APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1994, p. 64-70 0099-2240/94/$04.

00+0 Copyright 1994, American Society for Microbiology

Vol. 60, No. 1

Isolation and Properties of an Extracellular 1-Glucosidase from the Polycentric Rumen Fungus Orpinomyces sp. Strain PC-2
HUIZHONG CHEN, XINLIANG LI, AND LARS G. LJUNGDAHL* Center for Biological Resource Recovery and Department of Biochemistry, Athens, Georgia 30602 University of

Georgia,.

Received 30 July 1993/Accepted 24 October 1993

An extracellular 13-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a Mr of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pl of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-13-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40C are 0.25 mM and 27.1 ;Lmol- min-' mg', respectively; with PNPG as the substrate, the corresponding values are 0.35 mM and 27.7 ,umol minm- mg 1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10-2 and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50C. The enzyme has high activity against sophorose (13-1,2-glucobiose) and laminaribiose (,B-1,3-glucobiose) but has no activity against gentiobiose (1B-1,6-glucobiose). The activity of the ,3-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.

,-Glucosidase (,-D-glucoside glucohydrolase; EC 3.2.1. 21) is common among plants, fungi, and bacteria. ,-Glucosidase has aroused considerable interest primarily because of its involvement in the biological conversion of cellulosic material. The enzyme catalyzes the hydrolysis of various compounds with ,3-D-glucosidic linkages and plays a crucial role in large-scale saccharification of cellulose by removing cellobiose, which is an inhibitor of exo- and endoglucanases (11, 42). Since the discovery of the obligately anaerobic fungi, there has been increasing interest in the properties and functions of these unusual microorganisms. Anaerobic fungi are part of the natural microflora within the rumen (5, 34, 38). Several types of these fungi have been isolated (8, 23). They differ by being monocentric or polycentric and by zoospore flagellation (3, 19, 38, 49). Electron microscopic studies have revealed that the rumen fungi colonize plant fragments, which they are able to degrade (1, 4). The rumen fungi also interact with the rumen bacteria and methanogens, and mixed cultures rather effectively ferment cellulose to methane (6, 32, 36). The anaerobic fungi produce a variety of enzymes that degrade plant materials (30, 35, 50). Recently, P-xylosidase (21), xylanase (44), 3-glucosidases (20, 25, 26, 45), and p-coumaroyl and feruloyl esterases (9, 10) were purified and characterized. All of these enzymes were isolated from monocentric Neocallimastix or Piromyces species. Here we report the purification and characterization of an extracellular 1-glucosidase from a polycentric fungus that produces multiflagellated zoospores, has been designated Orpinomyces sp. strain PC-2 (8), and produces high levels of 3-glucosidase and xylanase. This work is part of our pro* Corresponding author. Mailing address: Center for Biological Resource Recovery, Department of Biochemistry, University of Georgia, Life Sciences Building, Athens, GA 30602-7229. Phone: (706) 542-7640. Fax: (706) 542-2222. Electronic mail address: Ljungdah@bscr.uga.edu.

study enzymes, which are important for the degradation of plant materials in the rumen, secreted by Orpinomyces sp. strain PC-2.
gram to

MATERUILS AND METHODS

Fungal culture conditions. Orpinomyces sp. strain PC-2 isolated from a cow rumen was maintained as described previously (8). For enzyme production, the fungus was grown at 39C for 8 days in 20-liter Pyrex carboys, each containing 14 liters of the basal medium described by Barichievich and Calza (2) and 42 g (0.3%) of Avicel PH-101 (microcrystalline cellulose; Fluka Chemie AG, Buchs, Switzerland). The medium was autoclaved for 1 h, after which cysteine-HCl (0.03%) was added. The medium was then cooled under a stream of CO2. Penicillin (334 U ml-1), streptomycin sulfate (80 ,ug ml-1), and chloramphenicol (10 ,ug ml-') were filter sterilized (0.22-,um-pore-size filter unit: Millipore Corp., Bedford, Mass.) and added just prior to inoculation. Each carboy was inoculated with 400 ml of a 72-h culture previously carried two consecutive times on the basal medium containing 0.2% cellobiose plus antibiotics. Enzyme assays. 13-Glucosidase (p-nitrophenyl-13-D-glucosidase [PNPGase]) and cellobiase activities were determined by the following standard procedures. With p-nitrophenylP-D-glucoside (PNPG) as the substrate, the reaction mixture of 1.2 ml contained 0.3 ml of appropriately diluted enzyme solution, 0.6 ml of 50 mM sodium phosphate (pH 6.0), and 0.3 ml of 12 mM PNPG. The reaction was carried out for 10 min at 50C and stopped by the addition of 2.4 ml of 1 M Na2CO3. The liberatedp-nitrophenol was measured spectrophotometrically at 405 nm (22). Cellobiase activity was determined by using a reaction mixture of 2 ml containing 1 ml of appropriately diluted enzyme solution in 50 mM sodium phosphate (pH 6.0) and 1 ml of 2 mM cellobiose. The reaction was carried out at 50C for 30 min and stopped by placing the assay tubes in boiling water for 5 min. Liberated
64

VOL. 60, 1994 V-GLUCOSIDASE FROM ORPINOMYCES SP. STRAIN PC-2

65

glucose was measured with a glucose determination kit no. 510 (Sigma Chemical Co., St. Louis, Mo.) as described in the
manufacturer's instructions. One unit of 3-glucosidase or cellobiase activity was defined as the amount of enzyme required for the hydrolysis of 1 ,umol of substrate per min under assay conditions. Specific activity was expressed as units per milligram of protein. Enzyme purification. The culture (about 150 liters), grown for 8 days, was filtered through a 50-mesh nylon net to remove residual Avicel and fungal mycelia. The filtrate was concentrated 150-fold and dialyzed against 20 liters of 10 mM piperazine-HCl (pH 5.5) by using tangential-flow ultrafiltration (Pellicon cassette system with a PTGC 10,000 WMWL cassette; Millipore Corp.). The final concentrate (1,000 ml) was centrifuged at 20,000 x g for 20 min to remove precipitated material. The concentrated supernatant was applied in 200-ml portions to a column of Q Sepharose Fast Flow (2.6 by 20 cm; Pharmacia Fine Chemicals, Uppsala, Sweden). Elution with 100 ml of 10 mM piperazine-HCl buffer (pH 5.5) removed unbound proteins. This was followed by elution with 1 liter of an NaCl gradient (0 to 0.7 M) in the same buffer. Fractions 42 to 60 (see Fig. 1) containing the main peak of ,B-glucosidase were pooled and concentrated to a volume of about 50 ml by using an ultrafiltration cell (Amicon Co., Beverly, Mass.) equipped with a PM 10 membrane. Solid ammonium sulfate was added to 70% saturation. The precipitate was removed by centrifugation at 20,000 x g for 20 min. The supematant containing 13-glucosidase activity was dialyzed for 24 h at 4C against 1,000 ml of 25 mM triethanolamine-iminodiacetic acid buffer (pH 8.1; changing three times) containing 0.02% (wt vol-1) NaN3. The final volume of the enzyme solution was 60 ml.
were

TABLE 1. Summary of the purification of 3-glucosidasea

Step
Total protein

(mg)

Total U

Sp act (U mg-')

Yield (%)

Culture filtrate Concentrated supernatant Q Sepharose Fast Flow (NH4)2SO4 fractionation Chromatofocusing and

7,119 5,097 1,429 157 5.9

1.9

2,207 2,141
929 519 115 86

0.31 0.42 0.65 3.3 19.5 45.3

100 97 42.1 23.5 5.2

ion-exchange Superose 12 gel filtration

3.9

a Activities were measured with PNPG as the substrate (PNPGase), and the purification was performed with about 150 liters of 8-day cultures.

Chromatofocusing and ion-exchange chromatography done on a Mono P HR 5/20 column (Pharmacia) equilibrated with 25 mM triethanolamine-iminodiacetic acid buffer (pH 8.1). The enzyme solution from the previous step was applied in 20-ml portions to the column, and elution was done first with 48 ml of Polybuffer-iminodiacetic acid (pH 5.0) and then with 80 ml of an NaCl gradient (0 to 0.8 M) in

the same buffer at a flow rate of 1 ml min-'. Fractions of 0.5 ml were collected, and those containing the main peak of 3-glucosidase were pooled and concentrated to 0.6 ml by using Centricon 10 (Amicon Co.). Final purification was achieved by gel filtration on a Superose 12 10/30 column (Pharmacia) equilibrated and run with 10 mM sodium phosphate (pH 6.0) containing 0.3 M NaCl. The enzyme solution in 200-,lI portions was injected into the column through a sample loop. Fractions of 0.3 ml were collected, and those containing ,-glucosidase activity were combined and stored at -20C. Analytical methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in Laemmli's buffer (24) with 10% polyacrylamide. Low-molecular-weight protein. standards (Bio-Rad Laboratories, Richmond, Calif.) were used as markers. Electrophoresis was performed in a Mini-PROTEIN II Cell (Bio-Rad). Gels were stained with Coomassie brilliant blue R 250 by the method of Fairbanks et al. (15). Analytical isoelectric focusing was done on a Phast System (Pharmacia) with a carrier ampholyte at pH 3 to 9 as described by the manufacturer. The carbohydrate content of the purified enzyme was determined by using the phenol-sulfuric acid method of Dubois et al. (14) with mannose as the standard. Protein content was measured by the method of Lowry et al. (31) with bovine serum albumin as the standard. For amino acid analysis, the purified enzyme sample was hydrolyzed in an

evacuated and sealed tube for 24 h at 110C with 6 N HCI containing 2 mg of phenol ml-1. After evaporation, the hydrolysates were analyzed with an Applied Biosystems amino acid analyzer. The pH optima of the 3-glucosidase and cellobiase activities were determined by performing assays at 50C with the following buffer systems: 0.1 M sodium acetate (pH 3.8 to 5.4), 0.1 M sodium phosphate (pH 5.8 to 7.8), and 0.1 M sodium borate (pH 8.2 and 8.6). Enzyme stability at different pH values was determined by measuring the residual activity after incubating the enzyme for 1 h at 22C at pH 3.0 to 8.6. The effect of temperature on 3-glucosidase and cellobiase activities was determined by assaying the enzyme at temperatures from 30 to 70C. Thermostability was measured by incubating the enzyme in 50 mM sodium phosphate buffer (pH 6.0) for 10 min to 24 h at temperatures from 30 to 65C. After incubation, the enzyme solution was chilled in an ice bath for 5 min after which residual activity was determined. The effects of various metal ions and reagents at 1 mM on the 0-glucosidase and cellobiase activities were determined by preincubating the enzyme with the individual reagent in 50 mM sodium phosphate buffer (pH 6.0) at 22C for 10 min. Activities were then measured at 50C in the presence of the metal ions or reagents. The activity assayed in the absence of metal ions or reagents was taken as 100%. Several a-, 3-glucosides (1 mM) and polysaccharides (0.5% [wt vol-1]) were tested as substrates for the purified enzyme. The assays were done by the standard procedures by determining eitherp-nitrophenol (22), glucose, or reducing sugars (33). The effect of the concentrations of PNPG (0.05 to 10 mM) and cellobiose (0.05 to 20 mM) on the reaction rate was determined at pH 6 at 30, 40, and 50C. The inhibitory effect of gluconolactone was determined with PNPG and cellobiose as substrates, whereas the inhibition by glucose was evaluated with only PNPG as substrate. Km. Vm., and K4 values were obtained from Lineweaver-Burk plots.

RESULTS

Purification of 13-glucosidase. A summary of the purification of the ,B-glucosidase from Orpinomyces sp. strain PC-2 is given in Table 1. The enzyme was purified about 146-fold with a yield of about 4%. During Q Sepharose chromatography, the major part of 3-glucosidase activity eluted as a wide peak between 0.3 to 0.5 M NaCl (Fig. 1). Two other active fractions eluted in the wash with the starting buffer and with 0.25 M NaCl were not further investigated. In these two fractions and the major fraction, about 70% of the ,-glucosidase activity applied to

66

CHEN ET AL.

APPL. ENVIRON. MICROBIOL.

1
1.20
0.40
t
.,.

2
AO

z
c

0~~~~~~~~~~~~~~~~~~~
0.90
0.30
_

97.4 66.2 -

0~~~~~~~~~~~~~~~~~ 20 60 0 0~~~~~~~~~~~ 40
OD

00

~~~~~~~~~~~~~~~~~N
w
0..

45

< .0

31
60

0.001
0 20 40

80

0.00 100

Fraction nurber

FIG. 1. Q Sepharose Fast Flow anion-exchange chromatography of ,B-glucosidase. The column was equilibrated with 10 mM piperazine-HCl buffer (pH 5.5). It was first washed with the equilibrating buffer and then eluted with a linear gradient of NaCl (0 to 0.7 M) in the same buffer. The flow rate was 3 ml min'-, and the fraction size was 10 ml. Symbols: -, A280; *, 13-glucosidase activity; - - -, NaCl gradient.

21.5 -_
14.4FIG. 3. SDS-PAGE of the purified ,B-glucosidase. Lanes: 1, molecular masses of protein standards, i.e., lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), bovine serum albumin (66.2 kDa), and phosphorylase b (97.4 kDa); 2, supernatant (30 ,ug of protein); 3, purified j3-glucosidase (1 ,ug).

the column was recovered. In the ammonium sulfate precipitation step, more than half (56%) of the ,B-glucosidase remained in solution; the rest of the activity was found in the pellet, where it appeared to agglomerate with other proteins and enzyme components. This step was important because almost all xylanase and carboxymethyl cellulase activities were removed as precipitate, and this simplified the further purification of the ,3-glucosidase. The next step was performed by using chromatofocusing and ion-exchange chromatography (Fig. 2). One sharp peak of 3-glucosidase activity containing about 22% of the activity applied to the column was eluted at pH 5 with 0.5 M NaCl. A number of smaller fractions with 3-glucosidase activity were also ob-

1.00

10
8

a
I

0.80

0.60

0 ~~~~~~~~~~~~~~~~.................3

0.40
0

<

0.20
0.00
0 40 80 120

a E
N

00
160 200 240 280 320

Fraction number

FIG. 2. Chromatofocusing and ion-exchange chromatography of 3-glucosidase. The Mono P HR 5/20 column was equilibrated with 25 mM triethanolamine-iminodiacetic acid buffer (pH 8.0). Elution was performed first with 48 ml of Polybuffer-iminodiacetic acid (pH 5.0) and then with a linear gradient of NaCl (0 to 0.8 M) in the same buffer. The flow rate was 1 ml min-', and the fraction size was 0.5 ml. Symbols: -, A280; *, P-glucosidase activity; . , pH value; - - -, NaCl gradient.

tained. Although the pIs of the 0-glucosidases representing these smaller peaks were different from the main 3-glucosidase, their molecular sizes determined by gel filtration results were similar (not shown). These 3-glucosidases were not further investigated. The main ,-glucosidase fraction (Fig. 2, fractions 242 to 245) was subjected to Superose 12 gel filtration. This step yielded an enzyme found to be homogeneous by SDS-PAGE (Fig. 3). The Vmax values with PNPG and cellobiose as substrates were 47.5 and 46.2 U mg-' (see Table 5), respectively, when measured at 50C and pH 6.0. Molecular weight, isoelectric point, carbohydrate content, and amino acid composition of the 13-glucosidase. The molecular weight of the purified 3-glucosidase was found to be 85,400 (Fig. 3) and 87,000 as determined by SDS-PAGE and gel filtration chromatography on a Superose 12 column, respectively. These results indicated that the 3-glucosidase is a monomeric protein. The isoelectric point was at pH 3.95 as found with analytical isoelectric focusing. The enzyme was glycosylated and contained 8.5% (wt wt-1) carbohydrate. The amino acid composition of the enzyme is given in Table 2. The enzyme contains high levels of Ser, Gly, Ala, and acidic amino acid residues which may contribute to the acidic pI of the enzyme. Attempts to sequence the N terminus of the 0-glucosidase were unsuccessful (100 pmol of protein was electroeluted from the SDS-polyacrylamide gel or the protein on the SDS-polyacrylamide gel was electroblotted onto a polyvinylidene difluoride membrane and then protein sample was subjected to automatic protein sequencing). It appeared that the N terminus of the 13-glucosidase was blocked. Effect of pH and temperature on enzyme activity and stability. The activity of the enzyme towards PNPG and cellobiose was determined at a pH of 3.8 to 8.6 at 50C. The maximum activity was at pH 6.2 with both substrates (Fig. 4A). No activity was observed below pH 4.2 and above pH 8.2. The enzyme was stable for at least 1 h when incubated at a pH of 5.0 to 8.0 at 22C. Under similar conditions, 40 and

VOL. 60, 1994 V-GLUCOSIDASE FROM ORPINOMYCES SP. STRAIN PC-2 TABLE 2. Amino acid composition of ,B-glucosidase
Amino acid residues'
No. of 100

67

80
u
>1

Asp ...........................................
Glu ........................................... Ser ...........................................

Gly ...........................................
His ...........................................

Arg ............................................
Thr ........................................... Ala ........................................... Pro ............................................

Tyr ...........................................
Val ........................................... Met ............................................

Cys ...........................................
Ile ........................................... Leu ............................................ Phe ...........................................

Lys ............................................ Trp ...........................................

31 41 68 173 6 28 18 70 40 31 57 10 45 45 71 38 18

60
40
20
o

a:

.4-

pH

100
80
O'

NDb

a Calculation is based on a molecular weight of 78,141 (without carbohydrate content). b ND, not determined.

u
.4_

60
40

0)

20% of the activity was lost at pH 4.2 and 8.6, respectively. Hydrolysis of PNPG and cellobiose by the P-glucosidase at pH 6.0 was highest at a temperature of 50C. Enzyme activity decreased rapidly above 50C and was totally lost at 65C (Fig. 4B). No activity loss was observed at 40C after preincubation for 24 h. The enzyme maintained .95% of its activity for 1 h at 50C, and at this temperature, 50% of the activity remained after 24 h. Rapid inactivation occurred at 60C, and the enzyme lost all of its activity after 15 min. Effects of metal ions and inhibitors on the activity. The P-glucosidase and the cellobiase were similarly affected by various metal ions and reagents (Table 3). Mg2", Mn2+, Co2+, and Ni2+ stimulated the enzyme activities from 10 to 30%. K+, Ca2+, Cd2+, Cs2+, Zn2+, dithiothreitol, ,3-mercaptoethanol, and EDTA did not significantly influence the enzyme activities, whereas Fe2+, Cu2+, and SDS were inhibitory. Ag+, Hg2+, andp-chloromercuribenzoate caused from 60 to 100% inhibition. Substrate specificity. The purified ,B-glucosidase was specific for aryl-3-glucosides and did not hydrolyze alkyl-,Bglucosides and ox-1,4-glucosides. The enzyme acted rapidly on sophorose (,-1,2-glucobiose), laminaribiose (P-1,3-glucobiose), and cellobiose (,B-1,4-glucobiose; Table 4). On the other hand, the enzyme had no measurable activity with gentiobiose (,B-1,6-glucobiose), methyl-a-glucoside, methyl3-glucoside, p-nitrophenyl-p-xyloside, salicin, maltose, sucrose, lactose, xylan, microcrystalline cellulose, or carboxymethyl cellulose as substrates. Kinetic constants. Km. K,, and Vma, values at 30, 40, and 50C were obtained from Lineweaver-Burk plots. The results are given in Table 5. Km values of the ,-glucosidase towards PNPG and cellobiose were similar and did not change much with the temperature. Vmax values, changing with the temperature, were also similar with both substrates. These values indicated a Qlo of about 2 for the enzyme. Lineweaver-Burk plots indicated that glucose and gluconolactone competitively inhibited PNPG hydrolysis. Similarly, gluconolactone competitively inhibited the hydrolysis of cellobiose. The gluconolactone was a particularly strong inhibitor with PNPG as the substrate.

a:1

a)

20
o
-

25

35

45

55
0

65

75

FIG. 4. Effects of pH and temperature on the activity of 1-glu-

TemperatLre ( C)

cosidase purified from Orpinomyces sp. strain PC-2. (A) Effect of the pH determined at 50C; (B) effect of the temperature determined at pH 6.0. Symbols: *, PNPGase activity; 0, cellobiase activity.

DISCUSSION
Because of aggregation of several hydrolytic enzymes (27), purification of the ,-glucosidase from Orpinomyces sp. strain PC-2 was somewhat cumbersome and low yielding. The occurrence of extracellular complexes of cellulolytic and hydrolytic enzymes in culture fluid of anaerobic fungi has been reported for Neocallimastix and Piromyces species (44, 47). It has even been suggested that cellulosome-like complexes are formed by anaerobic fungi (12, 44, 47). It seems that several types or multiple forms of ,-glucosidase are present in the culture supematant of Orpinomyces sp. strain PC-2 (Fig. 1 and 2). The presence of several 3-glucosidases appears common among cellulolytic microorganisms and has been demonstrated in, for example, Clostridium thermocellum (17), Phanerochaete chrysosporium (13, 43), Botryodiplodia theobromae (46), Bifidobacterium breve (40), Neocallimastix frontalis (25, 26), and Trichoderma reesei (11). In some microorganisms, different 3-glucosidases are coded by separate genes, but multiple forms of the enzyme having different pI values may also be due to differences in charge or in contents of carbohydrates (42). The ,B-glucosidase from Orpinomyces sp. strain PC-2 is a glycoprotein containing 8.5% carbohydrate. The molecular weight of the Orpinomyces 13-glucosidase is about 86,000, and this 3-glucosidase consists of a single

68

CHEN ET AL. TABLE 3. Effects of several compounds on 13-glucosidase activity

Relative activity (%)

APPL. ENVIRON. MICROBIOL.

TABLE 4. Substrate specificity of 13-glucosidasea

Substrate (1 mM) U mg-

Reagent'
PNPG

p-Nitrophenyl-13-glucosidase ....................
Cellobiose

K+
Ag+ Ca2+ Mg2+ Mn2+ Co2+

Fe2+
Ni2+

Cd2+
Cs2+ Cu2+ Zn2+ Hg2+ DTT"

PMCEd
PCMBe

EDTAf
SDS Noneg
b

101.0 8.9 96.2 131.9 117.1 130.9 75.8 120.4 95.2 101.4 75.9 96.6 0.0 98.5 99.3 36.2 96.9 70.8 100.0

98.5 4.7 91.8 122.1 112.6 120.0

o-Nitrophenyl-13-glucosidase ..... ............... Sophorose (13-1,2) ..................... Laminaribiose (13-1,3) .................... Cellobiose (,B-1,4) ..................... Gentiobiose (,B-1,6) .....................
a Conditions were 50C and pH 6.0.

33.5 53.1 61.8 68.9 42.8 0.0

84.7
113.3

95.8
99.3 86.6 91.6 0.0
_c

38.3 97.1 64.6 100.0

a All reagents were 1 mM. DTI, dithiothreitol. c Dithiothreitol and P-mercaptoethanol interfered with glucose determination.

IMCE, ,-mercaptoethanol. PCMB, p-chloromercuribenzoate. f The results were similar with 5 and 10 mM EDTA. g Enzyme used in this assay had specific activities of 48 and 46 U mg1 with PNPG and cellobiose as substrates, respectively.
d

polypeptide. This Mr seems to be about average for monomeric ,B-glucosidases. The enzyme from Piromyces sp. strain E2 has a Mr of 45,000 (45). A cellobiase from N. frontalis has a Mr of 85,000 (26), and the two 1-glucosidases from the same fungus have Mrs of 120,000 and 125,500 (20, 25). Mrs of 1-glucosidases from the rumen bacteria Ruminococcus albus and Butyrivibrio fibrisolvens are 82,000 and 91,800, respectively (28, 37). In aerobic fungi, monomeric (71-kDa; T. reesei) (11), dimeric (332-kDa; Aspergillus fumigatus) (39), and polymeric (311-kDa; Termitomyces clypeatus) (41) 1-glucosidases have been reported. The optimum pH (6.2) and the thermostability (40C) of the 1-glucosidase from Orpinomyces sp. strain PC-2 are similar to values reported for 1-glucosidases of N. frontalis (20) and Piromyces sp. strain E2 (45) and are close to the physiological pH and temperature in the rumen. The 1-glucosidase was strongly inhibited by a sulfhydryl reagent (p-chloromercuribenzoate) and sulfhydryl oxidant metals (Ag+ and Hg2+). It contains about 45 cysteine residues, which suggests that thiol may play an important role in the enzyme. It can be noted that the activity of an oxygensensitive 1-glucosidase from the rumen bacterium Fibrobacter (Bacteroides) succinogenes is stabilized by dithiothreitol (16). The 1-glucosidase was stimulated by several bivalent ions, including Mn2", Co2+, and Ni2+. Certain amounts of these ions are present in the rumen and probably contribute to an increase of the in situ efficiency of the enzyme. Similar temperature and pH curves for the PNPGase and cellobiase (Fig. 4) and the effect of different metallic ions and other agents on the two activities (Table 3) indicate the possibility that the binding site and catalytic center for the two substrates are the same. The ability of ,-glucosidases purified from anaerobic fungi

to hydrolyze disaccharides other than cellobiose has not been studied. 1-Glucosidases of aerobic fungi, such as those from T. reesei (11), Trichoderma koningii (48), Monilia sp. (7), and Schizophyllum commune (29) have wide substrate specificity and are able to hydrolyze gentiobiose (,B-1,6); some ,B-glucosidases from aerobic fungi even have the ability to hydrolyze alkyl-1-glucosides and a-1,4-glucobiose (11), but the 13-glucosidase purified from Orpinomyces sp. strain PC-2 is specific for aryl-13-glucoside and is not able to hydrolyze gentiobiose. In this respect, it is similar to a ,B-glucosidase purified from the anaerobic rumen bacterium R. albus (37). The Km of the 1-glucosidase with PNPG as the substrate is 0.35 mM at 40C and pH 6.0. It issimilar to the Kins of the 1-glucosidase and the cellobiase from N. fiontalis MCH3 (0.266 mM) (20) and EB188 (85-kDa enzyme; 0.36 mM) (26), respectively, but much lower than the Kms of the 1-glucosidases from N. frontalis EB188 (125.5-kDa enzyme; 2.5 mM) (25) and Piromyces sp. strain E2 (1.5 mM) (45). The Km for cellobiose was 0.25 mM, which was higher than those values of the cellobiase from N. frontalis EB 188 (0.053 mM; 85-kDa enzyme) (26) and the 1-glucosidase from Piromyces sp. strain E2 (0.05 mM) (45). The Kms (11, 42) of 13-glucosidases purified from aerobic fungi (0.75 to 3.6 mM) are generally much higher than those purified from anaerobic

fungi.
The ,B-glucosidase from Orpinomyces sp. strain PC-2 is inhibited by its substrate cellobiose at concentrations higher than 1.5 mM (result not shown). Substrate inhibition by cellobiose has also been reported previously for N. frontalis cellobiase (26) and 1-glucosidase (45). Inhibition at high substrate concentrations may result from the formation of transfer products, which has been reported to occur with a cellobiase from N. frontalis EB188 (26). Glucose and gluconolactone are inhibitors of the ,B-glucosidase from Orpinomyces sp. strain PC-2. The ratios between Km and K1 suggest that gluconolactone is an especially competitive inhibitor of the activity toward PNPG (KmIKj, 20.5). In contrast, cellobiose hydrolysis was less sensitive to gluconolactone inhibition (Km/K1, 0.098). Cellobiose is the main product of the hydrolysis of celluTABLE 5. Kinetic constants of
Substrate

1-glucosidase
Ki (mM)
Glucose Gluconolactone

Km

(temp)
PNPG (30C) Cellobiose (30C) PNPG (40C) Cellobiose (40C) PNPG (50C) Cellobiose (50C)

(mM)
0.39 0.25 0.35 0.25 0.39 0.28

(U mg-1)
13.5 12.9 27.7 27.1 47.5 46.2

8.43 8.75
9.73

1.35 x 10-2 2.16 1.68 x 10-2 2.57 1.44 x 10-2 2.09

V-GLUCOSIDASE FROM ORPINOMYCES SP. STRAIN PC-2 VOL. 60, 1994

69

lose by cellulolytic enzymes. This hydrolysis is inhibited by cellobiose. ,B-Glucosidases from anaerobic fungi are extracellular and released into the rumen fluid (25, 27; and present study). In contrast, 3-glucosidases of anaerobic rumen bacteria such as R. albus (37) and F. succinogenes (18) are cell associated. Therefore, it is possible that in the rumen the fungal ,B-glucosidases play a special role by removing cellobiose from the rumen fluid and thereby relieve the inhibition caused by the cellobiose produced in the hydrolysis of cellulose. It should be noted that the 3-glucosidases of the rumen fungi have low Km values and high cellobiase activity. They should be efficient catalysts in thisprocess. In addition, the rumen fungi have several 3-glucosidases that have yet to be studied and characterized. Clearly, further work on these enzymes, including their possible interactions with the cellulolytic enzymes of the anaerobic fungi and bacteria in the rumen, is warranted.
ACKNOWLEDGMENT This work was supported by project DE-FG09-86ER 13614 from the U.S. Department of Energy.
1. 2.

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