Purification and Characterization of a Major Secretory Cellobiase, Cba2, from Cellulomonas biazotea

Protein Expression and Purification 23, 159–166 (2001) doi:10.1006/prep.2001.1486, available online at http://www.idealibrary.com on

Purification and Characterization of a Major Secretory Cellobiase, Cba2, from Cellulomonas biazotea Andy T. Y. Lau and W. K. R. Wong1 Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China

Received March 29, 2001, and in revised form May 25, 2001; published online August 9, 2001

A novel cellobiase (Cba2) was purified from the culture supernatant of Cellulomonas biazotea and characterized. Cba2 appeared to be a major secretory cellobiase in C. biazotea as its enzymatic activity was estimated to represent over 40% of the total extracellular ␤-glucosidase activity. The enzyme was purified over 260-fold subsequent to ammonium sulfate precipitation, gel-filtration chromatography, anion-exchange chromatography, and reversed-phase high-performance liquid chromatography. Cba2 was shown by SDS–PAGE to have a large molecular mass of 109 kDa, which makes it one of the largest secretory cellobiases characterized. Its homogeneity was confirmed by Nterminal amino acid sequencing. The Km and Vmax values were 0.025 mM and 0.0048 mM minⴚ1, respectively, for the Cba2 hydrolysis of p-nitrophenyl-␤-D-glucopyranoside, and 0.73 mM and 0.00033 mM minⴚ1, respectively, for the hydrolysis of cellobiose (at 37ⴗC and pH 7.0). The purified enzyme has a pH optimum of 4.8 and the optimum temperature for activity is 70ⴗC. In view of the secretory nature of Cba2 and the fact that it is a major component of secretory cellobiases of C. biazotea, it is potentially important in the enzymatic degradation of cellulose, and its availability as a recombinant protein may facilitate the studies of its biotechnological applications. 䉷 2001 Academic Press Key Words: C. biazotea; cellulase; cellobiase; protein purification; enzyme kinetics.

Cellulose, which constitutes the highest proportion of municipal and plant wastes, represents a major 1 To whom correspondence and reprint requests should be addressed. Fax: (852)-2358-1552. E-mail: [email protected].

1046-5928/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.

source of renewable energy and raw materials. Therefore, the utilization of cellulosic wastes to produce energy is potentially of great importance. Complete enzymatic hydrolysis of cellulose requires the synergistic action of three types of enzymes: endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and cellobiases (EC 3.2.1.21) (1–3). Cellobiase is generally responsible for the regulation of the whole cellulolytic process and cellobiase activity is a rate-limiting factor during enzymatic hydrolysis of cellulose (2–4), as both endoglucanase and exoglucanase activities are often inhibited by cellobiose. Thus, cellobiase not only produces glucose from cellobiose, but also reduces cellobiose inhibition, allowing endoglucanase and exoglucanase enzymes to function more efficiently. Among the cellulolytic organisms, the Gram-positive soil bacterium, Cellulomonas fimi, has been one of the most extensively studied. Genes encoding all three types of cellulase have been cloned from C. fimi and expressed in a variety of microbial hosts (5–12). The cenA gene encoding an endoglucanase (Eng)2 and the cex gene encoding an exoglucanase (Exg) are the best characterized among the cellulase genes cloned from C. fimi (9, 12). Cellulases expressed from these genes are produced extracellularly in E. coli (8, 13) and the 2 Abbreviations used: Eng, endoglucanase; Exg, exoglucanase; Cba, cellobiase; cba, cellobiase gene; A, absorbance; HPLC, high-performance liquid chromatography; kDa, kilodalton; Km, Michaelis constant; CMC, carboxymethylcellulose; PB, sodium phosphate buffer; PMSF, phenylmethylsulfonyl fluoride; pNPG, p-nitrophenyl-␤-D-glucopyranoside; pNPGase, enzyme capable of hydrolyzing pNPG; PVDF, polyvinylidene difluoride; SDS–PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis; U, unit; v, initial velocity; Vmax, maximum velocity.

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brewers’ yeast (11), suggesting their potential application to enzymatic hydrolysis of cellulosic wastes. However, to hydrolyze native cellulose such as filter paper, the cenA and cex products have to work cooperatively with a competent cellobiase, which must also be expressed as an extracellular product. Although cellobiases have been identified from C. fimi, these enzymes appear to be cytoplasmic in location (14) and hence expression of them as extracellular proteins will be difficult. To search for a cellobiase which might work cooperatively with C. fimi Eng and Exg on cellulose hydrolysis, we screened close relatives of C. fimi for the target enzyme, since it has been shown that cellulase components from closely related microorganisms might also act synergistically on cellulose (15). Moreover, we sought a secretory cellobiase, with the view that the recombinant counterpart might also be expressed as an extracellular product. Recently, we have successfully cloned the C. biazotea cba gene (encoding an excreted cellobiase) in E. coli (16), while other groups have cloned DNA inserts encoding other C. biazotea cellobiases (17, 18). Purification and characterization of these isozymes may not only enhance our understanding of the cellobiase system in this organism, but also enable the cloning of genes other than cba that are capable of expressing extracellular cellobiase for use in cellulase reconstitution for cellulose hydrolysis. In this communication, we report the purification and characterization of a novel cellobiase, designated Cba2, from the culture medium of C. biazotea. Cba2 has a large molecular mass of 109 kDa and appears to be a major component of the secretory cellobiase complex of C. biazotea. Results of Nterminal sequencing and kinetic studies support the contention that Cba2 is a novel and genuine cellobiase. MATERIALS AND METHODS Bacterial Strain The C. biazotea ATCC 486 strain was obtained from the American Type Culture Collection (Rockville, MD) and used as the source of secretory cellobiases. Materials and Reagents All chromatographic media and columns were purchased from Amersham Pharmacia Biotech (Sweden) except for the reversed-phase HPLC column (Delta-Pak ˚ , 0.8 ⫻ 10 cm) which was from Waters (Milford, C18 300 A MA). Low-viscosity carboxymethylcellulose (CMC), pnitrophenyl-␤-D-glucopyranoside (pNPG), sodium azide (NaN3), D-(⫹)-cellobiose, and chemicals used for SDS– PAGE, protein precipitation, gel-filtration chromatography, ion-exchange chromatography, reversed-phase HPLC, and kinetic studies were purchased from Sigma

(St. Louis, MO), Bio-Rad (Richmond, CA), Life Technologies (Gaithersburg, MD), and Nacalai Tesque (Japan). The polyvinylidene difluoride (PVDF) membrane was purchased from Boehringer Mannheim GmbH (Germany). A sodium phosphate buffer (PB; 1.15 g Na2HPO4 and 0.228 g NaH2PO4 per liter, pH 7.4) was used in chromatographic procedures. A PB-saline (PB containing 1 M NaCl) buffer was used for anion-exchange chromatography, and buffer B (100 mL containing 70 mL acetonitrile and 30 mL PB) was used in reversedphase HPLC. Growth Conditions The 2⫻ YT medium used for growth of C. biazotea has been described (19). CMC was added to media to a final concentration of 1% (w/v) to induce expression of cellobiase. When solid media were required, Bacto agar was added to liquid medium to a final concentration of 1.5% (w/v). Shake-flask cultivations were carried out at 30⬚C (20, 21) until the A550 value was about 12. The cells were maintained by subculturing onto fresh agar plates every 4 weeks. Permanent stocks were stored at ⫺20⬚C in liquid culture medium containing 40% glycerol. Enzyme and Protein Assays Measurement of ␤-glucosidase (pNPGase) and cellobiase activities using pNPG and cellobiose as the substrates, respectively, has been described (16). Protein concentrations were determined by the method of Bradford (22) using the Bio-Rad protein assay with reference to the standard curve of bovine ␥-globulin (Bio-Rad Protein Standard I). Precipitation of Cba2 from the Culture Medium of C. biazotea Five hundred milliliters of medium from an induced C. biazotea culture were clarified by centrifugation, and then filtered through a 0.45-␮m filter. The ammonium sulfate precipitation step was carried out at 4⬚C. The supernatant was first brought to 40% saturation with (NH4)2SO4. After centrifugation and removal of the pellet, the supernatant was brought to 70% saturation with (NH4)2SO4. After centrifugation and removal of the supernatant, the pellet was resuspended in 1/30 the original volume of PB and filtered through a 0.45␮m filter. Purification of Cba2 All chromatographic procedures were conducted at room temperature. To perform gel-filtration chromatography, a Sephacryl S-200 (high resolution) matrix packed in two XK26/40 columns (2.6 ⫻ 30 cm) connected in series was employed. A batch of 10 mL (NH4)2SO4

SECRETORY CELLOBIASE FROM Cellulomonas biazotea

concentrate was chromatographed through the columns using PB as the eluting agent. Fractions (5 mL/fraction collected at a rate of 2 mL/min) that were pNPGaseactive were pooled and subjected to Q Sepharose (Fast Flow) matrix packed XK50/30 column (5 ⫻ 5 cm) anion exchange chromatography. The column (equilibrated with PB) was washed extensively with PB buffer after loading and the bound proteins were then eluted with a step gradient obtained by mixing PB with the PBsaline. The eluate was collected in 15-mL fractions at a rate of 2 mL/min, and the pNPGase-active fractions were located. The active fractions were pooled and concentrated by ultrafiltration using a PM10 membrane (Amicon, Beverly, MA) and dialyzed extensively against 0.1⫻ PB. The filtrate was then loaded onto a reversedphase HPLC column that had been equilibrated with PB. The bound proteins were eluted with an acetonitrile gradient of 0–80% buffer B at a rate of 1 mL/min and collected in 2-mL fractions. The fractions containing pNPGase activity were identified and then dialyzed and lyophilized. Electrophoretic Analysis and Sequencing of the Purified Products The purified products were resolved on a 7.5% (w/v) glycine–SDS–polyacrylamide gel (23). The Benchmark (Life Technologies, Gaithersburg, MD) protein markers were run alongside the samples and the protein bands were visualized by Coomassie blue staining. For Nterminal analysis, samples of 10 ␮g of HPLC purified products were resolved by SDS–PAGE, electrotransferred to PVDF membrane, which was stained with Coomassie blue and then destained. The bands corresponding to the purified products were excised and sent to the Molecular Biology Resource Facility at the University of Oklahoma Health Sciences Center (Oklahoma City, OK) for N-terminal amino-acid sequencing.

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velocity of Cba2-mediated hydrolysis was examined by varying the final substrate concentration from 0.01 to 10 mM in both pNPG and cellobiose assays. For the hydrolysis of pNPG, the final enzyme concentration used was 0.05 U/mL while that of cellobiose was 0.5 U/ mL. Both the pNPG and cellobiose assays were conducted essentially the same as described above and previously (16). The Km and Vmax values for both pNPG and cellobiose hydrolyses catalyzed by Cba2 were determined using Lineweaver–Burk analysis. The effects of pH and temperature on the hydrolysis of pNPG (2.5 mM) by Cba2 were studied by varying the pH from 2.6 to 8.0 and the temperature from 30 to 100⬚C. Citrate phosphate buffer (pH 2.6 to 7.0) and sodium phosphate buffer (pH 5.8 to 8.0) were employed to regulate the pH were prepared as described previously (24). RESULTS Concentration of the Extracellular pNPGase Activity Pilot experiments were conducted to study the application of (NH4)2SO4 precipitation to the concentration of secretory pNPGase from the culture supernatant of C. biazotea. Results showed that no detectable pNPGase activity was precipitated before 50% saturation of (NH4)2SO4 (unpublished data). A two-step precipitation protocol, first at 40% saturation of (NH4)2SO4 and then at 70% of saturation, was established to reduce supernatant volume and to efficiently recover pNPGase activity. When 1.2 L of culture supernatant were processed in this way, a satisfactory recovery of about 90.6% of total pNPGase activity, and a slight increase of 1.2-fold in specific activity, were attained in a much reduced volume of 40 mL, 30-fold less than the starting volume (Table 1). Purification of the Cba2 Cellobiase

Kinetic Studies of the Hydrolysis of pNPG and Cellobiose by Cba2 The effect of enzyme concentration on the initial velocity of Cba2-mediated hydrolysis was examined by varying the final enzyme concentration from 0.01 to 0.1 U/mL in the pNPG assay. The substrate mix was 20 ␮L of 20⫻ PB (pH 7.0), H2O (160 ␮L ⫺ x, where x is the enzyme volume), and 20 ␮L of 25 mM pNPG. Substrate mixes were first incubated at 37⬚C for 10 min. Then, different quantities of enzyme were added to 10 tubes of substrate mix, whereas the same volumes of H2O in lieu of enzyme were added to a further 10 tubes to form the blanks. After the contents were mixed by inversion, the tubes were incubated at 37⬚C for 10 min and the pNPGase activity were assayed as described previously (16). The effect of substrate concentration on the initial

The 40 mL of pNPGase containing solution were passed through a Sephacryl S-200 gel-filtration column and a few major protein peaks were resolved (Fig. 1A). One of the largest proteins, which constituted the first peak in the elution profile, was found to possess pNPGase activity (Fig. 1A). The pNPGase-active fractions, comprising fractions 25–35 (Fig. 1A), were pooled for anion-exchange chromatography. Despite a reduction in the concentration of pNPGase activity by 5.4-fold due to an increase in volume, there was an impressive recovery of 93.3% of total pNPGase activity and an increase in specific activity to 2.2-fold (Table 1). The pooled eluate from the gel-filtration chromatography was loaded onto a Q Sepharose anion-exchange column. With a step gradient of NaCl, the pNPGase activity was resolved to give two active peaks, which were eluted with 0.35 and 0.55 M of NaCl (Fig. 1B).

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TABLE 1 Summary of Purification of Types I and II Cba2 from the Culture Supernatant of C. biazotea Fraction

Volume (mL)

Total activity (Ua)

Supernatant Ammonium sulfate Gel filtration Anion-exchange Type I Cba2 Type II Cba2 Reversed-phase HPLC Type I Cba2 Type II Cba2

1200 40 220

47.3 42.9 44.1

94.1 70 39.8

225 315

8.9 24.9

2.5 1.6

30 30

5.1 14.2

0.039 0.11

a

Total protein (mg)

Specific activity (U/mg) 0.50 0.61 1.1 3.6 15.3 129 133

Recovery (%)

Purification

100 90.6 93.3

1 1.2 2.2

18.9 52.7

7.1 30.4

10.7 29.9

256 265

One unit of pNPGase activity is defined as the amount of enzyme that releases 1 ␮mol of p-nitrophenol min⫺1 at 37⬚C.

The pNPGase-active fractions of peak 1, comprising fractions 34–48 (Fig. 1B), and the pNPGase-active fractions of peak 2, comprising fractions 80–100 (Fig. 1B), were pooled separately. The total pNPGase activity of fractions pooled from peak 2 was about 2.8 times that of pooled fractions from peak 1 (Table 1). In comparison with the total activity in the culture supernatant, the recovery of pNPGase activity in peak 1 was only 18.9% (Table 1), whereas the recovery of pNPGase activity in peak 2 was 52.7% (Table 1). After the anion-exchange step, there was an increase in specific activity to 7.1fold for peak 1 and 30.4-fold for peak 2, respectively (Table 1). Peak 1 is termed type I Cba2 and peak 2 type II Cba2 below. The pooled active eluates of types I and II Cba2 from the anion-exchange column were concentrated and dialyzed. Each concentrate was loaded onto a reversedphase HPLC column and the bound proteins were eluted with an acetonitrile gradient of 0–80% buffer B. Fractions that were pNPGase-active were eluted at 20% buffer B for type I Cba2 (Fig. 2A) and at 15% buffer B for type II Cba2 (Fig. 2B). The pNPGase-active fractions of type I Cba2, comprising fractions 16–30 (Fig. 2A), and those of type II Cba2, comprising fractions 12–26 (Fig. 2B), were pooled separately for electrophoretic and sequencing analyses. The total pNPGase activity of the fractions pooled from type II Cba2 was about 2.8 times that pooled from type I Cba2 (Table 1). Of the original pNPGase activity loaded, the recovery of type I Cba2 after this step was only 10.7%, whereas that of type II Cba2 was about 29.9%. After this step, the specific activities of types I and II Cba2 were increased to 256and 265-fold, respectively (Table 1). Electrophoretic Analysis of the Purified Products Purified fractions of types I (Fig. 2A) and II (Fig. 2B) Cba2 were pooled, dialyzed, concentrated, and then analyzed by SDS–PAGE. Both fractions were shown to contain only a single protein (Fig. 3). The two proteins were indistinguishable in size, both with a large molecular mass of about 109 kDa.

Protein Sequencing Sequencing of the N-termini of types I and II Cba2 revealed that the former material was a mixture of at least two polypeptides, whereas the latter sample was apparently homogeneous. The major protein of the (mixed) type I Cba2 sample is apparently identical to the type II Cba2, as evidenced by the fact that the Nterminus of the type II product (Ser, Leu, Pro, Thr, Gln, Trp) is the major sequence from the type I sample. Kinetic Characteristics of Cba2 Since type II Cba2, or simply Cba2, was shown to be homogenous by N-terminal sequencing, it was used for kinetic studies. In studying the effect of Cba2 concentration on the hydrolysis of 2.5 mM pNPG, it was observed that the initial rate (v) of reaction increased in a linear fashion with enzyme concentration (unpublished results). This relation between the rate of reaction and enzyme concentration indicated that 2.5 mM pNPG constituted substrate excess. Therefore, to study the effect of pNPG concentration on the reaction rate, a much reduced range of substrate concentrations (from 0.01 mM) was used. To improve the sensitivity of the reaction, the final concentration of Cba2 employed was 0.05 U/mL. Initial rates were determined as a function of pNPG concentrations to yield data (data not shown), demonstrating the adherence of hydrolysis to the Michaelis– Menten equation. With a double reciprocal plot between 1/v and 1/[pNPG], a straight line was observed (Fig. 4A). The Michaelis constant Km and the maximum rate Vmax of pNPG hydrolysis catalyzed by Cba2 determined from the Lineweaver–Burk plot were 0.025 mM and 0.0048 mM min⫺1, respectively. Recalling that culture supernatant of C. biazotea is more active on pNPG than on cellobiose (16), a higher final concentration of Cba2 (0.5 U/mL) than that used in the pNPG assay was employed in the hydrolysis of cellobiose. As in the pNPG study, the initial rates of

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by Cba2 at 37⬚C, it was detected that the reaction rate increased as the pH increased from 2.6 to 4.8 and then decreased steadily from 4.8 to 8.0 (Fig. 5A). The curve follows a bell-shaped profile, suggesting that the pH optimum of this enzyme would be at 4.8. In studying the effect of temperature on the hydrolysis of pNPG by Cba2 at pH 7.0, the reaction rate was shown to increase as the temperature increased from 30 to 70⬚C (Fig. 5B). However, at 80⬚C or above, the rate dropped significantly and was totally abolished at 100⬚C (Fig. 5B). The curve also follows a bell-shaped profile, suggesting that the temperature optimum of this enzyme would be at 70⬚C.

FIG. 1. Purification of Cba2. (A) Gel-filtration chromatography of the pNPGase-active component in the supernatant of a C. biazotea culture using a Sephacryl S-200 column. (B) Anion-exchange chromatography of the pNPGase-active eluate collected from the gel-filtration column (A, fractions 25–35) using a Q Sepharose column. (I) and (II) indicate the locations of the type I and type II Cba2 peaks, respectively. The protein elution profile (—); the NaCl gradient (⭈⭈⭈); the fractions containing pNPGase activity (䉱).

hydrolysis increased as a function of cellobiose concentrations to yield a hyperbolic curve (data not shown), as expected from Michaelis–Menten kinetics. The Km and Vmax values of cellobiose hydrolysis catalyzed by Cba2 determined from the Lineweaver–Burk analysis (Fig. 4B) were 0.73 mM and 0.00033 mM min⫺1, respectively. In comparison with the kinetic coefficients of pNPG hydrolysis, the higher Km but lower Vmax values of cellobiose hydrolysis support the idea that pNPG is more susceptible than is cellobiose to Cba2-mediated degradation. In studying the effect of pH on the hydrolysis of pNPG

FIG. 2. Reversed-phase HPLC of the type I and type II Cba2. (A) Type I Cba2 (comprising fractions 34–48 in Fig. 1B) and (B) Type II Cba2 (comprising fractions 80–100 in Fig. 1B) were resolved using a Delta-Pak C18 column. The protein elution profile (—); the salt gradient (⭈⭈⭈); the fractions containing pNPGase activity (䉱). The composition of buffer B is given in materials and reagents.

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of two forms of Cba2 (types I and II) with small conformational differences. This slight variation might be responsible for the different binding affinities of the two to the positively charged gel matrix. On the other hand, the existence of the type I form (Figs. 1B and 2A) might be due to contamination of a minor fraction of Cba2 with other proteins so that the complex has different chromatographic behavior. Despite the high purity of type II Cba2, probably resulting from its large size, the sensitivity of the sequencing process diminished rapidly and only six residues were unambiguously determined from the use of 10 ␮g of sample. Kinetic studies of pNPG and cellobiose hydrolyses with pure Cba2 (purified type II enzyme) showed that Cba2 had a much higher affinity for pNPG than for

FIG. 3. SDS–PAGE of the HPLC purified pNPGase products. Lanes: 1, molecular weight markers (sizes are given in kDa); 2, purified type I Cba2; 3, purified type II Cba2. The arrow marks the location of the purified products in lanes 2 and 3.

DISCUSSION A native cellobiase, Cba2, was successfully purified from the culture supernatant of C. biazotea using four major chromatographic steps. The purified Cba2 represents a major cellobiase component, whose activity (types I and II Cba2 together) constitutes about 40% of the total supernatant pNPGase activity (Table 1). As viewed on SDS–PAGE, Cba2 appears to be composed of one polypeptide unit, which has a large molecular mass of 109 kDa (Fig. 3) and is thus one of the largest secretory ␤-glucosidases characterized (Table 2). The finding that the pNPGase activity of the culture supernatant of C. biazotea would not bind to concanavalin A (unpublished data) supports the idea that despite the large size of Cba2, it is nonglycosylated and composed of only protein. N-terminal sequencing showed that the first amino acid residue of Cba2 is serine, suggesting that mature Cba2 is secreted to the culture medium after processing by a signal peptidase. The fact that Cba2 is larger than other native or recombinant ␤-glucosidases from C. biazotea (16, 25), differences in published primary structures (16), and the unusually high level of its production all support the notion that Cba2 is a novel secretory cellobiase. The two peaks of activity obtained from anion-exchange chromatography (Fig. 1B) might be attributable to the large size of Cba2, resulting in the development

FIG. 4. Kinetic studies of Cba2. The Lineweaver–Burke analysis of (A) pNPG hydrolysis and (B) cellobiose hydrolysis by Cba2.

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FIG. 5. Factors affecting the hydrolysis of pNPG catalyzed by Cba2. (A) The effect of pH. (B) The effect of temperature. Citrate phosphate buffer (䉱) was used for the pH range 2.6 to 7.0 and sodium phosphate buffer (䉭) was used for the pH range 5.8 to 8.0.

cellobiose, as reflected by a 29-fold smaller Km value with the former substrate (Table 2). Also, at a saturating concentration of the substrate, the maximum rate of hydrolysis of pNPG (0.0048 mM min⫺1) is 14-fold higher than that of cellobiose (0.00033 mM min⫺1), indicating that pNPG is much more susceptible than cellobiose to hydrolysis by Cba2. The slower rate of hydrolysis of cellobiose might be attributable to a greater steric interference when interacting with the enzyme. The same substrate preference has also been shown by cellobiases purified from other microbial species (26–28). A comparison of the Km values of the hydrolysis of cellobiose catalyzed by various ␤-glucosidases (Table 2) reveals that the value of Cba2 is among the smallest, suggesting that Cba2 may be one of the more potent cellobiases among the enzymes compared. Our kinetic studies with Cba2 were conducted at neutral pH and 37⬚C, as were those published previously using cellulases from C. biazotea and other Cellulomonas species (5, 8, 11, 14, 16, 17, 29–32). However, it is surprising to find that Cba2 showed its maximum activity at a temperature as high as 70⬚C (Fig. 5B) and performed even more efficiently in an acidic range of pH (Fig. 5A). These results will be very useful to the identification of an optimum condition for the maximal action of Cba2 in cellobiose hydrolysis. On the other hand, the secretory nature, strong potency and stability over a wide range of pH and temperatures of Cba2 suggests a potential application to waste cellulose hydrolysis. The N-terminal sequencing of Cba2 will be valuable in the design of DNA probes for the isolation of the cba2 gene. A recombinant source of Cba2 may not only enable a detailed analysis of enzyme activities, but also facilitate studies of cooperativity between it and other cellulases in cellulose hydrolysis.

TABLE 2 Examples of Km Values of the Hydrolysis of pNPG or Cellobiose Catalyzed by ␤-Glucosidases from Various Microorganisms Location of enzyme

Molecular size (kDa)

Cellulomonas biazotea

Extracellular

109

Cellulomonas biazotea Streptomyces sp. strain QM-B814 Aspergillus niger CCRC 31494 Orpinomyces sp. strain PC-2 Trichoderma reesei

Intracellular Intracellular

355a 52.6

Extracellular

98b

Extracellular

85.4

Extracellular

71

Microorganism

a b

The size of a tetramer. The size of a dimer.

Substrate

Km (mM)

References

pNPG cellobiose pNPG pNPG cellobiose pNPG

0.025 0.73 4.25 0.27 7.9 21.7

Present work 25 27

pNPG cellobiose pPNG cellobiose

0.35 0.25 0.182 2.1

33 34 35

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ACKNOWLEDGMENTS We thank Dr. J. Hackett for reviewing the manuscript. This research was supported by Direct Allocation Grant DAG98/99.SC04 from the Hong Kong University of Science and Technology.

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