Jotnt~Az oF F~.V.M~ST,~TIOS AND BIOENGINEERING Vol. 73, NO. 1, 54-57. 1992

Purification and Properties of the Endo-l,4- -Glucanase III from Ruminococcus albus

YUICHI WATANABE, j RYUICHI MORIYAMA, 2 TSUKASA MATSUDA, 2 SHOICHI SHIMIZU, 3 AND KUNIO OHMIYA 4.

Niigata Research Laboratories, Mitsubishi Gas Chemical Company Inc., Niigata 950-31, ~ School of Agriculture, Nagoya University, 2 Professor Emeritus, Nagoya University, 3 Chikusa, Nagoya 464-01, Faculty of

Bioresources, Mie University, Kamihama, Tsu 514, 4 Japan

Received 9 August 1991/Accepted 17 September 1991

Endoglucanase III ( E G I ~ was purified from Ruminococcus albus culture supernatant. An enzyme having a molecular weight of 53,000 was stabilized by mercaptoethanoi and inhibited by sulfhydryl group-blocking reagents, and exhibited its highest CMC-degrading activity at pH 5.7 and 55°C. The enzyme hydrolyzed cellobiose (G2) and cellotriose (G3) only negligibly, but significantly hydrolyzed ceHotetraose (G4), cellopen- taose (G5) and cellohexaose (G6). The major hydrolysis reactions conducted by the enzyme were G4--+2G2, GS--+G2+ G3, G6--+G2+ G4 and G6--~2G3. The Vma., values of these reactions increased remarkably while the Km values decreased significantly with an increase in degree of polymerization of the substrate.

Ruminococcus albus F-40 is a useful anaerobic and cel- lulolytic rumen bacterium since the organism is capable of converting cellulose to ethanol and other valuable mate- rials. For enhancing cellulose utilization of R.albus, we have established anaerobic cultivation methods for R. albus (1) and purification methods for the mesophilic cel- lulases of the organism, resulting in endo-l,4-/~-gluca- nase (2), cellobiosidase (3) and fl-giucosidase (4) being puri- fied from the culture of R.albus to homogeneity and charac- terized. Only one endoglucanase was found in the R. albus culture supernatant using DEAE-Sephadex even though many cellulolytic bacteria (5, 6) and fungi (7) have several kinds of endo-l,4-fl-glucanases, all of which synergistically degrade cellulose. When DEAE-Bio Gel A was employed, we found two additional endo-l,4-~-glucanases, one of which has been purified (8). Its gene has been cloned into Escherichia coli HB101 using pBR322 (9) and the DNA se- quence of the gene was determined (10). In the present re- port, we purified the third cellulase, endo-l,4-fl-glucanase III (EG III) from the culture supernatant of R.albus and conducted further characterization studies.

Pure cellulose (3% suspension of KC flock W-300, from Sanyo Kokusaku Pulp, Tokyo) was used as bail-milled cel- lulose (BMC) after ball-milling for 3 d as the main carbon source in the medium. Carboxymethyl cellulose (CMC) with a degree of substitution of 0.6 and molecular weight of 180 kilodaltons was generously donated by Daiichi Kogyo Seiyaku (Kyoto). Glucose (G1), ceUobiose (G2), cel- lotriose (G3), cellotetraose (G4), cellopentaose (G5) and cellohexaose (G6) for high performance liquid chroma- tography were purchased from Seikagaku Kogyo Co. (Tokyo). Coating plates for enzyme linked immuno- sorbent assay (ELISA, Serocluster 96-well EIA plate, flat bottom) were obtained from Coaster Co. (Cambridge, Mass., USA ). Secondary antibody, horseradish peroxi- dase conjugated F(ab') 2 fragment goat anti-mouse im- muno globulin G was obtained from Cappel Co. (Phila- delphia, Mass., USA). All other reagents were commer- ciai products of the highest quality available.

* Corresponding author.

54

R. albus F-40 isolated from bovine rumen was cultivated in a medium containing 1.5% BMC at pH 6.5 and 37°C for 3 d. Details of the medium composition, the prepara- tion method and the cultivation conditions have been de- scribed previously (1).

Carboxymethyl cellulase (endoglucanase: EG) activity was measured by the viscosity reduction of a reaction mix- ture at 37°C and pH 6.8, consisting of 1% (w/v) CMC in 0.05 M sodium phosphate buffer (5 ml; pH 6.8) and en- zyme solution (1 ml). One unit of enzyme activity was de- fined previously as the amount of the enzyme needed to reduce the viscosity of CMC by I centipoise in 1 min at 37°C and pH 6.8 (2).

The endogiucanase was purified as follows: The super- natant of the R. albus culture (2.5/) was dialysed at 5°C with a cellophane membrane against 10 volumes of the 10 mM sodium phosphate buffer (pH 6.8) containing 10 mM 2-mercaptoethanol. Dialysis was repeated 3 times.

The dialyzate was loaded onto a DEAE-Bio Gel A column (Bio-Rad Laboratories, Richmond, Calif. USA) (2.6 by 40 cm) and then a linear gradient elution was ap- plied with the buffer containing sodium chloride (0 to 1.0 M). The fractions having the endoglucanase activity were concentrated with an ultrafiltration membrane (YM2, Amicon Co., Lexington, Mass., USA).

The concentrated active fractions from the DEAE-Bio Gel A column were gel-filtrated with a Sephacryl S-200 HR column (1.6 by 150 cm; Pharmacia Japan, Tokyo), using 10 mM phosphate buffer containing 0.5 M sodium chloride (pH 6.8). The fractions having the endogiucanase activity were pooled and diluted 50 times with the 10mM phos- phate buffer (pH 6.8).

The active fractions from a Sephacryl S-200 HR column were loaded onto a Mono Q column (0.5 by 5 cm; Pharma- cia Japan, Tokyo) and then equilibrated with 10 mM so- dium phosphate buffer (pH 6.8). A linear gradient elution was applied with the buffer containing sodium chloride (0 to 0.35 M).

The purification experiments involving column chro- matography were performed at 20°C in the presence of 10 mM 2-mercaptoethanol in the buffer.

VOL. 73, 1992 NOTES 55

Antiserum for endoglucanase II (EG II) purified previ- ously from the R. a lbus culture supernatant (2) was pre- pared by immunizing a mouse with an injection of a mix- ture consisting of 125 l~g of EGII and 125/d of Freund complete adjuvant (Difco, Michigan, USA). For a booster of the serum, EGII (125 ftg) was injected in the same man- ner 3 weeks after the first injection. One week after the booster injection, the mouse was bled and the serum was collected after centrifugation and stored as antiserum for EGII at - 8 0 ° C until used. Antiserum for endoglucanase I (Eg I) (8) purified from the t ransformant E. coli JM103 (pURA1) was also prepared in the same manner. The endo- glucanase active fractions from DEAE-Bio Gel A column chromatography were immunological ly identified by ELISA from both antisera.

The cellooligosaccharides generated by the enzyme were measured by analyzing the hydrolysates of cellooligo- saccharides (G2, G3, G4, G5 and G6) using high perform- ance liquid chromatography (BIP-1; Japan Spectroscopic Co., Tokyo) equipped with an Ultron NH2 column (Shinwa Kako Co., Kyoto). A refractive index detector (RID-300; Japan Spectroscopic Co., Tokyo) was used for detecting cellooligosaccharides after each saccharide (I ~ , 80/d) had been incubated with enzyme solution (11.6 mU, 10/~1) for a specified period of time at 37°C. The elution solvent was an acetonitrile-water mixture (6 : 4, v/v) and the flow rate was 0.7 ml /min .

Amino acid sequence analysis of endoglucanase was per- formed with a ABI 477A/120A sequence analyzer using 0.1 M Quadral buffer (pH 9.0) as a coupling buffer accord- ing to the details described in a previous paper (8).

Protein concentration in the fract ionated samples was determined by the absorbance at 280 nm. Sodium dodecyl sulfate (SDS)-polyacrylamide gel (14~) electrophoresis (PAGE) was carried out by the method of Laemmli (12). For molecular weight estimation, a polyacrylamide gel elec- trophoresis cal ibrat ion kit (Pharmacia Japan, Tokyo) was used for s tandards.

The DEAE-Bio Gel A column chromatographic pattern of the R. a lbus culture supernatant revealed three peaks having endoglucanase activity (Fig. 1). Immunological correspondence of the endoglucanases in the three active peaks was determined by ELISA (Table 1). Peak I eluted by 0.6 M NaC1 reacted remarkably with the antiserum against EGII( the EGII-ant iserum, OD: 0.679) as well as

I 1.5

E

v 1.0

. <

0.5

111 II 1

J

10 20 30 40 5O 60

Fraction number (12mr/tube)

2.0 1.0

i " i

LO ~: Q5

n z

0 0

FIG. 1. DEAE-Bio Gel A column chromatogram of the superna- tant of R. albus culture. Dotted line (---), broken line (-. -) and closed c i r c l e s ( o - - o ) represent protein concentration, NaCI concentration and endoglucanase (EG) activity, respectively. Bar represents the frac- tions pooled.

TABLE 1. Immunological correspondence ~ between endogluca- nase active peaks I, II and III and antisera of cloned endogluca-

nase I (EgI) and endoglucanase II (EG II) from R. albus

Endoglucanase EgI-antiserum EG II-antiserum

Eg I 0.623 0.050 EG II 0.031 0.702

Peak I 0.028 0.679 Peak II 0.428 0.159 Peak III 0.055 0.041

The extent of correspondence was determined by reading the op- tical density at 492 nm of ELISA.

TABLE 2. Summary of purification of EG III from R. albus

Total Total Specific Recovery of Purification Purification activity protein activity activity

step (U) (mg) (U/mg) (~) (fold)

Dialysis 862.6 4,870 0.2 100 1 DEAE-Bio 92.4 118.4 0.8 10.7 4

Gel A Sephacryl 34.6 4.9 7.1 4.0 36

S-200 HR Mono Q HR 8.4 0.5 16.6 1.0 83

EGII (0.702) but only negligibly with the EgI-antiserum, in- dicating that peak I contains mainly EGII . Peak II eluted by 0.4 M NaC1 reacted with the EGII-ant iserum with less than 1/3 the intensity of EGII (0.702) and reacted well with the Eg I-antiserum (0.428), indicating that peak II contains mainly EGI with some EGII . Peak III eluted by 0.25 M NaC1 reacted only negligibly with both EgI- and EGII-ant isera, indicating that the endoglucanase in peakl I I seems to be an enzyme which is different f rom EgI** (8), EGI*** (8) and EGII (2). Therefore, it was de- noted as endoglucanase III (EGIII) f rom R. a lbus . These results show that R. a lbus generates at least three endo- glucanases in the supernatant .

The EGIII active fractions from the DEAE-Bio Gel A col- umn chromatography were concentrated and gel-filtrated with a Sephacryl S-200 HR column. This step was very effective, with the specific activity increasing almost 10- fold (Table 2). The active fractions were then further purified by Mono Q HR column chromatography. The fraction from the Mono Q column had a specific activity of 16.6 U/mg. The final protein yield was 0.5 mg with 83- fold purification and a recovery of about I N . The puri ty of the enzyme at the final step was examined by SDS- P A G E (Fig. 2), showing that the preparat ion is electro- phoretically homogeneous. The N-terminal amino acid se- quence of the enzyme was determined to be as follows: Ala-Gly-Gly-Asp-Ala-Asn-Tyr-Ser-Asp-Ala-Leu-Ala-Leu- Ser-Leu-Tyr-Phe. This sequence is completely different from those of both EGI and EGII . The molecular weight of EGII I (Fig. 2) was 53,000, which is similar to that o f EGII (Table 3). Maximum activity of the enzyme was observed at about pH 5.7, a value which is lower than that of EGI (pH 6.8) and EGII (pH 6.7). R . a lbus is known to degrade cellulose to low molecular cellooligomers and glucose and to ferment them to volatile fat ty acids which acidify the broth. In such an acidified culture broth, EGII I

** EgI: Endoglucanase I translated from the gene of R. albus in E. coli.

*** EGI: Endoglucanase I isolated from the R. albus culture super- natant which is immunologically identical to EgI.

56 WATANABE ET AL. J. FERMENT. BIOENG.,

MW

94,000

67,000

43,000

30,000

20,100

14,400

<--

FIG. 2. SDS-polyacrylamide gel electrophoretogram of EG III purified from R. albus culture supernatant. Left lane: Standard pro- teins as molecular weight markers. Right lane: EG III from Mono Q column chromatography. Arrow shows the position of EG III.

may contribute to solubilizing cellulose. Maximum activity of the enzyme was observed at 55°C, a temperature that is higher than those (37°C and 44°C) of EGI and EGII. The optimal temperature of the enzyme for CMC-liquefy- ing activity was remarkably higher than that (37°C) of the R. albus growth. The enzyme was stable up to 35°C for 10rain of incubation and lost its activity completely at 70°C. To study the effects of chemical reagents on activity, each reagent was added to the substrate at 1 mM, follow- ing which the enzyme reaction was allowed to proceed for 5 rain at 37°C. Reducing reagents such as 2-mercaptoeth- anol, dithiothreitol, glutathione and cysteine-HC1 brought

TABLE 3. Properties of EG III and other endoglucanases from R. albus

Properties Endo-1,4-,8-glucanase

I • II b III,

Molecular weight 43,000 54,000 d 53,000 pH optimum 6.8 6.7 5.7 Temperature optimum (*C) 37 44 55 Specific activity (U/mg) 54 34 17 SH enzyme = Yes Yes Yes Transglycosidation Yes No No Substrate degradation

Crystalline cellulose - - - Ball-milled cellulose -- +~ - Carboxymethyl cellulose ~ ~ ~- Xylan -- t+ -- Cellotriose + + - Celiotetraose -~ ¢t- + Cellopentaose ~ tt+ tt+ Cellohexaose ~ t~- +1+

• ,b and c References (8), (2), and this paper, respectively. d Molecular weight determined by gel filtration was 50,000 in the

previous paper (2). • Enzyme stabilized by mereaptoethanol and inhibited by

sulfhydryl group-blocking reagents. --, +, ~-, and tt+: Negligible, low, medium, and high extents of degradation, respectively.

about slight activation as well as stabilization of the en- zyme. Sulfhydryl group blocking-reagents such as p-chlo- romercuribenzoic acid, N-ethylmaleimide and iodoaceto- amide strongly inhibited the activity (more than 40%). Therefore, the sulfhydryl group of the enzyme is most likely essential for activity. Cu ++, Zn ÷ +, Hg +÷ and Fe +÷ were found to depress the enzyme activity remarkably (more than 40%). These properties of the enzyme were also observed in EGI(8), EGII(2), i~-glucosidase (4) and cellobiosidase (3) from R. albus.

EGIII only negligibly hydrolyzed G2 and G3. It hydro- lyzed G4, although 70% of the initial amount of G4 re- mained even after the 50 h reaction (Fig. 3A). The enzyme hydrolyzed G5 completely within 25 h (Fig. 3B) and G6 within 15 h (Fig. 3C), suggesting that the rate of degra- dation of soluble cellooligosaccharides by the enzyme in- creased remarkably with an increase in the degree of glucose polymerization. The kinetic values (Kin and Vm~ of the degradation of cellooligosaccharides by the enzyme were evaluated by Lineweaver-Burk plots. The Km values for en- zyme action on G4, G5 and G6 were 8.85, 7.24 and 0.72 mM, respectively, while the Vm~ values for G4, G5 and G6 were 1.72, 8.01 and 13.0 pmol. m i n - ~. mg protein- t, respec- tively. As far as product formation was concerned, G4 was degraded mainly to G2 and to a lesser extent to G1 and G3, suggesting that G4---~2G2 may be a major reaction and G4--+G1 +G3 a minor one. G5 was degraded mainly to G2 and G3 and only slightly to GI and G4, suggesting that G5--*G2+G3 may be a major reaction and G5~G1 + G4 a minor one. G6 was degraded mainly to G2, G4 and G3, and slightly to G1, indicating that G6---~G2+G4 and G6--+2G3 are the main reactions and that G6--+G1 +

20 E

E g to

U

E

i tc ._~

u

On

o c.}

~ Q _ G4 o

G2_ I c~ GI'G3gl

0 15

B yF. oG2,G3

C

lC ~ • .G2

u aG4

5 . .G 3

0 10 20 30 /~0 50

Reaction time ( h ) FIG. 3. Degradation of eellotetraose (A), cellopentaose (B) and

cellohexaose (C) by EG Ill at 37°C, pH 6.8 for a given period.

VOL. 73, 1992 NOTES 57

G5 is a m ino r one. In addi t ion , G2 and G3 were degraded only negligibly. These results indicate that soluble sub- strates with a higher degree o f glucose po lymer iza t ion are bet ter substrates for hydrolysis by the enzyme, suggesting that E G I I I may be an endo- type cellulase in a more strict sense o f the word than the o ther two. Cons ider ing these results, it is concluded that the third endoglucanase purified f rom the R . a l b u s cul ture superna tan t is an endo-l ,4-/9- glucanase.

REFERENCES

I. Ohmiya, K., Nokura, K., and Shimizu, S.: Enhancement of cellu- lose degradation by Ruminococcus albus at high cellulose concen- tration. J. Ferment. Technol., 61, 25-30 (1983).

2. Ohmiya, K., Maeda, K., and Shimizu, S.: Purification and properties of endo-(l-~4)-~-D-glucanase from Ruminococcus albus. Carbohydrate Res., 166, 145-155 (1987).

3. Ohmiya, K., Shimizu, M., Taya, M., and Shimizu, S.: Purifica- tion and properties of cellobiosidase from Ruminococcus albus. J. Bacteriol., 150, 407-409 (1982).

4. Ohmiya, K., Shiral, M., Kurachi, Y., and Shimizu, S.: Isolation and properties of /~-glucosidase from Ruminococcus albus. J. Bacteriol., 161, 432-434 (1985).

5. Beguin, P., Millet, J., and Aubert, J. P.: The cloned eel (cellu-

lose degradation) genes of Clostridium thermocellum and their products. Microhiol. Sci., 4, 277-280 (1987).

6. McGavin, M., and Forsberg, C. W.: Isolation and characteriza- tion of endoglucanase l and 2 from Bacillus succinogenes $85. J. Bacteriol., 170, 2914-2922 0988).

7. Shoemaker, S. P. and Brown, R. D. Jr.: Enzymatic activities of endo-l,4-/~-D-glucanase purified from Trichoderma viride. Bio- chim. Biophys. Acta, 523, 133-146 (1978).

8. Deguchi, H., Watanabe, Y., Sasaki, T., Matsuda, T., Shimizu, S., and Ohmiya, K.: Purification and properties of the endo-l,4- ~-glucanase from Ruminococcus albus and its gene products in Escherichia coil. J. Ferment. Bioeng., 71, 221-225 (1991).

9. Ohmiya, K., Nagashima, K., Kajino, T., Goto, E., Tsukada, A., and Shimizu, S.: Cloning of the ceUulase gene from Ruminococ- cus albus and its expression in Escherichia coll. Appl. Environ. Microbiol., 54, 1511-1515 (1988).

10. Ohmiya, K., Kajino, T., Kato, A., and Shimizu, S.: Structure of a Ruminococcus albus endo-l,4-~-o-glucanase gene. J. Bacteriol., 171, 6771-6775 (1989).

11. Engvall, E. and Perhann, P.: Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobin G. Immuno- chemistry, 8, 871-874 (1971).

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