JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 76, NO. l, 14-18. 1993

Purification and Properties of Extracellular -Mannanases Produced by Enterococcus casseliflavus FL2121 Isolated from

Decayed Konjac YUJI ODA,* TOSHIAKI KOMAKI, AND KENZO TONOMURA

Department of Food Science and Technology, Fukuyama University, Fukuyama, Hiroshima 729-02, Japan

Received 10 February 1993/Accepted 3 April 1993

Two fl-mannanases (M-I and M-II) were purified from the culture filtrate of Enterococcus cassellflavus FL2121 by ammonium sulfate precipitation, column chromatography of DEAE-Toyopear1650M and Phenyl- Toyopearl 650M and preparative polyacrylamide gel dectrophoresis to homogenlties. The molecular weights of M-I and M-H were estimated to be 142,000 and 137,000 by SDS-polyacrylamide gel electrophoresis and 127,000 and 113,000 by gel filtration, respectively. M-I and M-H exhibited an optimum pH at 6.0 and an op- timum temperature at 50°C. The enzymes were activated slightly by CoCI2 and MnCI2, and inhibited strongly by AgNO3, HgCI2, EDTA, and N-bromosuccinimide. The Km values of M-I and M-II for konjac fl-l,4- glucomannan were 0.14 and 0.30 (mg/ml), and the maximum velocities were 1,110 and 1,700 (U/rag protein), respectively. Both enzymes were endo-type ~.mannanases hydrolyzing mannosides larger than fl-l,4-D-man- notetraose.

/3-Mannanases (/~-l,4-D-mannan monohydrolyase, EC 3.2.1.78) are enzymes which catalyze the random hydro- lysis of/3-1,4-mannosidic linkages in mannans, glucoman- nans, and galactomannans (1). Although/3-mannanase has been found in bacteria (2--4), actinomycetes (5), fungi (1, 6), and higher plants (1, 7), little is known about that from lactic acid bacteria.

On the other hand, increasing attention is being paid to mannooligosaccharides produced from konjac glucoman- nan since these sugars have been found to stimulate the growth of Bifidobacterium (8). However, /3-mannanases adequate for the production of mannooligosaccharides from/3-mannan are not commercially available.

From decayed Konjac, therefore, we isolated a lactic acid bacterium, Enterococcus casseliflavus FL2121, which produced extracellular /3-mannanase. The present paper reports on the purification and properties of two /3- mannanases in the culture filtrate of E. casseliflavus FL2121.

MATERIALS AND METHODS

Preparation of Konjac Konjac powder (6 g) was sus- pended in 220 ml of distilled water and mixed for several minutes. After 20ml of 1% Ca(OH)z were added with vigorous stirring, 5 g of the mixture was poured into a 15 ml centrifuge tube and sterilized at 121°C for 20 min.

Isolation of microorganism Decayed Konjac was do- nated by a local manufacturer. To enrich microorganisms producing/3-mannanase, 0.1 ml of the decayed Konjac was aseptically transferred to 5 g of freshly-prepared Konjac and incubated at 30°C. Konjac was liquefied for 3 to 5 d and its pH was reduced from 8.5 to 5.0. After enrichment culture was repeated once more, liquefied Konjac was di- luted and spread on agar plates containing the two media as described below and incubated at 30°C for 2 d. Seven and fourteen microorganisms were isolated from the me-

* Corresponding author.

14

dium containing 1% konjac powder, 0.5% Bacto-yeast extract, 0.5% Polypepton, 0.1% K2HPO4, and 0.02% MgSO4.7H20 (pH 6.8) and that supplemented with 0.5% Na2CO3 (pH 9.7), respectively. Among these isolates, five strains derived from the alkaline medium could liquefy Konjac, and one strain FL2121, which started growing actively with the production of/~-mannanase under alkaline conditions, was selected. This strain was identified as E. casseliflavus by the National Collection of Industrial and Marine Bacteria, Ltd. (Scotland) (9). The bacterium was a facultatively anaerobic, Gram-positive, non-motile, and yellow-pigmented strain. The cells showed coccoid cells in pairs and short chains and did not form spores. It responded negatively to the reactions of catalase and oxidase. L-Lactic acid was the major product of glucose fermentation. FL2121 was atypical of E. casseliflavus with respect to nonmotility, but was more atypical of E. mundtii by exhibiting positive assimilation of gluconate and a-methyl-D-glucoside.

Culture The bacterium was grown on 100 ml of the medium supplemented with 0.5% Na2CO3 as described above in a 500 ml Erlenmeyer flask at 37°C for 24 h with shaking (220 rpm). During cultivation the pH of the cul- ture medium was reduced from 9.7 to 8.7. The supernatant obtained by centrifugation at 100,000 x g for 10 rain was used for enzyme purification.

Enzyme assay A reaction mixture contained 1 mg of purified konjac glucomannan in 0.1 ml of distilled water, 0.1 ml of 0.1 M phosphate buffer (pH 6.0), and 0.05 ml of the enzyme. After incubation at 37°C for 10 min, the reac- tion was stopped by the addition of 3,5-dinitrosalicylic acid reagent, and the reducing sugars produced were deter- mined (10). One unit was defined as the amount of enzyme which releases 1/~mol of reducing sugar as equivalent to D- mannose per minute under the above conditions. Protein was determined by the method of Bradford with bovine serum albumin as a standard (11).

Gel electrophoresis Polyacrylamide gel electrophore- sis (PAGE) was performed on 7.5% gel according to the

VOL. 76, 1993 PURIFICATION OF E. CASSELIFL.4 VUS ~-MANNANASES 15

method of Laemmli (12). The gel was stained with 0.25% Coomassie Brilliant Blue R-250 to locate the protein bands.

Molecular weight Enzyme molecular weight was estimated by sodium dodecyl sulfate (SDS)-PAGE and gel filtration. The SDS-PAGE was carried out on 7.5% gel using the method of Laemmli (12). Gel filtration was performed on a column of Toyopearl HW-55S (1.5 × 95 cm, Toso Co., Tokyo) equilibrated with 0 .2M phos- phate buffer (pH 7.0). Standards for molecular weight determination were obtained from Bio-Rad Laboratories (Richmond, CA, USA).

High performance liquid chromatography (HPLC) Mannooligosaccharides were analyzed with a high per- formance liquid chromatograph (Hitachi model L-6000, Tokyo) equipped with a Gelpack GL-C610 column (Hitachi) and a refraction monitor (Hitachi L-3000).

Materials Bacto-yeast extract and Polypepton were obtained from Difco Laboratories (Detroit, MI, USA) and Nippon Seiyaku Co. (Tokyo), respectively. Purified konjac glucomannan was prepared by the method of Sugiyama et al. (13). 8-1,4-D-Mannooligosaccharides were a kind gift from Professor I. Kusakabe of Tsukuba University, Japan.

RESULTS

Purification of two ~-mannanases Unless otherwise stated, 20 mM phosphate buffer (pH 7.2) was used through- out the following purification experiments.

Step 1. Ammonium su~ate precipitation To 3,730 ml of culture filtrate, solid ammonium sulfate was added to produce 40% saturation. After 1 h, the resulting pre- cipitate was removed by centrifugation and additional ammonium sulfate was added to the supernatant to give 80% saturation. The resultant precipitate was collected, dissolved in a small volume of buffer, and dialyzed.

Step 2. DEAE-Toyopear! 650M The dialyzed en- zyme solution was applied to a column (2.5 x 18 cm) of DEAE-Toyopear1650M equilibrated with the buffer. After the column was washed with the buffer, the enzyme was eluted with a linear gradient of NaCI concentration from 0 to 0.7 M in the buffer. The active fraction eluted with 0.3 M NaCI was collected and dialyzed against the buffer containing 20% ammonium sulfate.

Step 3. Phenyl-Toyopear! 650M The enzyme solu- tion was loaded on a Phenyl-Toyopearl 650M column (1.5 x 6 cm) equilibrated with the buffer containing 20% ammonium sulfate. The column was washed with the same buffer, and successively eluted with a decreasing gradient of ammonium sulfate from 20% to 0%. The enzyme was recovered in the fractions elnted using buffer containing 0% ammonium sulfate. From the above procedures, the enzyme was purified 240-fold with a recovery of 38.3% (Table 1).

When the active fraction was subjected to PAGE, two stained bands appeared (Fig. 1A). /~-Mannanase activity

-I-

:E ~1.0 (9 E

0

B

B

I I I I I I 0 1 2 3 z, 5 6

Distance (cm) FIG. 1. Locations of protein and ~-mannanase activity on the

electrophoresed gel. (A) About 4 pg of the purified enzyme was loaded on the PAGE gel (1 x 85 x 60ram), and protein was stained. (B) Separate electrophoresis was carded out with 12 P8 of the purified enzyme. The enzyme activity in each fraction was determined after the gel plate was cut at intervals of 1 mm and extracted with 0.1 ml of 20 mM phosphate buffer (pH 7.2) overnight.

was detected in both of the bands (Fig. 1B). The one which moved more slowly was named ~-mannanase I (M-I), and the other ~-mannanase II (M-II).

Step 4. Preparative PAGE To separate M-I from M-II, the purified enzyme was loaded on a slab gel of poly- acrylamide (3 x 140x 100mm) and electrophoresed. The gel plate was cut at intervals of 2 mm and extracted with the buffer overnight. The two active fractions were col- lected and dialyzed against the buffer. Two enzymes pro- duced single bands on PAGE, indicating electrophoretic homogeneity (data not shown).

Molecular weights of the enzymes The molecular weights of M-I and M-II were estimated to be 142,000 and 137,000 by SDS-PAGE, and 127,000 and 113,000 by gel filtration, respectively (Fig. 2). Since the values obtained from the two methods were approximately the same, both enzymes were thought to be monomeric proteins.

Effects of pH and temperature on activity and sta- bility The activities of M-I and M-II were assayed in the pH range of 4.0 to 7.8 at 37°C. Maximal activity was found around pH 6.0 in both enzymes (Fig. 3A). M-I was inactivated over pH 7, and M-II was stable between pH 4 to pH 7 (Fig. 3B).

The effects of temperature were examined in the range of 30°C to 60°C at pH 6.0 (Fig. 3C). The highest activities of the enzymes were obtained at 50°C. The enzymes were relatively stable up to 40°C, but rapidly inactivated at tem- peratures above 40°C (Fig. 3D).

Effects of metal ions and chemical reagents The ac- tivities of M-I and M-II treated with various metal ions and chemical reagents were assayed (Table 2). Both en- zymes were activated by Co 2+ and Mn 2+, and strongly in- hibited by Ag 2+, Hg 2+, EDTA, and N-bromosuccinimide.

TABLE 1. Purification of/~-mannanases from the filtrate of E. casseliflavus FL2121

Step Total activity Total protein Specific activity Purification Yield (U) (mg) (U/mg) (fold) (%)

Culture filtrate 2,272 410 5.54 1 100 (NH+)2SO + precipitation 2,096 200 10.5 1.9 92.3 DEAE-Toyopearl 650M 1,365 4.22 323 58.3 60.1 Phenyl-Toyopearl 650M 870 0.65 1338 242 38.3

16 ODA ET AL.

° a A c ( B r ,

'~Mul_i I - 5.5 5.2

.-~ o ~ b -I "c 5.o ~ ,4-xz s.o

4.5 4.5

/,.6 I I I I I /,.0 O.5 1.0 70 80 9O I00

Relative mobility Elution volume(mD

FIG. 2. Estimation of molecular weights of M-I and M-II by SDS-PAGE (A) and gel filtration (B). Standard marker proteins; a, myosin (200,000); b, /%galactosidase (116,250); c, phosphorylase b (97,400); d, bovine serum albumin (66,200); e, ovalbumin, hen egg white (45,000); f, thyoglobulin (670,000); g, gamma-globulin (158,000); h, ovalbumin, chicken (44,000); i, myoglobin (17,000).

Since N-e thylmale imide and iodoacet ic acid had little effect on the activities of bo th enzymes, sulfhydryl groups are most l ikely not involved at the active sites.

Kinet ic propert ies The effects o f various concentra- tions o f substrate on enzyme activity were examined. Lineweaver-Burk double reciprocal plots were used to cal- culate the Km values for konjac g lucomannan. The Km values o f M-I and M-II were 0.14 and 0.30 (mg/ml) , respec- tively. Max imum velocities (Vm~) were 1,110 (U/mg pro- tein) in M-I and 1,700 in M-II .

Hydro lys i s o f k o n j a c g l u c o m a n n a n and m a n n o o l i g o s a e - charides The react ion products o f konjac glucoman- nan hydrolyzed by M-I were analyzed using high per form-

.•I00

._~

0

A

.A

l. 5 6 7 8

B

\

i 5' 5 10

pH

A

100 -~

50

f f l

l 0 ~:

i

~ 1 0 0

N 50

o

FIG. 3.

C

I t i 30 /-,0 50 60

D !100 .~

¢ ' r

t t 0

Temperature ( 'C)

Effects of pH on the activity (A) and stability (B) of the enzymes and those of temperature on activity ((2) and stability (D). Buffer used: glycine-NaOH-HCl ( [] ), sodium acetate (zx), phosphate (©), MOPS (<>), borate (v). Open and closed symbols indicate M-I and M-II, respectively, pH and temperature stabilities were evaluated after preincubation at 45°C for 30min with various pHs and at various temperatures in 20 mM phosphate buffer (pH 7.2) for 30 min, respectively.

J. FERMENT. BIOENG.,

TABLE 2. Effects of various metal ions and chemical reagents on the activities of two/~-marmanases, M-I and M-II

Compound (1 mM) Residual activity (%)

M-I M-II

None 100 100 MgCI2 96 100 CaC12 101 86 BaCI2 96 101 CuC12 102 108 NiC12 100 103 FeCI2 87 89 Pb(CH~COO)2 100 98 COC12 128 121 MnC12 135 120 AgNO3 19 20 HgC12 4 9

2-Mercaptoethanol 100 104 SDS (0.1%) 92 92

N-Ethylmaleimide 90 l01 Iodoacetic acid 89 100

EDTA 51 44 N-Bromosuccinimide 0 0

The enzymes were preincubated at 30°C for 30 rain in the presence of the above compounds at I raM, except for SDS (0.1%), and the residual activities were assayed as described in the Materials and Methods.

ance l iquid ch romatography (Fig. 4). Authent ic manno- ol igosaccharide and mannose s tandards eluted in order according to molecular weight. The retent ion times were 9 .1 ra in for mannopentaose (M5), 9 . 9min for manno- tetraose (M4), 11.1 rain for mannot r iose (M3), 13.0min for mannobiose (M2), and 15.8 min for mannose (M1). The peak for glucose appeared at 14.7m in (data not shown). Before reaction, a single peak derived f rom the substrate and solvent was observed. As the react ion proceeded, delayed and successive peaks emerged due to the degradat ion o f g lucomannan. The peak at 10.7 min between M4 and M3 was the highest among those which appeared in 24 h-hydrolysis , and its area reached a cons- tant . Since the p ropor t ion o f mannose content in konjac g lucomannan is about 0.6 (6), the peaks which were not detected in the s tandard assay seemed to be derived f rom heterool igosaccharides that were composed o f mannose and glucose. These results suggest that M-I is an endo-type and not an exo- type/3-mannanase.

To clarify the substrate specificity o f M-I, the enzyme was incubated with an authentic mannool igosacchar ide , and the react ion products were examined with H P L C (Fig. 5). M-I hydrolyzed M5 to M4, M3, and M2, and M4 to M3, M2, and M I , but not M3 and M2. Similar results were observed with H P L C chromatograms for hydrolysis by M-II .

D ISCU SSIO N

/3-Mannanase activity in the culture filtrate o f E. cas- seliflavus FL2121 was based on the two enzymes, M-I and M-II . Mult iple forms of /3-mannanases have been found in Bacillus sp. (4) and Streptomyces sp. (5), and the molecu- lar weights were found to be less than 70,000. The molecu- lar weights o f M-I and M-II were est imated to be 142,000 and 137,000 by S D S - P A G E and 127,000 and 113,000 by gel fi l tration, respectively, values which are much larger than those o f other organisms. It is unknown whether two enzymes are individual ly synthesized or if one of the en-

VoI.. 76, 1993 PURIFICATION OF E. CASSELIFLA VUS/3-MANNANASES 17

Reaction period lh

8h ~ _ _

I I I I I 0 5 10 15 20

Time (rain)

FIG. 4. High performance liquid chromatograms of the products from konjac glucomannan hydrolyzed by ~5-marmanase I. M-I (5 U) in 0.5 ml was added to 10 ml of 1% konjac glucomarman in 40 mM phosphate buffer (pH 6.0, autoclaved previously), and incubated at 37°C. A portion of the sample was withdrawn and the reaction was stopped by placing in boiling water for 10 min. Reaction products in 10/~1 were analyzed by HPLC as described in the text. Mannooligo- saccharides were applied at 3/~g; MS, mannopentaose; M4, man- notetraose; M3, mannotriose; M2, mannobiose.

zymes is processed f rom the other with certain modifica- t ions.

The op t imum p H values o f M-I and M-II were a round 6.0. Lit t le act ivi ty was detected at p H 9.7, the initial p H of the culture medium. In strain FL2121, konjac glucoman- nan seemed to induce the produc t ion of /3-mannanase and not to be util ized as the carbon source. The bacter ium was assumed to grow on carbon compounds other than the g lucomannan in konjac powder . However , the activity of the crude enzyme in the culture filtrate was relatively high under alkal ine condi t ions; the activity at p H 8.5 was about 70% of that at p H 6.0 (data not shown). The enzymes may be par t ly involved in the assimilat ion o f g lucomannan under alkal ine condi t ions. The effects o f culture condi- t ions on the growth o f strain FL2121 and the produc t ion of /3-mannanase and/3-mannos idase will be repor ted else- where (14).

The thermal stabilities of M-I and M-II were less than those o f o ther microorganisms, but both enzymes were more stable under acidic condi t ions than those isolated f rom other sources (1). The p H stabilit ies o f the enzymes may account for the characterist ics o f E. casseliflavus, a lactic acid bac ter ium (9).

The Vm~x values o f the two enzymes for konjac g lucomannan were similar to those of Bacillus sp. (4). The Km values o f M-I and M-II were 0.14 and 0.30 (mg/ml) ,

M5 I~N3H2 M1

M5 digest

N4 digest ~ ~

_2 M3 digest

M2 digest

I I I I I 0 5 10 15 20

Time (min)

FIG. 5. High performance liquid chromatograms of the products from four t3-1,4-mannooligosaccharides hydrolyzed by/~-mannanase I. The mixture containing 1 mg of/~-l,4-mannooligosaccharide and 1 U of enzyme in 0.1 ml of 40 mM phosphate buffer (pH 6.0) was in- cubated at 37°C for 2 h, and the products in 3 pl of reaction mixture were analyzed as in Fig. 4.

respectively, values which are 10-fold smaller than those o f Bacillus subtilis (2) and Bacillus sp.(4).

F r o m the analyses o f react ion products , M-I and M-II were found to be endo-/3-1,4-mannanases which hydrolyze sugars larger than mannote t raose . These substrate spe- cificities were similar to those o f Rhizopus niveus (6) and B. subtilis (2), and different f rom those o f Aspergillus niger (1) and Bacillus sp. (4). In conclusion, /3-mannanases produced by E. casseliflavus FL2121 were shown to be appl icable for prepar ing mannool igosacchar ides f rom /3- mannan without further degradat ion to monosacchar ides .

ACKNOWLEDGMENTS

We thank Professor I. Kusakabe, Tsukuba University, for provid- ing ~5-1,4-mannooligosaccharides, and M. Horii and C. Matsuo for their technical assistance. This work was supported in part by a Grant-in-Aid (Glyco-technology Program) from the Japanese Minis- try of Agriculture, Forestry and Fisheries (MAFF).

R E F E R E N C E S

1. McCieary, B.V.: ~-Mannanase. Methods Enzymol., 160, 596- 610 (1988).

2. Emi , S . , F u k u m o t o , J . , and Y a m a m o t o , T.: Crystallization and some properties of mannanase. Agric. Biol. Chem., 36, 991-1001 (1972).

3. Araki, T. and Kitamikado, M.: Purification and characterization of a novel exo-~-mannanase from Aeromonas sp. F-25. J. Biochem., 91, 1181-1186 (1982).

18 ODA ET AL. J. FERMENT. BIOI~NG.,

4. Akino, T., Nakamura, N., and Horlkoshi, K.: Characterization of three/~-mannanases of an alkalophilic Bacillus sp. Agric. Biol. Chem., 52, 1459-1464 (1988).

5. Takahashi, R., Knsalkabe, L, Kobayashi, H., Mnrakami, K., Maekawa, A., and Suzuki, T.: Purification and some properties of mannanase from Streptomyces sp. Agric. Biol. Chem., 48, 2189-2195 (1984).

6. Hashimoto, Y. and Fuknmoto, J.: Studies on the enzyme treat- ment of coffee beans. I. Purification of mannanase of Rhizopus niveus and its action on coffee mannan. Nippon Nogei- kagaku Kaishi, 43, 317-322 (1969).

7. Shimahara, H., Suzuki, H., Sngiyama, N., and Nisizawa, K.: Partial purification of ~-mannanases from the konjac tubers and their substrate specificity in relation to the structure of konjac glucomannan. Agric. Biol. Chem., 39, 301-312 (1975).

8. Kobayashi, Y., Eehizen, R., Mada, M., and Mntal, M.: Effect of hydrolyzate of Konjac mannan and soybean oligosaccharides on intestinal flora in man and rats, p. 69-90. In Mitsuoka, T. (ed.),

Intestinal flora and dietary factors. Japan Scientific Societies Press, Tokyo (1983).

9. Collins, M., Farrow, J . A . E . , and Jones, D.: Enterococcus mundtii sp. nov. Int. J. Syst. Bacteriol., 36, 8-12 (1986).

10. Bernfeld, P.: Amylases. a and ~. Methods Enzymol., 1, 149-158 (1955).

I I. Bradford, M. M.: A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976).

12. Laemmli, U. K.: Cleavage of structural proteins during the assem- bly of the head of bacteriophage T4. Nature, 227, 680-685 (1970).

13. Sugiyama, N., Shlmahara, H., and Andoh, T.: Studies on man- nan and related compounds. I. The purification of konjac man- nan. Bull. Chem. Soc. Japan, 45, 561-563 (1972).

14. Oda, Y., Komaki, T., and Tonomura, K.: Production of/~-man- nanase and/~-mannosidase by Enterococcus casseliflavus FL2121 isolated from decayed Konjac. Food Microbiol., in press.