Enzymatic Degradation of Insoluble Carbohydrates - American

Chapter 13

Thermostable β-Glucosidases 1

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Badal C. Saha , Shelby N. Freer, and Rodney J . Bothast Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, EL 61604

Interest in β-glucosidase has increased in recent years because of its application in the conversion of cellulose to glucose for the subsequent production of fuel alcohol. Cellulolytic enzymes in conjunction with β-glucosidase act sequentially and cooperatively to degrade cellulose to glucose. Product inhibition, thermal inactivation, low product yield and high cost of the enzyme constitute some problems to develop enzymatic hydrolysis of cellulose as a commercial process. A thermostable β-glucosidase from Aureobasidium pullulons, a yeast-like fungus, was optimally active at 75°C and pH 4.5 against p-nitrophenyl-β-D-glucoside, cellobiose and a series of higher cellooligosaccharides. Recent developments in thermostable β-glucosidase research particularly the biochemical and kinetic properties of the enzyme, mode of action and its use in the conversion of cellulose to glucose are described.

The search for lower cost raw materials for the production of biofuels has led to increasing interest in the enzymatic hydrolysis of cellulose. Currently, over one billion gallons of ethanol are produced annually in the United States, with approximately 95% derived from the fermentation of corn starch. With increased attention to clean air and oxygenates for fuels, opportunities exist for expansion of the fuel ethanol industry. Lignocellulosic biomass, particularly corn fiber, represents a renewable resource that is available in sufficient quantities from the Permanent address: Department of Biochemistry, Michigan State University, East Lansing, MI 48824

0097-6156/95/0618-0197$12.00/0 © 1995 American Chemical Society

In Enzymatic Degradation of Insoluble Carbohydrates; Saddler, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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com wet milling industry to serve as a low cost feedstock. Advances in enzyme technology are necessary if conversion of cellulosic biomass to ethanol is to te a commercial reality.


Role of β-Glucosidase in Enzymatic Hydrolysis of Cellulose Cellulose is a linear polymer of 8,000-12,000 D-glucose units linked by 1,4-B-Dglucosidic bonds. The enzyme system for the conversion of cellulose to glucose comprises endo-1,4-B-glucanase (EC 3.2.1.4), exo-1,4-B-glucanase (EC 3.2.1.91) and B-glucosidase (β-D-glucosidic glucohydrolase, E C 3.2.1.21). Cellulolytic enzymes in conjunction with β-glucosidase act sequentially and cooperatively to degrade crystalline cellulose to glucose. Endoglucanase acts in a random fashion on the regions of low crystallinity of the cellulosic fiber whereas exoglucanase removes cellobiose (β-1,4 glucose dimer) units from the non-reducing ends of cellulose chains. β-Glucosidase hydrolyzes cellobiose and in some cases cellooligosaccharides to glucose. The enzyme is generally responsible for the regulation of the whole cellulolytic process and is a rate limiting factor during enzymatic hydrolysis of cellulose as both endoglucanase and cellobiohydrolase activities are often inhibited by cellobiose (7,2,5). Thus, the β-glucosidase not only produces glucose from cellobiose but also reduces cellobiose inhibition, allowing the cellulolytic enzymes to function more efficiently. However, like Bglucanases, almost all β-glucosidases are subject to end-product (glucose) inhibition. Problems of Current β-Glucosidase Product inhibition, thermal inactivation, substrate inhibition, low product yield and high cost of β-glucosidase constitute some barriers to commercial development of the enzymatic hydrolysis of cellulose. There is an increasing demand for the development of a thermostable, environmentally compatible, product and substrate tolerant β-glucosidase with increased specificity and activity for application in the conversion of cellulose to glucose in die fuel ethanol industry. A thermostable β-glucosidase offers certain advantages such as higher reaction rate, increased product formation, less microbial contamination, longer shelf-life, easier purification and better yield. Production of Thermostable β-GIucosidases β-Glucosidases having temperature optima of at least 60°C will be considered thermostable. Thermostable β-glucosidases reported to date include those purified from thermophiles such as Clostridium thermocellum (4), Thermoascus aurantiacus (5,6), Talaromyces emersonii (7,8), Thermotoga sp. (9), Microbispora bispora (10), Mucor miehei (11), Thermoanaerobacter ethanolicus (12), Sporotrichum thermophile (13), a thermophilic cellulolytic anaerobe Tp8 (14), an extremely thermophilic anaerobic strain Wai21W.2 (15) and the hyperthermophilic archaeon Pyrococcusfiiriosus (16). Some thermophilic organisms such as Thermomonospora



13. SAHA ET AL.

Thermostable β-Glucosidases

199

sp. produce less thermostable β-glucosidase (17,18). On the other hand, some mesophilic fungi such as Aspergillus sp. and Sclerotium sp. produce fl-glucosidase having an optimum temperature at 60-70°C (19,20). Aureobasidium pullulons, a yeast-like fungus, produces a thermostable fl-glucosidase (231 mU/ml culture broth) when grown on corn bran (21). Lactose was also a good carbon source for the production of fl-glucosidase. A time course study of fl-glucosidase production by A. pullulons grown on corn bran at 28°C showed that the fl-glucosidase production increased gradually up to 4 days, after which it remained constant. P. funosus cells grown on cellobiose contained very high levels of fl-glucosidase (18 U/mg at 80°C) (16). Physico-chemical Characteristics The specific activity of purifiai extracellular fl-glucosidase from A. pullulons was 315 U/mg protein at pH 4.5 and 75°C using p-nitrophenyl fl-D-glucoside (pNPflG) as assay substrate (21). The specific activity of purified fl-glucosidase from various microorganisms varies from 9 to 468 U/mg protein with pNPflG as substrate. (9, 22-24). The native fl-glucosidase from P. funosus had a molecular weight of 230,000 and was composed of four subunits each with a molecular weight of 58,000 (16). The native fl-glucosidase from Pisolithus tinctorius had a molecular weight of 450,000 with three subunits each of molecular weight 150,000 (25) . The fl-glucosidase from A. pullulons was a glycoprotein having a molecular weight of 340,000 with two similar subunits of molecular weight of 165,000 (21). The fl-glucosidase from S. thermophile had a molecular weight of 240,000 with two similar subunits (13). The fl-glucosidase from an extremely thermophilic anaerobic bacterium had a molecular weight of 43,000 (15). A fl-glucosidase from Clostridium stercorium was a monomer with a molecular weight of 85,000 (26) . The cloned fl-glucosidase (BglB) from M. bispora had a molecular weight of 52,000 (10). Thus, there is considerable diversity in enzyme structure for different thermostable fl-glucosidases. Thermostability and Thermoactivity The half-lives of some thermostable fl-glucosidases are given in Table I. The purified fl-glucosidase from P. funosus showed optimum activity at pH 5.0 and 102-105°C, and was remarkably thermostable with a half life of 85 h at 100°C and 13 h at 110°C (16). The thermostability of a fl-glucosidase from the thermophilic bacterium Tp8 cloned in Eschenchia coli was greatest at pH 6.0-6.5, and the enzyme had a half-life value of 11 min at 90°C, 105 min at 85° and 900 min at 80°C (14). The fl-glucosidase from an extremely thermophilic anaerobic bacterium strain Wai21W.2 was inactivated with a half-life of 45 h at 65°C, 47 min at 75°C and 1.4 min at pH 6.2 and 85°C (15). At pH 7.0, which was the optimum pH for thermostability, half-life of the enzyme was 130 min at 75°C. The thermostability of this enzyme was enhanced 8 fold by 10% glycerol, 6-fold by 0.2 M cellobiose and 3 fold by 5 m M dithiothreitol and 5 m M 2-mercaptoethanol at pH 6.2 and 75°C. A partially purified fl-glucosidase from Thermotoga sp. had a half-life of


200


Table I. Half-Eves of some thermophilic 0-glucosidases B-Glucosidase

Half-life

Partially pure Pure Pure

8 h at 90°C (pH 7.0) 2.5 h at 98°C (pH 7.0) 11 min at 90°C (pH 6.0-6.5)

Thermophilic anaerobic bacterium Wai21W.2 (15)

Pure

Pyrococcusfitriosus(16)

Pure

Aureohasidium

Crude

45 h at 65°C (pH 6.2) 47 min at 75°C (pH 6.2) 130 min at 75°C (pH 7.0) 85 h at 100°C (pH 5.0) 13 h at 110°C (pH 5.0) 72 h at 75°C (pH 4.5) 24 h at 80°C (pH 4.5)

Source

Thermotoga

sp. (9)


Thermophilic bacterium Tp8 enzyme clonal in Escherichia

coli (14)

pullulons

(21)

8 h at 90°C (9). The pure enzyme had a half-life of 2.5 h at 98°C in the presence of bovine serum albumin (40 ^g/ml). The thermostability of the enzyme was increased further by addition of either trehalose or betaine. Immobilization of Bglucosidase from A. phoenicis increased the half-life at 65°C from 0.5 to 252 h (27). One B-glucosidase (BglB) from M. bispora expressed in E. coli showed an optimum activity at 60°C and pH 6.2 (10). The cloned enzyme was thermostable retaining about 70% activity after 48 h at 60°C. In our work (27), the optimum temperature of the crude B-glucosidase from A. pullulons was 80°C (Figure 1). The crude enzyme had a half-life of 72 h at 75°C and 24 h at 80°C. However, the optimum temperature of the purified B-glucosidase was 75°C. Similar findings were reported for the B-glucosidase from Neocallimastix frontalis, in which case the pure enzyme had an optimum temperature at 45°C, whereas the crude enzyme showed optimum activity at 55-60°C (28). While the crude B-glucosidase from C. stercorarium exhibited a half-life of 3 h at 60°C, the purifiai enzyme was rapidly inactivated at this temperature (26). However, the thermostability of the purifiai enzyme could be increased by M g , C a or DTT. By adding M g C l and DTT, the half-life of the purifiai enzyme at 60°C was increased to more than 5 h. The enzyme showed optimal activity at p H 5.5 and 65°C. A B-glucosidase from 5. thermophile was optimally active at pH 5.4 and 65°C (75). The B-glucosidase from Trichoderma reesei Q M 9414 exhibited optimal activity towards cellobiose at p H 4.5 and 70°C (29). Characteristics of some recently described thermostable B-glucosidases are presented in Table II. + 2

+ 2

2

Catalytic Properties B-Glucosidases constitute a very diverse family of enzymes capable of hydrolyzing



13. SAHA ET AL.

201


120

0 I

20

ι

ι

ι

i

ι

ι

30

40

50

60

70

80

i 90

1

100

Temperature (oC) Figure 1. Effect of temperature on activity of crude β-glucosidase from Aureobasidium pullulons. The enzyme activity was assayed at various temperatures at pH 4.5 using p-nitrophenyl β-D-glucoside as substrate (30 min reaction).


202


Table Π . Characteristics of some recently described thermostable β-glucosidases


Organism

Specific activity (U/mg protein)*

Thermotoga sp. (9) 195 Sporotrichum thermophile (13) 89 Pyrococcusfiiriosus(16) 389 Candida cacaoi (22) 9 Clostridium thermocellum (23) 125 Aspergillus nidulans (24) 468 Pisolithus tinctorius (25) 128 Aspergillus niger (30) -

Optimum Optimum temp. PH (°Q

na 65 102-105 60 65 60 65 65

Glucose inhibition (K , mM)) t

0.42 0.5 300 8 na 5.48 na 3.22

7.0 5.4 5.0 4.0-5.5 6.5 5.5 4.0 4.6

* Specific activity was determined using p-nitrophenyl β-D-glucoside (pNPBG) as substrate. One unit (U) of β-glucosidase was defined as the amount of enzyme required to liberate 1 μπιοί p-nitrophenol per min from pNPBG under certain assay conditions. na, not available.

a broad range of β-glucosides. The hydrolytic mechanism of a β-glucosidase is considered to be by general acid catalysis (31). β-Glucosidases may be divided into three groups on the basis of substrate specificity: 1) aryl-B-glucosidases hydrolyze exclusively aryl-fi-glucosides, 2) cellobiases hydrolyze cellobiose and cello-oligosaccharides only, and 3) broad specificity β-glucosidases hydrolyze both substrate types and are most common in cellulolytic microbes (7,2,52). The βglucosidase from P.fiiriosushad higher affinity for pNPfiG than cellobiose with K values of 0.15 and 20 m M , respectively (16). It also exhibited β-galactosidase, β-xylosidase and some β-mannosidase activity. The β-glucosidase from C. stercorarium hydrolyzed a variety of substrates including pNPBG, cellobiose and disordered cellulose (26). K values were determined to be 0.8 m M for pNPBG and 33 m M for cellobiose. The K values of β-glucosidase from thermophilic anaerobic strain Wai21W.2 were 0.15 and 0.73 m M for hydrolysis of pNPflG and cellobiose, respectively (15). Purified β-glucosidase from A. pullulons hydrolyzed cellobiose and pNPBG effectively (27). The purified enzyme had very little (< 5%) or no activity on lactose, maltose, sucrose and trehalose. It also had no or very little activity on pNP-a-D-glucoside, ρΝΡ-β-D-xyloside, ρΝΡ-β-Dcellobioside, pNP-a-L-arabinofuranoside and ρΝΡ-β-D-glucuronide (< 5%). K values of 1.17 and 1.00 m M and values of 897 and 800 U m g protein of βglucosidase from A. pullulons were obtained for the hydrolysis of pNPBG and cellobiose, respectively, at pH 4.5 and 75°C. The purified β-glucosidase m

m

m

m

1


13. SAHAETAL.

203


hydrolyzed cello-oligosaccharides. K values for hydrolysis of cellotriose, cellotetraose, cellopentaose, cellohexaose and celloheptaose by this enzyme were 0.34, 0.36, 0.64, 0.68 and 1.65 m M , respectively. The enzyme preparation did not hydrolyze lactose, although the organism produced very high level of flglucosidase when grown on lactose. It, however, hydrolyzed pNP-fl-D-galactoside at 7.6% of that of pNPflG. The fl-glucosidase from P.fiiriosushydrolyzed lactose very well (16). The intracellular fl-glucosidase from Evernia prunastri was considered to be a true cellobiase because of its great affinity toward cellobiose (33). The K values for the hydrolysis of cellobiose and pNPflG were 0.244 and 0.635 m M , respectively. K values for the hydrolysis of cellobiose and pNPflG by fl-glucosidase from T. reesei strain Q M 9414 were 0.5 m M and 0.3 m M , respectively (29). This enzyme hydrolyzed cellodextrins by sequentially splitting off glucose units from the non-reducing end of the oligomers. It is interesting that the major role of a thermolabile fl-glucosidase from Butyrivibrio fibrisolvens cloned in E. coli in the degradation of cellulose was the cleavage of cellodextrins rather than cellobiose (34). The K values for the hydrolysis of pNPflG and cellobiose by some thermophilic fl-glucosidases are presented in Table III. The fl-glucosidase from Bacillus potymyxa expressed in E. coli produced glucose and cellotriose from cellobiose (35). The cellotriose was formed by transglycosylation. Metal ions such as C a , M g , M n or C o (5 mM) did not stimulate or inhibit the fl-glucosidase activity of A. pullulons, and thiol was not essential for activity (21). The fl-glucosidase from P.fiiriosuswas also unaffected by thiol-specific inhibitors (16). Galactose, mannose, arabinose, fructose, xylose m


m

m

m

2 +

2 +

2 +

2 +

Table Ι Π . Comparison of K values of some thermostable β-glucosidases for the hydrolysis of p-nitrophenyl β-D-glucoside (pNPfiG) and cellobiose m

Source

K value (mM) m

Thermotoga sp. (9) Sporotrichum thermophile (13) Pyrococcus funosus (16) Aureobasidium pullulons (21) Candida cacaoi (22) Clostridium thermocellum (23) Aspergillus nidulans (24) P-l P-II Pisolithus tinctorius (25) Trichoderma reesei QM9414 (29) Aspergillus niger (30) USBD 0827 USBD 0828

pNPflG

Cellobiose

0.10 0.29 0.15 1.17 0.44 2.20 0.84 0.47 0.87 0.30 0.75 1.23

19 0.83 20 1 87 77 1 0.8

-

0.5 0.89 1.64


204

ENZYMATIC DEGRADATION O F INSOLUBLE CARBOHYDRATES

and lactose at 1 % (w/v) concentration did not inhibit β-glucosidase activity from A . pullulons. Some β-glucosidases preferentially utilize alcohols rather than water as acceptors for the glycosyl moiety during catalysis, yielding ethyl β-D-glucoside in the reaction (9,35-37). The β-glucosidase of Dekkera intermedia is activated by 2 M ethanol using pNPBG as substrate, suggesting that ethanol increases the hydrolysis rate of pNPflG by acting as an acceptor molecule for the intermediary glucosy cation (37). The effect of ethanol can be attributed to β-glucosyl transferase activity - the ethanol acting as a suitable acceptor for this reaction (9). The initial activity of the β-glucosidase from A. pullulons was slightly stimulated by ethanol. H P L C analysis of the reaction products in the presence of 6% ethanol after 24 h indicated the formation of an additional unidentified peak (21).


1

Substrate Inhibition Substrate inhibition by cellobiose is a common property of β-glucosidase from Trichoderma sp. and other microorganisms (29,38,39). Cellobiose strongly inhibited its own hydrolysis by β-glucosidase from Pyromyces sp. at concentrations above 0.2 m M (38). The inhibition constant (Ki) for cellobiose was 0.62 m M . The β-glucosidase from A. pullulons was not inhibited by up to 20 m M pNPBG or 6% (w/v) cellobiose (21). It was shown that the β-glucosidase from A . niger normally inhibited by cellobiose concentration greater than 10 m M , was not apparently subject to inhibition by cellobiose concentration as high as 100 m M i f it was immobilized and entrapped with calcium alginate gel spheres (40). Glucose Inhibition Inhibition by glucose, a common characteristic of β-glucosidases (1,9,41,42) although there are exceptions (10,43,44), is an important constraint to overcome if this enzyme is to have industrial applications. Most of the β-glucosidases studied were competitively inhibited by glucose. Glucose inhibited the flglucosidase catalyzed reaction of cellulase of T. viride in a mixed inhibition pattern with a competitive character (45). The fl-glucosidase from A. pullulons was competitively inhibited by glucose with an inhibition constant (/Q of 5.65 m M (21). The intracellularfl-glucosidasefrom E. prunastri was competitively inhibited by glucose with a K value of 1.26 m M (33). fl-Glucosidase from S. thermophile was competitively inhibited by glucose with a K of 0.5 m M (13). A fl-glucosidase from Streptomyces sp. was not only resistant to glucose inhibition but it was stimulated two-fold by 0.1 M glucose (44). A cloned fl-glucosidase (BglB) from M. bispora was also activated two to three fold in the presence of 2-5% (0.1 0.3M) glucose and did not become inhibited until the glucose concentration reached about 40% (10). The fl-glucosidase from thermophilic anaerobic bacterium strain Wai21W.2 was insensitive to glucose inhibition (15). The inhibition of fl-glucosidase activity from P. fiiriosus by glucose was almost negligible with a K of 300 m M (16). t

t

t


13.

SAHA E T AL.


205


Synergism with Cellulases Supplementation of β-glucosidase from S. thermophile stimulated cellulose hydrolysis by cellulases where there was no accumulation of cellobiose in the reaction mixture (73). The β-glucosidase from A. pullulons showed synergistic interaction with cellulase to increase the efficiency of glucose production from cellulose by converting cellobiose to glucose (Table IV). This β-glucosidase may have utility in the enzymatic hydrolysis of cellulose from corn fiber and other cellulosic biomasses for the subsequent production of fuel ethanol. The addition of cloned β-glucosidase from C. thermocellum increased the degradation of crystalline cellulose by C. thermocellum cellulase complex (3). Addition of βglucosidase from A. niger to a simultaneous saccharification/fermentation resulted in a 20% increase in percent conversion of cellulose to ethanol while addition to saccharification resulted in a 53% increase in percent conversion (36). The presence of immobilized β-glucosidase from A. phoenicis during enzymatic hydrolysis of cellulosic materials significantly increased the concentration of glucose by converting cellobiose effectively to glucose (45).

Table I V . Cellulose hydrolysis by a commercial cellulase preparation supplemented with purified β-glucosidase from Aureobasidiumpullulans Enzyme

Hydrolysis (%)"

Cellulase Cellulase + β-Glucosidase β-Glucosidase

58.5 66.4 0.0

•At pH 4.5 and 50°C. Reaction time, 48 h. Substrate used: Sigmacell 50 2% (w/v). Enzyme used: cellulase, 5 U/ml; β-glucosidase, 0.1 U/ml. The reaction products were quantified by analyzing in H P L C (27).

Concluding Remarks In recent years, a lot of research effort has been directed toward finding a suitable β-glucosidase for application in the enzymatic conversion of cellulose to glucose. A number of β-glucosidase genes have been cloned from different organisms and more than fifteen nucleotide sequences are now available, allowing for the identification of enzyme families, domain, putative catalytic sites, evolutionary traits, and other features (10,47,48). Our approach has been to develop an improved β-glucosidase that is temperature and pH compatible with process conditions and to screen for a glucose tolerant β-glucosidase. The high activity of the A. pullulans β-glucosidase on cellobiose, its ability to hydrolyze a variety of cellooligosaccharides, high substrate tolerance, its non-dependence on metal


206

ENZYMATIC DEGRADATION O F INSOLUBLE CARBOHYDRATES

ions or thiol compounds, and the high thermoactivity make the enzyme a suitable candidate for application in the enzymatic hydrolysis of cellulose to glucose. The glucose inhibition of β-glucosidase could possibly be overcome by employing a combined saccharification and fermentatation process using a glucose fermenting organism.


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Enzymatic Degradation of Insoluble Carbohydrates - American

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