Purification and Some Properties of Sucrose Phosphorylase from Leuconostoc Mesenteroides

Agric. Biol. Chem., 55 (7), 1805-1810, 1991 1805

Purification and SomeProperties of Sucrose Phosphorylase from Leuconostoc mesenteroides Takuro Koga, Kazuo Nakamura, Yoshio Shirokane, Kiyoshi Mizusawa, Satoshi Kitao and MamoruKikuchi Research and Development Division, KikkomanCorporation, 399 Noda, Noda-shi, Chiba 278, Japan Received January 17, 1991

Sucrose phosphorylase (EC 2.4.1.7) was purified to homogeneity from Leuconostoc mesenteroides cells with a specific activity of 173.8 units per nig protein by ammoniumsulfate fractionation, anion exchange HPLC on TSKgel DEAE-5PW,and hydrophobic HPLCon TSKgel Ether-5PW. The purified enzyme was an acidic protein having an isoelectric point of pH 4.6 and sJo.w °f 4.34 S. The molecular weight of this enzyme was estimated to be 56,400 by sedimentation equilibrium, 55,000 by SDS-polyacrylamide gel electrophoresis, and HPLCgel filtration on TSKgel G3000SW, suggesting that the enzyme is a monomeric protein. With regard to molecular weight, amino acid composition, and N-terminal amino acid sequence of 30 residues, this enzyme is close to the glucosyltransferase A of Streptococcus mutans.

Sucrose phosphorylase (EC 2.4.1.7) was first produced by P. saccharophila has been purified reported to be found in Leuconostoc mesenter- and fully characterized. 1 3) However, relatively oides by Kagan et al.,^ and subsequently found little is known about the physico-chemical in a few microorganisms.2) Recently Russell et properties of sucrose phosphorylase from L. al. reported that glucosyltransferase A pro- mesenteroides, which could be most useful in duced by Streptococcus mutans was identified industrial use. as sucrose phosphorylase,3) and a complete In this report, we studied some physico- nucleotide sequence was identified.4) chemical properties of a sucrose phosphorylase Sucrose phosphorylase has plenty of bio- purified from L. mesenteroides, and found that technological uses in sucrose production from this enzymewas different from P. saccharophila starch,5'6) in enzymatic assay for sucrose,7) in enzyme, and close to glucosyltransferase A of synthesis of 14C-labeled sucrose,8) in the S. mutans. specific measurement of inorganic phos- phate,2'^ and so on. L. mesenteroides has a Materials and Methods significant advantage in the microbiological Chemicals. Except as noted otherwise, all chemicals were production of sucrose phosphorylase, because from Wako Pure Chemical Industries, Osaka. Glucose- 1 ,6- crude extracts of this organism had much diphosphate, phosphoglucomutase, D-gluconate-6-phos- higher total and specific activities than those phate, and Mr marker proteins for SDS-polyacrylamide of Pseudomonas saccharophila and Pseudomo- gel electrophoresis were obtained from Boehringer nas putrefaciens. 10) Furthermore, Vandamme MannheimYamanouchi, Tokyo. Nicotinamide adenine et al.xl) investigated the fermentation condi- dinucleotide phosphate oxidized form (NADP+), glucose- tions that affect sucrose phosphorylase produc- 6-phosphate dehydrogenase, and Mr marker proteins for HPLCgel filtration were obtained from Oriental Yeast tion by L. mesenteroides in detail, and Guibert Co., Tokyo. TSKgel DEAE-5PW, TSKgel Ether-5PW, and et al. applied it to glucose-1-phosphate pro- TSKgel G3000SW columns were products of Tosoh, duction by the immobilized enzyme techni- Tokyo. que.12) Onthe other hand, sucrose phosphorylase Organism and cultivation. L. mesenteroides ATCC12291 1806 T. Koga et al. was used in this study. The organism was grown at 30°C for the enzyme was calculated from the amino acid for 16hr with slow agitation in a 30-liter jar fermenter composition. 19) containing 20 1 of culture medium modified that of De Moss et al.14) It consists of2% sucrose, 1.5% polypepton, Amino acid composition and N-terminal amino acid 1.5% yeast extract, 1.5% K2HPO4, 0.5% L-lysine mono- sequence. Amino acid analysis of the enzyme was done hydrochloride, 0.04% MgSO4à"7H2O, 0.001% FeSO4- using a Hitachi amino acid analyzer model L-8500. The 7H2O, 0.02% MnSO4-7H2O, 0.001% thiamine hydro- enzyme was hydrolyzed by the method of Simpson et al.21) chloride, and 0.005% ascorbic acid (pH 7.8). The cells of in 4n methanesulfonic acid under vacuum at 110°C for an overnight culture were harvested by centrifugation at 24, 48, and 72hr. The sequence of the first 30 amino acid 8,000rpm for 30min and stored at -20°C. residues at the N-terminus of the enzyme was analyzed on an Applied Biosystems 470A gas phase sequencer. Enzymeassay. Sucrose phosphorylase activity was Phenylthiohydantoin derivatives were identified by reverse measured by the method of Silverstein et al.,13) in which phase chromatography with an on-line Applied Biosystems the production of glucose-1-phosphate from sucrose and 120A PTH-analyzer. inorganic phosphate is coupled to the reduction ofNADP+ in the presence of phosphoglucomutase and glucoses- phosphate dehydrogenase. The standard assay medium Results contained 50mMof potassium phosphate buffer (pH 6.8), 140mMsucrose, 1mMEDTA-2Na, 150mMMgCl2, 1 mg Purification of sucrose phosphorylase of NADP+, ljug of glucose-1,6-diphosphate, 100fig of All the procedures for isolation and pu- phosphoglucomutase, 20 units of glucose-6-phosphate de- rification of the enzyme were done at 0^°C. hydrogenase, and the enzyme solution (20/A) in a final volume of 3.3ml. Increase in adsorption of NADPHat Cells were harvested from 10 1 of culture broth 340nm was measured at 25°C. One unit of sucrose by centrifugation at 8,000 rpm and washed with phosphorylase activity was defined as the amount of 50mMpotassium phosphate buffer (pH 6.5). enzyme which caused the reduction of 1 /rniol of NADP+ The cells were suspended in 1 1 of the same per min under the above assay conditions. An extinction buffer. After addition of0.1% lysozyme, 0.1% coefficient of6.22 x 103 m" 1 cm" * was used forcalculation. Triton X-100, and 10mM EDTA-2Na, the Measurement of protein. Protein was measured by the solution was incubated at 37°C for 1hr and method of Lowryet al.,15) using bovine serum albumin as centrifuged for lOmin at 12,000rpm. the standard protein. Nucleic acids in the supernatant solution were precipitated by forming an aggregate with HPLCgel filtration. The estimation of the molecular polyethyleneimine. The precipitate was re- weight of sucrose phosphorylase by HPLCgel filtration moved by centrifugation. The supernatant on TSKgel G3000SW (7.15mm i.d. x 60cm x 2) were done by the method of Fukano et al.16) solution was brought to 35% saturation of ammoniumsulfate by addition of neutral Electrophoresis. The molecular weight was measured saturated ammoniumsulfate solution. After using sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis by the method of Laemmli.17) A 1 hr, the precipitate was removed by centrifuga- SDS-polyacrylamide gradient slab gel (10-20%; Daiichi tion and the supernatant was brought to 75% Kagaku Yakuhin, Tokyo) was used. The gel was stained saturation of ammoniumsulfate. Theresulting with 0.25% Coomassie Brilliant Blue R-250. precipitate wascollected by centrifugation and dissolved in 10 ml of 10 mM2-(iV-morpholino)- Isoelectric focusing. Isoelectric focusing on poly- ethanesulfonic acid (MES) buffer (pH 6.0). The acrylamide gel was done using carrier ampholite (pH 3-10, solution was dialyzed against the same buffer Ampholine, LKB, Bromma, Sweden) by the method of Catsimpoolas. 1 8) overnight and centrifuged. The precipitate was discarded. Ultracentrifugal analysis. All sedimentation experiments The supernatant (1ml) was put onto a were done with a Hitachi UCA-1A analytical ultra- TSKgel DEAE-5PW column (21.5mm i.d. x centrifuge. Sedimentation coefficients were corrected to 15cm) previously equilibrated with 10mM standard conditions and extrapolated to infinite dilution as described by Schachman. 19) Sedimentation equilibrium MESbuffer (pH 6.0). The column was washed study was analyzed by interference optics by the method with 10mM MES buffer containing 0.1 m KC1, of Richards and Schachman.20) The partial specific volume and eluted with a linear gradient of KC1from Purification and Properties of Sucrose Phosphorylase 1807

0.1m to 0.3m for 60min in the same buffer at A typical purification is summarized in Table a flow rate of 6ml/min. The active fractions I. The overall purification was approximately obtained from 20 runs of the DEAE-5PW 25-fold with a yield of 17%. chromatography were pooled and concentrat- ed to about 20ml by ultra filtration, and were Homogeneity dialyzed overnight against 0.1 m potassium The purified enzyme preparation gave a phosphate buffer containing 1.5 mammonium single protein band on SDS-polyacrylamide gel sulfate (pH 7.0), and centrifuged. electrophoresis (Fig. 2A). Sedimentation ve- The supernatant (1ml) was put onto a locity experiments showed that the protein TSKgel Ether-5PW column (21.5mm i.d. x 15 cm) previously equilibrated with 1.5 mammoni- umsulfate in the samebuffer. The columnwas washed with the same buffer, and eluted with a linear gradient of ammoniumsulfate from 1.5m to zero for 60min in the same buffer at a flow rate of 6ml/min. The elution pattern is shown in Fig. 1. The active fractions from 30 runs of this chromatography were pooled and concentrated to about 10 ml by ultra filtration, and used as the purified enzyme preparation.

Fig 2. SDS-Polyacrylamide Gel Electrophoresis (A) and Sedimentation Pattern (B) of Purified Sucrose Phosphor- ylase. (A) About 20 fig of the purified enzyme was electrophor- esed on lane 2. Lane 1 was maker proteins; a2 macroglobulin (Mr= 170,000 reduced), phosphorylase b (Mr= 97,400), gluta- Fig. 1. Hydrophobic Chromatography of Sucrose Phos- mate dehydrogenase (Mr = 55,400), lactate dehydrogenase phorylase from L. mesenteroides through TSKgel Ether- (Mr= 36,500), trypsin inhibitor (Mr=20,100, soybean). 5PW. (B) The protein concentration was ll.9mg/ml in 10mM Fractions of 6ml were collected from a linear gradient potassium phosphate buffer containing 0.1 m potassium ( ) of ammonium sulfate (1.5-0.75m), and enzyme chloride. The temperature was 20°C and bar angle was activity (O-O) and absorbance at 280nm ( ) were 70°. The photograph was taken at 80min after attainment measured. of 55,430 rpm.

Table I. Purification of Sucrose Phosphorylase from L. mesenteroides Total protein Total activity Specific activity Yeild P (mg) (units) (units/mg) (% ) Crude extracts0 2605.6 18,500 7. 1 100 (NH4)2SO4 (35-75% satn.) 680.9 8492 12.5 45.9 DEAE-5PW HPLC 41.5 5275 127.1 28.5 Ether-5PW HPLC 18.2 3145 173.8 17.0

a Crude extracts were from cultured cells from 10 1 of broth. 1808 T. Koga et al

Table II. Amino Acid Composition of Sucrose Phosphorylase of L. mesenteroides and Glucosyltransferase A of S. mutans . Residues A à" 'A m01(%) Ammo acid :. ^L SPL* gtfAb

Aspartic acid+Asparagine 16.0 77 71 Threonine 8. 1 40 29 Serine 5.0 25 22 Glutamic acid+Glutamine 10.1 48 52 Glycine 5.0 28 23 Fig. 3. Measurement of the MolecularWeight of Sucrose Alanine 7.9 41 33 Phosphorylase by HPLC Gel Filtration on TSKgel Cysteine + Cystine n.d.c n.d. ld G3000SW. Valine 5.4 26 28 Isoleucine 5.9 29 33 Closed circle, (1) glutamate dehydrogenase (Mr = 290,000): Leucine 8. 1 39 41 (2) lactate dehydrogenase (Mr= 142,000); (3) enolase (Afr=67,000); (4) adenylate kinase (Mr=32,000); (5) Tyrosine 5. 1 24 3 1 Phenylalanine 4. 7 22 24 cytochrome c (Mr*= 12,400); open circle, sucrose phos- Lysine 6.6 3 1 33 phorylase. Histidine 2.0 10 1 1 Tryptophan 1.4 6 6 Arginine 3.2 1 5 1 9 Proline 3.4 1 7 1 5 Methionine 2.0 1 0 9 Total 488 48 1

a SPL: sucrose phosphorylase Calculated based on a molecular weight of 55,000. b gtfA: glucosyltransferase A by Ferretti et al.A) c n.d.: not detected. d Cysteine residue.

ing a partial specific volume, 0.73ml/g, estimated from the amino acid composition (Table II), the molecular weight was found to Fig. 4. Isoelectric Focusing of Sucrose Phosphorylase in be 56,400 by sedimentation equilibrium studies. a Polyaerylamide Gel Column. The molecular weight was also estimated to be About 60ng of purified enzyme was put on the column. 55,000 by HPLC gel filtration on a TSKgel After the electrophoresis, the gel was cut into 2 mmslices. G3000SW column as shown Fig. 3. From Each slice was extracted with 0.5 ml of distilled water for 4hr at 5°C, and then pH and absorbance at 280nmof the electrophoresis in the presence of 1 % SDS, the extract weremeasured. molecular weight of sucrose phosphorylase was Opencircles, absorbance at 280nm;closed circles, pH. estimated to be 55,000 (Fig. 2A). Isoelectric point was a single component with ^o,w °f 4.34 S, The isoelectric point of the enzymewas pH as shown in Fig. 2B. 4.6 by isoelectric focusing, as shown in Fig. 4. Molecular weight The molecular weight of the purified enzyme Aminoacid composition and N-terminal amino was measured by sedimentation equilibrium acid sequence ultracentrifugation, HPLC gel filtration, and The amino acid composition of the enzyme SDS-polvacrvlamide 2el electrophoresis. Tak- is shown in Table II. Cysteine and cystine were Purification and Properties of Sucrose Phosphorylase 1 809 transferase A of S. mutans, having a molecular weight of 55,665 composed of 481 amino acids,4) but distinct from the enzymes of P. saccharophila (Mr = 84,000)13) and Clostridium pasteurianum (Mr = 35,600).2) Silverstein et al. reported that sucrose phosphorylase of P. Fig. 5. Comparison of N-Terminal Amino Acid Se- saccharophila was a dimeric protein having a quence of 30 Residues. sedimentation constant of 5.2 S,13) but that of The upper sequence (SPL, sucrose phosphorylase) is the L. mesenteroides was 4.34 S. In a comparison result of this report. The lower sequence (gtfA, of the sequence of the first 20 amino acid glucosyltransferase A) is the deduced sequence based residues at the N-terminus on this enzymewith on the nucleotide sequence.4) Positions where identical those on glucosyltransferase A showed that 17 amino acids are found in the alignment of two enzymes of 20 residues were identical. We further are boxed. compared30 residues on this enzymewith the deduced amino acid sequence based on not detected. The N-terminal amino acid of the nucleotide sequence of glucosyltransferase A; enzymewas methionine. The sequence of the 18 of 30 residues were identical. Furthermore first 30 amino acid residues at the N-terminus amino acid composition of this enzyme was of the enzyme is given and compared with the quite similar to that of glucosyltransferase A. deduced amino acid sequence of glucosyl- In addition, cysteine and cystine were not transferase A in Fig. 5. detected in amino acid analysis of sucrose phosphorylase, on the other hand only one Discussion cysteine residue was detected on glucosyl- transferase A,4) suggesting that both enzymes In this study, sucrose phosphorylase was have no disulfide bonds. These results indicate purified from L. mesenteroides and some of its that this enzymeis similar to glucosyltransfer- physico-chemical properties studied. The pro- ase A, but not to P. saccharophila enzyme. teins eluted from TSKgel DEAE-5PWdis- Comparison of the complete amino acid played two main bands on SDS-polyacryl- sequence with the sucrose phosphorylase of L. amide gel electrophoresis (data not shown). mesenteroides appears to be untimely because One corresponded to sucrose phosphorylase, only limited information is available. and the other was a 79kDa protein. In further We have purified this enzyme from L. purification, it was hard to separate these two mesenteroides to use for the enzymatic proteins by HPLCgel filtration on TSKgel measurement of inorganic phosphate. A large G3000SW and hydroxylapatite chromatog- scale fermentation is required for supplying the raphy on TSKgel HA-1000. As a result, we enzymefor commercial diagnostic use, because succeeded in the purification of the enzyme of its low productivity. Therefore we are by HPLC hydrophobic chromatography on cloning the sucrose phosphorylase gene from TSKgel Ether-5PW. The purity of this enzyme L. mesenteroides for constructing an high- was demonstrated in both SDS-polyacrylamide producing strain to resolve this problem. gel electrophoresis and sedimentation velocity analysis. Acknowledgments. Wethank Dr. T. Horiuchi and Mr. These results showed that sucrose phosphor- H. Takei for their support and encouragement. Thanks ylase of L. mesenteroides was a monomeric are also due to Ms. E. Yamazaki and Ms. T. Kurokawa protein with a molecular weight of 55,000 for their help in the amino acid analysis and isoelectric composed of 488 amino acids. Its isoelectric point was at pH 4.6. With regard to molecular weight, this enzyme was close to glucosyl- 1810 T. Koga et al.

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