Purification and some properties of β-phosphoglucomutase from Lactococcus lactis subsp. cremoris IFO 3427

JOURNALOP FERMENTATION ANDBIOEN~INBERIN~ Vol. 85, No. 3, 350-353. 1998

Purification and Some Properties of ,&Phosphoglucomutase Lactococcus lactis subsp. cremoris IF0 3427 KAZUO

NAKAMURA,

YOSHIO

SHIROKANE,*

AND MASARU

from

SUZUKI

Research & Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278, Japan Received 10 November 19971Accepted 3 December 1997

,%Phosphoglucomutase (BPGM, EC 5.4.2.6) was isolated to homogeneity from a cell-free extract of Lactococcus tit& subsp. cremorzFIF0 3427 by chromatographies with QAE-Sephadex A-50, phenylhpharose CL-4B, hydroxylapatite, and Bio-Gel A-1Sm. The enzyme was puritied about 260-fold with a yield of 7.2% and a specific activity of 113 units/mg protein. The molecular weight was estimated to be 34,000 and 25,000 by HPLC gel filtration on TSKgel G~OOOSWXL and SDS-PAGE, respectively. The enzyme showed optimum activity around pH 7.0 and its optimum temperature was about 40°C. The enzyme was stable over a pH range from 5.0 to 9.5 and retained its activity up to 45%. It was activated by four divalent cations (Co2+ >MnZ+ > Mg*+ >Ni*+ at 1.0 mM concentration). The K, value was 0.23 mM for ,%D-glucose l-phosphate. The enzyme activity was strongly inhibited by other divalent cations (Cu*+, Cd*+, Zn*+, and Hg*+). ADP and ATP also greatly inhibited the enzyme activity, whereas AMP hardly did. a-~-Glucose l-phosphate and o-glucose 6phosphate were not potent inhibitors of the enzyme. A comparison of its characteristics with the properties of other known BPGMs indicated that the ,EPGM from Lactococcas lactis subsp. cremoti IF’0 3427 is a new type of enzyme. [Key words: ,9-phosphoglucomutase,

Lactococcus 1acti.ssubsp. cremoris IF0 3427, purification,

In the maltose metabolism of lactic acid bacteria, maltose is phosphorylated by maltose phosphorylase to fi-~-glucose l-phosphate and D-glucose. f3-D-Glucose l-phosphate is reversibly converted to /-D-glucose 6-phosphate by a specific mutase, p-phosphoglucomutase (I-PGM, EC 5.4.2.6). The presence of ,Q-PGM was first shown in Neisseria meningitidis in 1960 (l), and the reaction and some properties of the partially purified enzyme of Neisseria perflava were studied by Ben-Zvi and Schramm (2). ,3-PGM has since been reported from several origins, including Euglena gracilis (3), Lactobacillus brevis (4), Lactococcus lactis 65.1 (S), and Entamoeba histolytica (6). The properties of the partially prepared enzymes from E. gracilis and L. brevis have also been investigated in detail. Recently, the properties of the enzyme from L. lactis subsp. lactis purified to more than 90% homogeneity were studied (7), and the gene encoding ,9-PGM was cloned from a genomic library of L. lactis ATCC 19435 using antibodies (8). P-PGM has been used as a coupled enzyme for the measurement of cr-amylase activity in clinical practice (9). However, in general, the properties of the known enzymes, especially concerning their temperature stability and pH optima, have not yet been sufficiently elucidated with respect to their practical application. This paper describes the purification and some properties of a new type of ,B-PGM from Lactococcus la&is subsp. cremoris IF0 3427. The assay of fl-PGM activity was carried out at 37°C with a reaction mixture (3.0ml) containing 2.2mM ,&Dglucose l-phosphate, 0.3 mM D-glucose 1,6-diphosphate, 1.2 mM NADP+ , 12 U/ml glucosed-phosphate dehydrogenase, 20 mM KCl, 2.0mM MgCl*, and 0.8% Triton X-100 in 50mM HEPES-NaOH buffer (pH 7.0). The reaction was started by the addition of 0.03 ml of a properly diluted enzyme solution, and the increase in the absorbance due to NADPH formation was measured

properties]

at 340nm, using a Hitachi U-2000A spectrophotometer. One unit (U) of ,!3-PGM is defined as the amount of enzyme that catalyzes the conversion of 1 ,umol of P-Dglucose l-phosphate to ,8-D-glucose 6-phosphate per min. The protein concentration was monitored by measuring the absorbance at 280nm or measured with a Bio-Rad Protein Assay kit (Bio-Rad, USA; standard protein, bovine plasma gamma globulin). SDS-PAGE was done by the method of Laemmli (10) with an SDS-polyacrylamide gradient slab gel (4-20%; Daiichi Kagaku Yakuhin, Tokyo). A MW-marker kit (Daiichi) was used for polypeptide standards. The molecular weight of the native enzyme was estimated by HPLC gel filtration on TSKgel G3000SWxL by the method of Fukano et al. (11) using a MW-marker kit (Oriental Yeast, Tokyo). L. lactis subsp. cremoris IF0 3427 was cultured without aeration at 30°C for 24 h in a 500-l tank containing 400 1 of the medium (0.5% maltose.HzO, 2.0% Polypepton, 1.0% yeast extract, 0.5% KH2P04, 0.5% CH$OONa. 3Hz0, 0.02% MgS04.7H20, and 0.0002% MnC12.4H20, pH 7.0). All operations were carried out below 5°C throughout the purification steps. The cells were harvested from 400 1 of culture broth with a hollow-fiber concentrating apparatus (Microza; Asahi Kasei Kogyo, Tokyo.) and washed with 1OmM phosphate buffer (pH 7.0) containing 2.0 mM EDTA (Buffer A). To the cell suspension (12 I), 25 g lysozyme, 60ml Triton X-100, and 160 g (NH&SO4 were added. After leaving the mixture to stand for 2 h at 35”C, 10% polyethyleneimine solution (pH 7.5) was added dropwise, and the precipitation formed was removed by filtration. The filtrate was concentrated by ultrafiltration and dialyzed against Buffer A containing 0.05 M KCl. To the dialyzed enzyme solution, QAE-Sephadex A-50, previously equilibrated with Buffer A containing 0.05 M KCl, was added, and washed with the same buffer. The adsorbed enzyme was eluted with Buffer A containing 0.4M KCl, concentrated by

* Corresponding author. 350

NOTES

VOL. 85, 1998 TABLE 1.

Step

Summary of purification of ,B-phosphoglucomutase from L. lactis subsp. cremoris IF0 3421 Total protein (mg) 98600 46500

Crude extract QAE-Sephadex A-50 (Batchwise) QAE-Sephadex A-50 1040 Phenyl-Sepharose CL4B 281 Hydroxylapatite 78.6 Bio-Gel A- 1.5m 27.0

Total activity (u) 42100 37900

Specific activity Wmg) 0.43 0.82

30100 16900 7100 3050

29.0 60.0 91.0 113

1

**-‘.* 97.4k * 66.3k

100 90

s+-w~ 42.4k

72 40 17 7.2

rrr*

w

PI-I

12

14.4k

FIG. 1. SDS-PAGE of purified ,Q-phosphoglucomutase. The gel was stained with Coomassie briiliant blue (R-250), and destained in 7% acetic acid. Lane 1, Purified enzyme (about 10 /I&; lane 2, marker proteins.

subsp. Iactis (18 U/mg). The purified enzyme preparation gave a single protein band on SDS-PAGE (Fig. 1). The molecular weight of the native enzyme was estimated to be 34,000 from the retention time of HPLC gel filtration with TSKgel G3OOOSWxr. From the mobility of SDSPAGE, the molecular weight of the enzyme protein monomer was found to be 25,000 (Fig. 1). Thus, /?-PGM appeared to have a monomer structure. The molecular weight of the enzyme resembled those of the monomeric enzymes from several origins (3, 4, 7). The enzyme showed optimum activity around pH 7.0 (Fig. 2A), and was stable over a pH range from 5.0 to 9.5 on incubation at 30°C for 30min (Fig. 2B). The optimum pH of the enzyme was higher than those of L. lactis subsp. lactis (pH 6.3-6.7), N. pe&va (pH 6.4-6.8), and L. brevis (pH 6.3-6.8), but resembled that of E. gracilis (about pH 7.0). The optimum temperature of the enzyme was estimated to be about 40°C (Fig. 3A). It retained its activ-

I

0

10

30.0k

20.lk

m

8

2

Yield (%)

ultrafiltration, and dialyzed against Buffer A containing 0.05 M KCI. The enzyme solution was applied onto a QAE-Sephadex A-50 column (16 x 50 cm) equilibrated with Buffer A containing 0.05 M KCl. The column was washed with Buffer A containing 0.2 M KCl, and eluted with Buffer A containing 0.25 M KCI. The eluted active fractions were pooled, and concentrated by ultrafiltration. To the concentrate, (NH&SO4 was added up to a final concentration of 20% saturation. The enzyme solution was applied onto a phenyl-Sepharose CL-4B column (2.5 x 30 cm) equilibrated with Buffer A containing (NH&SO4 (20% saturation). The enzyme was eluted with a reverse linear concentration gradient of (NH&SO4 (20% to 0% saturation) in Buffer A. The active fractions were concentrated by ultrafiltration and dialyzed against 2mM phosphate buffer (pH 6.8). The dialyzed enzyme solution was applied onto a hydroxylapatite column (2.5 x 16 cm) equilibrated with 2 mM phosphate buffer (pH 6.8). The column was eluted with a linear concentration gradient of phosphate buffer, pH 6.8 (2 to 30 mM). The active fractions were pooled and concentrated by ultrafiltration. The concentrated enzyme solution was put on a Bio-Gel A-l .5m column (2.5 x 96 cm) equilibrated with Buffer A containing 0.1 M KCI, filtered with the same buffer, and the active fractions of the single peak were combined. The purification of B-PGM is summarized in Table 1. At the final stage, the enzyme was purified up to homogeneity 260-fold with a yield of 7.2% from the cell-free extract, and a specific activity of 113 U/mg protein, which is significantly higher than that of L. Iactis

6

351

’ 2

I

I

I

4

6

/

8

I

10

I

12

PH

FIG. 2. Effects of pH on p-phosphoglucomutase activity. (A) Optimum pH. The enzyme activity was measured in 50 mM buffer solution at various pHs. The buffer systems used were: A, sodium acetate; q , MES-NaOH; W, HEPES-NaOH; 0, Tris-HCl; 4, CHES-NaOH. (B) pH stability. The enzyme solution was kept at 30°C for 30 min in 50 mM buffer solution at various pHs and the remaining activity was measured. The buffer systems used were: A, sodium acetate; q , MES-NaOH; n , HEPES-NaOH; 0, Tris-HCl; V, CAPS-NaOH.

352

NAKAMURA ET AL.

J. FERMENT.

BIOENG.,

b

0

20

a 30

\

I

0 40

50

60

70

20

30

I

/

40

50

,

60

70

“C

“c

FIG. 3. Effects of temperature on ,¶-phosphoglucomutase activity. (A) Optimum temperature. The enzyme activity was measured at various temperatures in 50 mM phosphate buffer @H 7.0). (B) Thermal stability. The enzyme solution was kept at various temperatures for 15 min in 50 mM phosphate buffer @H 7.0) and the remaining activity was measured.

ity up to 45°C at pH 7.0 for 15 min, and the remaining activity at 50°C was 88% (Fig. 3B). No significant loss of activity was observed on incubation at 37°C for one week (data not shown). The enzyme was demonstrated to be more thermostable than the enzymes of L. lactis subsp. lactis (remaining activity, 4% at 50°C for 15 min, pH 7.3, and about 50% at room temperature for one night) (7), L. brevis (almost no activity at 4’C for 2 h above pH 8.0) (4), and E. gracilis (70% at 0°C for 2 d, pH 7.0) (3). The enzyme was inactive in the absence of an added divalent cation. Four divalent cations (Co2+ >Mn2+ > Mg2+ > Ni2+ at 1.0 mM concentration) were found to effectively activate the enzyme (Fig. 4), although the enzyme of L. lactis subsp. lactis was reported to require Mg2+ for activity (7). The results of activation by these four divalent cations were similar to those for the enzyme of L. brevis, but Co2+ and Ni2+ did not activate the enzyme of N. pery7ava and Ni2+ was not a potent activator for the enzyme of E. gracilis (2-4). The K, value for ,9-D-glucose l-phosphate was estimated to be 0.23 mM from a Lineweaver-Burk plot (data not shown), which resembled that of N. perflava (0.3 mM) (2). The effects of various reagents on the enzyme activity were

examined (Table 2), because no such data for the enzyme of L. lactis subsp. Iactis have been reported. a-D-Glucase l-phosphate and n-glucose 6-phosphate were not potent inhibitors of the enzyme, whereas n-fructose 1,6diphosphate significantly inhibited the enzyme activity. ADP and ATP were also inhibitory, but AMP showed relatively little inhibition. Among various other cations tested, Cu2+, Cd2+, Zn2+, and Hg2+ were strong inhibitors. Sulfhydryl-group blocking agents @-chloromercuribenzoic acid, iodoacetic acid, and N-ethylmaleimide; 1 .O mM concentration), reducing agents (L-cysteine, dithiothreitol, and 2-mercaptoethanol; 2.0 mM concentration), phenylmethylsulfonyl fluoride (0.1 mM concentration), and iodine (1 .O mM concentration) had almost no effect (data not shown). The characteristics of the enzyme from L. Iactis subsp. cremoris IF0 3427, especially its specific activity, optimum pH, thermal stability, and pattern of activation by divalent cations, clearly indicate that it is a new type of /3-PGM, distinct from other known I-PGMs (2-4, 7), and that it is applicable as a coupled enzyme for the measurement of a-amylase activity in clinical practice. TABLE 2.

Effects of various reagents on ,9-phosphoglucomutase activity

Concentration (mM) None a-n-Glucose l-phosphate 10 D-Glucose 6-phosphate 10 o-Fructose 1,6-diphosphate 10 AMP 10 ADP 10 ATP 10 FeC12 1.0 1.0 CaC& 1.0 BaCl, 1.o cuso4 CdCl* 1.0 ZnS04 1.0 1.0 HgClz 1.0 AgNG, Reagent added

1.0

2.0

3.0

4.0

Divalent cation (mM) FIG. 4. Dependence of p-phosphoglucomutase activity on the concentrations of various divalent cations. The reaction was carried out using divalent cations (Mg*+, CoZf , MnZf , and N?+) at the various concentrations (0 to 4.0 mM). The activity with a Mg*+ concentration of 2.0 mM is expressed as 100%. Symbols: 0, Mg2+; A, Coz+., 0 1Mn*+.,, n Ni*f .

RelatiyY&ivity 0 100 88 89 37 86 7 2 32 23 88 5 6 4 7 44

The enzyme activity was measured after a preincubation period of 10 min at 5°C in the presence of each reagent. The activity without any additive was taken as 100%.

NOTES

VOL. 85, 1998 We would like to thank Drs. S. Ishii, M. Kikuchi, and Y. Imai for their support and encouragement. REFERENCES 1. Ben-Zvi, R. and Schramm, M.: A phosphoglucomutase for ,%glucose l-phosphate. Biochem. Biophys. Res. Comnmn., 2, 8-11 (1960). 2. Ben-Zvi, R. and Schmmm, M.: A phosphoglucomutase specific for j-glucose l-phosphate. J. Biol. Chem., 236, 2186-2189 (1961). 3. Belocopitow, E. and Marechai, L. R.: Metabolism of trehalose in Euglena gracilis (partial purification and some properties of phosphoglucomutase acting on ,%glucose l-phosphate). Eur. J. Biochem., 46, 631-637 (1974). 4. Marechal, L. R., Oliver, G., Veiga, L. A., and Roiz Ho&ado, A. A. P.: Partial purification and some properties of /%phosphoglucomutase from Lactobacillus brevis. Arch. Biochem. Biophys., 228, 592-599 (1984). 5. Sjoherg, A. and Hahn-Hagerdal, B.: ,L?-Glucose-l-phosphate, a possible mediator for polysaccharide formation in maltoseassimilating Lactococcus lactis. Appl. Environ. Microbial., 55, 1549-1554 (1989). 6. Vargas, M. A. and Orozco, E.: Entamoeba histolytica: changes

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in the zymodeme of cloned nonpathogenic trophozoites cultured under different conditions. Parasitol. Res., 79, 353-356 (1993). 7. Qian, N. Y., Stanley, G. A., Hahn-Hagerdal, B., and Radof two phosStrom, P.: Purification and characterization phoglucomutases from Lactococcus lactk subsp. lactis and their regulation in maltose-utilizing cells. J. Bacterial., 176, 5304-5311 (1994). 8. Qian, N. Y., Stanley, G. A., Bunte, A., and Radstrom, P.: Product formation and phosphoglucomutase activities in Lactococcus lactis: cloning and characterization of a novel phosphoglucomutase gene. Microbiology, 143, 855-865 (1997). 9. Makise, J., Ito, M., and Kaaayama, M.: Investigation of the optimum conditions for measurement of a-amylase activity with maltopentaose and maltose phosphorylase. Seibutsu Shiio Bunseki, 9(2), 21-29 (1986). (in Japanese) 10. Laemmli, U.K.: Cleavage of structure proteins during the assembly of the head of bacteriophage 4. Nature, 227, 680-685 (1970). 11. Fnkano, K., Komiya, K., Sasaki, H., and Hasimoto, T.: Evaluation of new supports for high-pressure aqueous gel permeation chromatography: TSK-gel SW type columns. J. Chromatogr., 166, 47-54 (1978).