacillussp.T3株 由 来 の トレ ハ 1) A. D. ELBEIN: Adv. Carbohydr. Chem. Biochem., 30, ラ ー ゼ を 精 製 し た.本 酵 素 の 分 子 量 はSDS-tｰリ ア ク 227-256 (1976). リ ル ア ミ ドゲ ル 電 気 泳 動 法 で58,000,ゲ ル 濾 過 法 で 2) A. R. S. PRASAD and R. MAHESHWARI: Biochim. Biophys. Acta, 525, 162-170 (1978).3 52,000と 推 定 さ れ た.等 電 点 は4.8で あ っ た.本 酵 ) M. SUMIDA,S. OGURA,S. MIYATA,M. ARAIand S. 素 の 最 適pHは7.8,最 適 温 度 は50℃ で あ っ た.ま た, MURAO: J. Ferment. Bioeng., 67, 83-86 (1989) pH6.0-9.5の 範 囲 で,温 度55℃ 以 下 で 安 定 で あ っ た. 4) P. J. KELLEYand B. J. CATLEY: Anal. Biochem., 72, 本 酵 素 の ト レ ハ ロ ー ス に 対 す る.Km値 は3.OmMで 353-358 (1976). あ っ た.本 酵 素 はCu2+,Hg2+に よ り不 活 化 さ れ た. 5) E. GUILLOUX,M. A. ARCILA,J. E. COURTOISand V.