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P-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose D. STERNBERG, P. V I J A Y A K U M A R ,A N D E. T . REESE U . S . Arrny Natick Reserirch otrd D r ~ ~ e l o p m e nCommritrri, r Pollutio~lAbr~tetnetrtDirisiotz, FoodScirtlces Lrrbornrotg, Norick, M A , U . S . A . 01760 Accepted October 26, 1976 STERNBERG, D., P. VIJAYAKUMAR, and E. T . REESE. 1977. P-Glucosidase: microbialproduction and effect on enzymatic hydrolysis of cellulose. Can. J. Microbiol. 23: 139-147. The enzymatic conversion of cellulose is catalyzed by a multiple enzyme system. T h e Triclrorlertnrt enzyme system has been studied extensively and has insufficient 0-glucosidase ( E C 3.2.1.21) activity for the practical saccharification of cellulose. The black aspergilli ( A . t ~ i g e r and A . phoetricis) were superior producers of P-glucosidase and a method for production of this enzyme in liquid culture is presented. When Triclroclermo cellulase preparations are supplemented with Pglucosidase from Aspergilllrs during practical saccharifications. glucose is the predominant product and the rate of saccharification is significantly increased. The stimulatory effect of P-glucosidase appears to be due to the removal of inhibitory levels of cellobiose. STERNBERG, D., P. VIJAYAKUMAR et E. T . REESE. 1977. P-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Can. J. Microbiol. 23: 139-147. La conversion enzy matique de la cellulose est catalysee par un systeme enzymatique multiple. Le systeme enzymatique de Tricl~orlermoa ete etudie de f a ~ o nintensive, et on a trouve que I'activite de la Pglucosidase (EC 3.2.1.21) est insuffisante pour la saccharification industrielle de la cellulose. A ~ p ~ r g i l l ~t ~t isg e r et Asper;~illtrsp11crtlic.i~sont de meilleurs producteurs d e P-glucosidase et on presente une methode pour la production de cet enzyme dans un milieux d e culture liquide. Lorsque pendant la saccharification industrielle on ajoute de la P-glucosidase obtenue d'Aspergilllts, a des preparations de cellulase provenant de Tricl~orlet.tncr,le glucose est le produit rnajeur et le taux de saccharification est augment6 de f a ~ o nsignificative. Cette stimulation par la P-glucosidase semble attribuable a la suppression des taux inhibiteurs de la cellobiose. [Traduit par le journal]
The enzymatic saccharification of cellulose has been proposed as a means of producing glucose on an industrial scale. The advantage of such a process is that cellulose is one of the world's most abundant resources. The main disadvantage is that a high enzyme concentration is required to obtain a reasonable reaction rate. Crystalline cellulose is attacked by several different enzymes whose concerted action releases cellobiose as a major soluble product (4, 5, 15, 23). Cellobiose is hydrolyzed to glucose by P-D-glucoside glucohydrolase (Pglucosidase, EC 3.2.1.21). While Trichoderma viride is one of the better sources for cellulosesolubilizing enzymes, its production of P-glucosidase is relatively low (20). Therefore, cellobiose is a significant product when practical saccharifications are carried out with T. viride 1,4-(1,3; 1 ,4)-P-D-glucan 3(4)-glucanohydrolase (cellulase, EC 3.2.1.4). Because cellobiose is an inhibitor (5, 7, 23) of the cellulases, the addition of P-glucosidase to T. viride cellulase may in-
crease the rate of cellulolysis. Yamanaka and Wilke (24) recently reported some preliminary results involving the addition of P-glucosidase to saccharification reactions. The purpose of this paper is (a) t o select a microbe which produces (8, 14) relatively large quantities of P-glucosidase; (b) to determine some of the kinetic properties of the selected P-glucosidase as they relate to cellulolysis, and (c) to determine the optimal level of P-glucosidase in saccharification reactions containing T. viride cellulase.
Methods Screening for a-Glrrcosirlose Organisms from the Q M culture collection* were grown o n the salts solution previously used for the production of various enzymes (13, 17). T h e cultures were incubated for 2 weeks at 28 "C o n a reciprocating shaker, and assayed on the 7th and 14th days. Many of the cultures were also grown by the Koji method (wheat *Department of Botany, University of Massachusetts, Amherst, MA, U.S.A. 01001.
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bran 1 part 2 parts of water or salts solution) in unshaken culture. This is similar to the commercial method used for production of various hydrolytic enzymes. The enzyme was extracted from the bran on a shaker (30°C, 1 h) in the presence of toluene, using 10 parts of 0.025 M citrate buffer (pH 4.5) per part of bran.
VOL.
23. 1977
pellets. The mixture was incubated a t 50 OC for 1 h, a n d the supernatant assayed for enzymatic activity.
Results
A. Soeenirlg of Microorganisms for Ability to Produce P-Glucosidase Assn~js Over 200 strains of fungi, a n d 15 of bacteria P-Glucosidase activity was nieasured using either salicin or cellobiose as substrates. Salicinase activity was were grown for 2 weeks at 28 "C on various nieasured by incubating 0.5-1111 aniounts of enzyme a n d carbon sources in shake flasks, or on bran of substrate (4 mg/ml, p H 4.5) for 30 min at 50 "C. (Koji). The n o s t productive organisms are Reducing sugar produced was measured a s glucose by listed in Table 1. The black aspergilli were the dinitrosalicylic acid method (13). Cellobiase activity superior to all others, and 22 additional strains was measured by incubating 1.0-nil amounts of enzyme a n d cellobiose (7.5 m M in reaction mixture) in 0.05 M of these were further evaluated. Aspergill~rs citrate buffer a t p H 4.8 for 30 min a t 50 OC. Glucose niger QM877 and A. phoenicis QM329 were released was measured by the Glucostat procedure selected for further work. (Worthington Biocheniical Corp., Freehold, NJ). CelluThe black aspergilli produced a P-glucosidase lase activities were measured by reducing sugar released that is several times more active on cellobiose from filter paper (FP'ase) o r 0.5% carboxyniethylcellulose (CMC) solution (CMC'ase) (13). A unit (U) of enzyme than on salicin. Of the A . nigcr. group, only the is the amount producing 1 pniol of product (glucose) A . luchuensis strains lacked this high specificity per minute under the assay conditions. Protein was for cellobiose, and in this, resembled most of measured by the method of Lowry el 01. (11). the other fungi examined. Submerged growth in shake flasks was superior SnccIrnrifica/iot~s Cellulase was produced by growing T. uiricle QM9414 t o the Koji method for production of P-glucosiin 15-litre laboratory fermentors. Saccharification of dase in that the specific activity and yield per cellulose (15%) was carried out in flasks containing ceilulase in 0.05 IM citrate buffer a t p H 4.8 a n d at 50 "C gram of carbon source is greater in shake with constant rotary shaking at 200 rpnl. Substrates were flasks, e.g., 1.2 U/mg starch versus 0.2 U / m g Avicel p H 102 ( F M C Corp., American Viscose Div., bran. Furthermore, the amount of endo- 1,4-PNewark, DE), a niicrocrystalline cellulose, and Solka glucanase (CMC'ase) (EC 3.2.1.4) was inuch Floc BW 200 (Brown Co., Berlin, NH), a purified wood lower in shake-flask culture. O n the other hand, cellulose which has been ball-milled and passed through a 200-mesh screen. During hydrolysis, samples were if an economical way of producing high levels removed from the flasks, immersed in a boiling water of crude P-glucosidase was desired, Koji may be bath for 5 niiin, centrifuged, and the supernatants assayed the method of choice. i c method (3) for total sugar by the phenol - s u l f ~ ~ r acid Many cominercial enzyine preparations conand for glucose by theGlucostat method with the addition tain P-glucosidase. More than 60 crude preparaof 6-gluconolacrone to inhibit j3-glucosidase activity in tions of different enzymes, primarily fungal, were the Glucostat reagent. Some samples were analyzed by evaluated for their P-glucosidase content. Sehigh-pressure liquid chromatography using pBONDAPAK/carbohydrate column (Waters Associates, Milford, lected examples are listed in Table 2. One can MA) and a n acetonitri1e:water (80:20) solvent (15). almost predict which of these come from A . Cot~cet~/rcr/iotz ntlcl Frnc/iot~n/iotzof Etlzyt~les niger grown o n bran. They had a high ratio of Culture filtrates (12-14 days) were adjusted to p H 4.5 cellobiose activity to salicin activity; and a with citric acid and concentrated 10-fold by ultrafiltration, high 1,4-P-glucanase content. Almond emulsin with washing. T h e concentrate was adjusted to p H 3.5 showed the opposite relationship; it had low a n d kaolin added (6 g/100 ml). After 10 min of stirring, cellobiase and high aryl P-glucosidase activities. the mixture was centrifuged a n d the supernatant discarded. The precipitates were washed once with water Most of the commercial proteases and amylases and the enzyme eluted with 30 nil of 0.02 M K-PO, tested were free of P-glucosidase (not shown). (pH 7.0). This product was precipitated with 1 volume of acetone, centrifuged, redissolved in 0.02 M K-PO, buffer (pH 7.0), and added to a diethylaminoethyl (DEAE) cellulose c o l u n ~ n equilibrated with the same buffer. The c o l ~ ~ n iwas n eluted with a NaCl gradient (0-0.5 M ) a t the same p H (16, 17).
Ex-/rac/iot~o j ' E t ~ z ~ ~Jiot~l m e Myceli~rt~r Washed mycelium was ground for 1 niin in a Waring Blendor in 0.05 M citrate buffer ( p H 4.5) to break up the
B. Factors A.ffectirig the Prod~rctior7of P-Gl~~cosidose b y Black Aspergilli P-Glucosidase often remains cell-bound in microorganisms such as Sclzizopl1ylhm7 commune (22), Chuetotni~rrn tiler-r?iopliile (12), a n d Alcaligenes faecalis (6). T h e proportion of mycelial-bound t o cell-free enzyme was studied
STERNBERG E T A L
TABLE1. Organisms selected for ability to produce good yields of a-glucosidase -
-
Yield, U/ml, grown in shake flasks
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Name
QM No.
Salicinase
Cellobiase
Aspergillus niger A. phoenicis A. luchuensis Arrreobasidirmz pullula~zs Cizlot~idium7virirle Cladosporiunr resinae Fusarirn~~ moniliforme Mucor oerrrln~nsii Pe~~icilliron variable P. brefeldianurr~ P. worfir~a~nri P. parv~rn~ Pesfalotiopsis wesferclijkii
TABLE2. Commercial enzyme preparations containing P-glucosidase U/mg powder Source Cellulase concn. 9 x Cellase 1000 P-Glucosidase No. 1 Hemicellulase concn. F1329 Cellulase 19AP Snail enzymes* p-Glucosidase Cellulase P-Glucosidase Our acetone powders
Organism
Cellobiase
Salicinase
Miles Lab Wallerstein Rohm & Haas Miles R&H Miles-Seravac Onozuka Rohm
Helix Almonds T. viride A. ~ve~rtii A. niger A. piroenicis
*Snail enzyme comes as a liquid. The values shown are for Ujmg protein.
in the two aspergilli (Fig. I). After 6 days of growth tlie P-glucosidase level reached its peak with the enzynie being more or less evenly distributed between the myceliuin and the culture fluid. After this time, the mycelial level decreased while the filtrate level increased, indicating that the increased level in the filtrate was due to its release and not to further synthesis. After about 12 days most of the P-glucosidase was found in the culture filtrate. These results are similar to those noted earlier (17) for P-xylosidase (EC 3.2.1.37) production by A . 12 iger . P-Glucosidase could be induced in shakeflask cultures by the addition of P-glucosides. A s p e l g i l l ~ iplloenicis, ~ unlike A . niger, produced
appreciable quantities of P-glucosidase on a starch medium without inducer. Enzyme levels were increased in both organisms by the addition of inducer. 0-Glucosides which induced were methyl P-glucoside, amygdalin, salicin, and phenyl-P-glucoside. P-Xylosides, e.g., methyland phenyl-P-xylosides were also good inducers. Cellobiose, lactose, cellobiitol, and methyl 0cellobioside were not effective. P-Glucosidase was also produced on simple sugars, but addition of an inducer was necessary for both species and yields of tlie enzynie were less than with starch. N o inducer was required with the Koji method o r when xylan o r 1,3-P-glucan were used as carbon sources in shake-flask cultures. T h e addition of sodium oleate or Tween 80
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CAN. J. MICROBIOL. VOL. 23, 1977
Incubation
FIG.1. Location of p-glucosidase duringgrowth on starch F = filtrate; 877 = A. niger; 329 = A . plzoenicis.
+ inducer + surfactant. M = mycelium;
TABLE 3. Purification of B-glucosidase of A. phoenicis QM329 U/mg protein Fraction
p-Glucosidase*
Culture filtrate Kaolin eluate Acetone ppt. (1 vol.) DEAE fraction
1.9 9.1 13.3 57.0
Amylase 24.2 11.4 1.8 0.7
CMC'ase 0.5 2.0 0.4 0.3
1,3-p-glucanase 1.3 2.6 3.9 13.8
p-Glucosidase, yield % 100 45 8 3
*As measured against salicin.
at 1 to 2 mg/ml doubled the yields of P-glucosidase in shake-flask cultures. As the time of addition was unimportant, surfactant was usually sterilized with the medium. Nearly as effective as Tween were Carbopol 934 (B. F. Goodrich Chem. Co., Cleveland, OH) and Triton X- 100. Sucrose monopalmitate, Igepol Co630, and polyethylene glycol 1000 had no effect. Quaternary ammonium compounds were toxic. Best P-glucosidase yields (about 12 U/ml as cellobiase) were obtained with A. pl~oe/iicisin shake-flask culture containing 1 % starch, 0.2% methyl P-glucoside, and 0.2% Tween 80.
C. Purification of P-Glucosidc~seof A. phoenicis The increase in specific activity during purification of P-glucosidase is given in Table 3. T h e final product came off the DEAE-cellulose column, when the salt gradient began, as a double P-glucosidase peak which had a constant activity ratio of salicinase t o cellobiase over the range. The increase in specific activity repre-
sents a 30-fold purification. T h e final P-glucosidase preparation had 57 U/mg as salicinase and 160 U/mg as cellobiase. These values compare favorably with cellobiase values for A, crystalline P-glucosidase of A. ~ventii, 113 U/mg (1, lo), and of Alcaliget~esfaecalis, 18 U/mg (6), both of which had been purified more than 100-fold. I ,3-P-Glucanase activity paralleled the increase in P-glucosidase activity. It appeared that activity on the 1,3-P-glucan substrate was dependent on P-glucosidase in that it was strongly inhibited by nojirimycin, i.e., the ratio of inhibitor t o substrate, for 50% inhibition was 0.0005, the same for both 1,3-P-glucan a n d salicin. Exo- and endo- 1,3-P-glucanases are relatively resistant to nojirimycin inhibition (18).
D. Properties of t l ~ eP-Glucosidase of A. pkoenicis Q M329 The D E A E P-glucosidase fraction (Table 3) acts rapidly both on aryl P-glucosides and o n glucosyl P-glucosides and much less rapidly o n alkyl P-glucosides (Table 4). The high activity
STERNBERG ET AL.
TABLE4. Specificity of e-glucosidase of A. phoenicis QM329 RS mg/min per ml enzyme*
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Substrate Esculin Cellobiose Cellotriitol Cellobioside, e-methyl Amygdalin Arbutin Phenyl p-glucoside
Aryl e-glucoside 1,4-e-Diglucose Reduced cellotriose Alkyl-p-cellobioside Aryl p-gentiobioside Aryl a-glucoside
>PNOzCellobiitol Salicin Glucoustilic acid Methyl e-glucoside Phloridzin Phenyl 0-xyloside Stevioside
Reduced cellobiose Aryl p-glucoside 0-Cellobioside Alkyl p-glucoside Aryl a-glucoside Aryl-a-xyloside A a-sophoroside
*Substrate 7.5 :i lo-' M in 0.05 M citrate pH 4.5; 5 0 ° C ; RS
=
7.6 5.8 3.7 3.4 2.6 2.3 2.2 2.0 0.64 0.60 0.46 0.34 0.06 0.01 0.01
r e d u c i n ~sugar.
salicin (not shown). According to the plots, maximum velocities for cellobiose and cellotetraose were 164 and 95 pmol glucose released min-' mg-', respectively. The inhibition constant ( K , ) for nojirimycin was 0.64 x l o F 3mM with cellobiose as the substrate. Substrate inhibition occurred when cellobiose concentration exceeded 10 mM and cellotetraose concentration exceeded 4 mM. The optimum pH for activity of A. phoaiicis P-glucosidase (50 "C) is about 4.3, and between pH 3 and 6 there was more than 5 0 z of the nlaximum activity. Stability at 50 "C was optimal at pH 5.5, with 5 0 x inactivation in 22 h at pH 3.7 and 6.1. The enzyme was stable under the conditions (pH 4.8 and 50 " C ) used for the enzymatic hydrolysis of cellulose by the T. viride system, 85% of the enzyme activity being retained after 4 days. Inactivation was more rapid at higher temperatures, the half-life of 60 "C and 70 "C being 6.5 and less than 0.5 h, TABLE 5. Relative susceptibility of various gli~cose respectively. The crude P-glucosidase of A. tiiger. oligosacchar~desto e-glucosidase of A. phoer~icis was compatible with the cellulase system of T. viride. Neither system adversely affected the Substrate, 7.5 x M Rate, relative enzymic activity of the other over the 4-day period tested.
on cellobiose is the characteristic of the enzymes of the black aspergilli that makes them more applicable to our problems than the P-glucosidases of other fungi. Like typ~cal0-glucosidases (16), they act on all of the variously linked P-glucosyl glucoses, though at different rates; and less rapidly on tetramers than on diniers (Table 5). They are strongly inh~bitedby nojiri~nycin(R J / S 5 , = 0.0005). They are active on P-glucopyranosides but not on P-glucofuranosides. The P-glucosidase had no action on methyl P-xyloside, methyl P-galactoside, methyl 0thioglucoside, methyl a-glucoside, or rutinosides (hesperidin, rutin). The very low act~vity on stevioside niay ~ndicatea structural impedance. The K,, of A. phoe17icl~P-glucosidase as determined by Lineweaver-Burk plots (Fig. 2) was 0.75 mM for cellobiose, 0.36 nlM for cellotetraose, and 44 niM for the aryl glucoside,
Cellobiose p,B-Trehalose Gentiobiose Laminari biose Sophorose
1,4-p-Diglucose 1,l " 1,6 " 1,3 " " 1,2
Laminaritetraose Cellotetraose
G-3G-3G-3Gz G-4G-4G-4G4
'Reduc~ng cnd of molecule.
100 75 72 66 54 46 39
E. Effect of Addition of P-Glucosid~iseto T. uiride Cellulase 011 the Hyrl,olysis of Cell~ilose The addition of 0-glucosidase to saccharification reactions containing a fixed co~~centration of T. viride cellulase enhanced both glucose and total sugar production (Fig. 3). The T. uil.ide
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CAN. J. MICROBIOL. VOL. 23, 1977
FIG.2. Lineweaver-Burk plots of A . plroenicis P-glucosidase, and standard plots (insert). Symbols: m M nojirimycin; !J (---), cellotetraose. cellobiose; A (---), cellobiose + 0.96 x
0 (--),
cellulase preparation contained 0.5 U/ml pglucosidase (as cellobiase); for higher levels of this enzyme, Aspergillus p-glucosidase was added. By comparing values in Fig. 3 it can be seen that the time required to reach any sugar concentration with T. viride cellulase alone was reduced at least 50% when the p-glucosidase activity was increased to 2.7 U/ml of digestion mixture. Enhancement was nearly maximal at 3 U/ml (Fig. 4). The amount of stimulation by Aspergill~rs P-glucosidase was the same with either the crude or purified enzyme, from either A. niger or A. phoetzicis. In digests with T. viride cellulase alone, the concentration of cellobiose was reached and remained at about 12 mg/ml in hydrolysates of BW 200 and about 8 mg/ml in those of Avicel (Fig. 5). The addition of Aspergill~rs p-glucosidase (6 U/mI) reduced the cellobiose levels to about 2 mg/ml and 1 mg/ml in BW 200 and Avicel hydrolysates, respectively. Xylose (not shown) was present at about 8 mg/ml during the course of BW 200 hydrolysis and was either absent or nearly so in Avicel hydrolysates.
Discussion For the enzymatic conversion of cellulose to glucose on a commercial scale it is necessary to have all cellulolytic components at the optimal level. Since P-glucosidase activity in T. viride cellulase preparations is suboptimal for the saccharification process (20, 24), it is essential to find a way of supplying additional P-glucosidase to such reactions. The approach used here was to search for an alternate source of pglucosidase and to maximize its production. Aspergillus tziger QM877 and A. phoetzicis QM329 were the most active P-glucosidase producers of the organisms tested in the QM culture collection. The former organism, already recognized as a producer of p-glucosidase (9), is cultured on bran (Koji method) for commercial preparations of this enzyme. Our work has emphasized P-glucosidase production in liquid culture (shake flasks). We have obtained maximum yields (about 12 U/ml as cellobiase) with soluble starch as the carbon source, methyl P-glucoside as an inducer, and surfactants. We estimate the yields of cell-free enzymes to be
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STERNBERG E T AL.
Hwn
Hwn
FIG.3. Effect of the addition of crude a-glucosidase (as cellobiase) on the saccharification of BW 200 T. uirirle cellulase alone (FP'ase = 2.0 U/rnl, CMC'ase = and Avicel by T. uiridecellulase. Symbols: 0, 95 U/ml, !3-glucosidase = 0.5 U/ml); A, T. uiricle cellulase as above plus 1.1 U/ml A . niger a-glucosidase (total !3-glucosidase = 1.6 U/ml); 0, T. uirirle cell~llaseas above plus 2.2 U/ml A. nyer glucosidase (total a-glucosidase = 2.7 U/ml); 0 , T. uirirle cellulase as above plus 5.5 U/ml A. niger a-glucosidase (total 0-glucosidase = 6.0 U/ml; e, A. niger preparation alone (P-glucosidase = 6 U/ml).
a-
> 100-fold those obtained from Alccrligerzes (6), Schizopl~llutncornnurne (22), Chaetomi~rmthermophile (12), Botryodiplodia theobror?zae (2 l), or Aspergillrrs ~ventii(1 , 10). The specificity of the P-glucosidase of A. phoerzicis is similar to that of A. niger (16) in having good activity on all five of the P-linked dimers of glucose. The P-glucosidase of A. funzigntus (19) differs in that its activity on laminaribiose is greater than on sophorose, and there is very little action on cellobiose or on gentiobiose. All act on aryl P-glucosides. The affinity of Aspergill~rs P-glucosidase for cellobiose (K, = 0.76 mM) is somewhat greater than that reported for the Triclzodertnn enzyme ( K , = 1.5 and 1.8 mM) (2, 4, 20). But, like the P-glucosidase of Trichoderma, the K, for larger
oligosaccharides (trimer and tetramer) is lower than that for the dimer, and the enzymes are inhibited by cellobiose concentrations greater than 10 m M (3.4 mg/ml). Therefore, the Aspergillus enzyme should behave similarly to that of Trichoderma in saccharification reactions. The highest P-glucosidase activity obtained from ~richodermain this laboratory has been around 0.5 U/ml (as cellobiase). On the basis of results presented here it appears that the optimum P-glucosidase activity for the saccharification of 1 5 x cellulose is much higher, i.e., at least 3 to 4 U/ml. When the saccharification reaction contains 0.5 U/ml P-glucosidase, substantial levels of cellobiose persist throughout the course of the hydrolysis, whereas when activity is 3 U/ml or greater, cellobiose levels
146
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VOL.
23. 1977
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crystalline cellulose (Avicel) is of particular interest. T h e hydrolysis rate is markedly increased and may even exceed that of ball-milled cellulose, when the latter is saccharified without additional cellobiase.
2
4
6
2
8
4
8
6
Celloblas. U/ml
FIG. 4. Effect of the addition of (3-glucosidase (as cellobiase) o n rates of cellulose hydrolysis by T. virirle cellulase. Times required to reach 4% glucose ((I) and 5% total sugar (b) for each cellobiase concentration. Symbols: 0, 15% BW 200; A, 15% Avicel (conditions as in Fig. 3).
Acknowledgements We thank Drs. Mary Mandels, Hillel Levinson, and Gabriel Mandels for their assistance in the preparation of this manuscript. We a r e grateful to D r . Derek Ball of the Natick Laboratories for his preparation of sophorose and t o Dr. S. Inouye of Meiji Seika Co., for the gift of nojirimycin. This research was conducted while one of us (D. S.) was sponsored by the National Research Council's (U.S.A.) Research Associateship Program at the U.S. Army Natick Research a n d Development Command.
I. BAUSE,E., and G . LEGLER.1974. Isolation and amino acid sequence of a peptide from the active site of P-glucosidases A, fi-omA. i ~ ~ ~ t z Hoppe-Seyler's tii. Z. 80 Physiol. Chem. 355: 438-442. 1974. 2. BERGHEM.L . E. R.. and L . G . PETTERSSON. p-Glucosidase from Tric.lroder~t71c,~,it.itle.Eur. J . Biochem. 46: 295-305. 3. DUBOIS,M..'K. A. G I L L E SJ, . K. H A M I L T O NP.. A. RERERS, and F. S M I T H 1956. . Colorimetric method for determination of sugars and related substances. Anal. 10 20 30 40 50 10 20 30 40 50 Chem. 28: 3 5 c 3 5 6 . 4. EMERT.G. H., E. K. G U M ,J . A. LANG.T. H . L I U , and R. D. BROWN.J R . 1974. Cellulases. Irr Advances FIG. 5. Effect of the addition of (3-glucosidase (as in chemistry. Series No. 136. Etlitrtl by J . Whitaker. cellobiase) o n the accumulation of cellobiose and glucose Am. Chem. Soc.. Washington. DC. pp. 79-100. as a result of T. viride cellulase activity. Glucose and 5. H A L L I W E L G. L , 1975. Action of the componentsof the cellobiose levels during saccharification of 15% cellulose. cellulase complex. Itr Symposium on enzymatic hyglucose; A, cellobiose. (Closed symbols, Symbols: 0, drolysis of cellulose. Erlitetl hy M. Bailey, T. M. 0.5 U/ml (3-glucosidase; open symbols, 6.0 U/ml (3Enari. and M. Linko. SITRA. Helsinki, Finland. pp. glucosidase). T. viride enzyme levels same as in Fig. 3. 319-336. . P6. H A N , Y . W., and V . R. S R I N I V A S E N1969. are very low. Because cellobiose inhibits celluGlucosidase of A l t ~ r t l i ~ ~ c t r e s f i ~J~. ~Bacterioi. c ~ ~ ~ I i . ~100: . lolytic enzymes and decreases the rate of saccha1355-1363. 7. HOWELL,J . A , , and J. D. STUCK.1975. Kinetics of rification (5, 7, 23), the stimulatory effect of Solka Floc cellulose hydrolysis by Tr.icl~odertt~o ~,it.itle additional P-glucosidase is probably due to the cellulase. Biotech. Bioeng. 17: 873-893. removal of inhibitory levels of cellobiose. 8. J E R M Y NM. . A. 1961. Glycosidases. Rev. Pure Appl. For the development of a n economically Chem. 11: 92-1 15. feasible process, it is necessary t o reduce the time , and B . A. ST.ONE. 196 1. Fmc9. K R I S H N A - M U RCT.IR., tionat ion of p-glucosidases from A. tligpr.. Biochem. J. required to hydrolyze the cellulose. Addition 78: 7 15-723. o f P-glucosidase t o the T. viride system has this 10. LEGLER,G. 1968. Isolation a n d enzymatic activity of effect (Fig. 4). The time can be reduced t o onetwo P-glucosidases of Aspergillrrs n.c,t~tii. Hoppehalf by the addition of about 3 P-glucosidase Seyler'sZ. Physiol. Chem. 348: 1359-1366. A. L. FARR,and U/ml of digestion mixture. As the A . pl~oer~icis 11. LOWRY,0 . H., N. J. ROSEBROUGH. R. J. RANDALL. 1951. Protein measurement with Folin culture filtrates contain about 12 U/ml, one part phenol reagent. J . Biol. Chem. 193: 265-275. of this conveniently could be added to three 12. L.usls. A. J . , and R. R. BECKER.1973. p-Glucosidases parts of the T. viride filtrates. The effect of the ofClrnc~ror~~ir117r tl1er111o/211ile. Biochim. Biophys. Acta, 329: 5-16. addition of P-glucosidase on the digestion of l00L
'
I
'
I
'
--
I
Avicel
I
I
I
I -
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STERNBERG ET AL.
13. MANDELS, M., L . HONTZ,and J . NYSTROM.1974. Enzymatic hydrolysis of waste cellulose. Biotech. Bioeng. 16: 1471-1493. 14. NISIZAWA, K., and Y . HASHIMOTO. 1970. Glycoside hydrolases. 111 The carbohydrates, v2A. Etliretl by W. Pigman and D. Horton. Academic Press, Inc.. NY. 15. PALMER, J . 1975. Liquid chromatogl-aphyfor monitoring the conversion of cellulosic wastes to sugars. Appl. Poly. Symp. No. 28. John Wiley & Sons. NY. pp. 237-245. 16. REESE,E. T . , A. MAGUIRE. and F. W. PARRISH. 1968. Glucosidases and exo-glucanases. Can. J . Biochern. 46: 25-34. E , F. W. PARRISH. 1973. 17. REESE.E. T.. A. M A G U I R and Production of P-xylosidases by fungi. Can. J . Microbiol. 19: 1065-1074. 1971. Nojirimycin 18. REESE.E. T., and F. W. PARRISH. and gluconolactone as inhibitors of carbohydrases. Carbohydr. Res. 18: 381-388. 19. R U D I C K , MJ.. . and A. D. ELBEIN.1973. Glycoprotein
20. 21. 22. 23.
24.
147
~ r t rChern. ~ s . 248: enzyme of Aspcygilllrs f i ~ ~ ~ ~ i J~. Biol. 6506-65 13. D. 1976. P-Glucosidase: its biosynthesis STERNBERG, and role in saccharification of cellulose. Appl. Enviran. Microbiol. 31: 64S654. U M E Z U R I KG. A . M. 1970. Purification and properties of P-glucosidase of Bof~yodiploitlio rllcobrotrlrrr. Biochirn. Biophys. Acta. 227: 419. WILSON,R. W., and J. J. NIEDERPRUEM. 1967. P-Glucosidases of Schi:ol~h\llirtt~ co11itl7rtt1e.Can. J. Microbiol. 13: 1009-1020. WOOD,T.. and S . I. MCCRAE.1975. Cellulase complex of Trichodrrtr~cikotlitlgii. 111 S y m p o s i ~ ~on m enzymatic hydrolysis of cellulose. Erlitttl by M. Bailey, T . M. Enari, and M. Linko. SITRA. Helsinki, Finland. pp. 23 1-254. Y A M A N A K Y., A , and C. R. WILKE.1976. Cellulose hydrolysis with a mixed enzyme system. AIChE 81st Ann. Meet. Abstr. Kansas City, MO. April 11-14, 1976.
P-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose D. STERNBERG, P. V I J A Y A K U M A R ,A N D E. T . REESE U . S . Arrny Natick Reserirch otrd D r ~ ~ e l o p m e nCommritrri, r Pollutio~lAbr~tetnetrtDirisiotz, FoodScirtlces Lrrbornrotg, Norick, M A , U . S . A . 01760 Accepted October 26, 1976 STERNBERG, D., P. VIJAYAKUMAR, and E. T . REESE. 1977. P-Glucosidase: microbialproduction and effect on enzymatic hydrolysis of cellulose. Can. J. Microbiol. 23: 139-147. The enzymatic conversion of cellulose is catalyzed by a multiple enzyme system. T h e Triclrorlertnrt enzyme system has been studied extensively and has insufficient 0-glucosidase ( E C 3.2.1.21) activity for the practical saccharification of cellulose. The black aspergilli ( A . t ~ i g e r and A . phoetricis) were superior producers of P-glucosidase and a method for production of this enzyme in liquid culture is presented. When Triclroclermo cellulase preparations are supplemented with Pglucosidase from Aspergilllrs during practical saccharifications. glucose is the predominant product and the rate of saccharification is significantly increased. The stimulatory effect of P-glucosidase appears to be due to the removal of inhibitory levels of cellobiose. STERNBERG, D., P. VIJAYAKUMAR et E. T . REESE. 1977. P-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Can. J. Microbiol. 23: 139-147. La conversion enzy matique de la cellulose est catalysee par un systeme enzymatique multiple. Le systeme enzymatique de Tricl~orlermoa ete etudie de f a ~ o nintensive, et on a trouve que I'activite de la Pglucosidase (EC 3.2.1.21) est insuffisante pour la saccharification industrielle de la cellulose. A ~ p ~ r g i l l ~t ~t isg e r et Asper;~illtrsp11crtlic.i~sont de meilleurs producteurs d e P-glucosidase et on presente une methode pour la production de cet enzyme dans un milieux d e culture liquide. Lorsque pendant la saccharification industrielle on ajoute de la P-glucosidase obtenue d'Aspergilllts, a des preparations de cellulase provenant de Tricl~orlet.tncr,le glucose est le produit rnajeur et le taux de saccharification est augment6 de f a ~ o nsignificative. Cette stimulation par la P-glucosidase semble attribuable a la suppression des taux inhibiteurs de la cellobiose. [Traduit par le journal]
The enzymatic saccharification of cellulose has been proposed as a means of producing glucose on an industrial scale. The advantage of such a process is that cellulose is one of the world's most abundant resources. The main disadvantage is that a high enzyme concentration is required to obtain a reasonable reaction rate. Crystalline cellulose is attacked by several different enzymes whose concerted action releases cellobiose as a major soluble product (4, 5, 15, 23). Cellobiose is hydrolyzed to glucose by P-D-glucoside glucohydrolase (Pglucosidase, EC 3.2.1.21). While Trichoderma viride is one of the better sources for cellulosesolubilizing enzymes, its production of P-glucosidase is relatively low (20). Therefore, cellobiose is a significant product when practical saccharifications are carried out with T. viride 1,4-(1,3; 1 ,4)-P-D-glucan 3(4)-glucanohydrolase (cellulase, EC 3.2.1.4). Because cellobiose is an inhibitor (5, 7, 23) of the cellulases, the addition of P-glucosidase to T. viride cellulase may in-
crease the rate of cellulolysis. Yamanaka and Wilke (24) recently reported some preliminary results involving the addition of P-glucosidase to saccharification reactions. The purpose of this paper is (a) t o select a microbe which produces (8, 14) relatively large quantities of P-glucosidase; (b) to determine some of the kinetic properties of the selected P-glucosidase as they relate to cellulolysis, and (c) to determine the optimal level of P-glucosidase in saccharification reactions containing T. viride cellulase.
Methods Screening for a-Glrrcosirlose Organisms from the Q M culture collection* were grown o n the salts solution previously used for the production of various enzymes (13, 17). T h e cultures were incubated for 2 weeks at 28 "C o n a reciprocating shaker, and assayed on the 7th and 14th days. Many of the cultures were also grown by the Koji method (wheat *Department of Botany, University of Massachusetts, Amherst, MA, U.S.A. 01001.
140
CAN. J. MlCROBlIOL.
+
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bran 1 part 2 parts of water or salts solution) in unshaken culture. This is similar to the commercial method used for production of various hydrolytic enzymes. The enzyme was extracted from the bran on a shaker (30°C, 1 h) in the presence of toluene, using 10 parts of 0.025 M citrate buffer (pH 4.5) per part of bran.
VOL.
23. 1977
pellets. The mixture was incubated a t 50 OC for 1 h, a n d the supernatant assayed for enzymatic activity.
Results
A. Soeenirlg of Microorganisms for Ability to Produce P-Glucosidase Assn~js Over 200 strains of fungi, a n d 15 of bacteria P-Glucosidase activity was nieasured using either salicin or cellobiose as substrates. Salicinase activity was were grown for 2 weeks at 28 "C on various nieasured by incubating 0.5-1111 aniounts of enzyme a n d carbon sources in shake flasks, or on bran of substrate (4 mg/ml, p H 4.5) for 30 min at 50 "C. (Koji). The n o s t productive organisms are Reducing sugar produced was measured a s glucose by listed in Table 1. The black aspergilli were the dinitrosalicylic acid method (13). Cellobiase activity superior to all others, and 22 additional strains was measured by incubating 1.0-nil amounts of enzyme a n d cellobiose (7.5 m M in reaction mixture) in 0.05 M of these were further evaluated. Aspergill~rs citrate buffer a t p H 4.8 for 30 min a t 50 OC. Glucose niger QM877 and A. phoenicis QM329 were released was measured by the Glucostat procedure selected for further work. (Worthington Biocheniical Corp., Freehold, NJ). CelluThe black aspergilli produced a P-glucosidase lase activities were measured by reducing sugar released that is several times more active on cellobiose from filter paper (FP'ase) o r 0.5% carboxyniethylcellulose (CMC) solution (CMC'ase) (13). A unit (U) of enzyme than on salicin. Of the A . nigcr. group, only the is the amount producing 1 pniol of product (glucose) A . luchuensis strains lacked this high specificity per minute under the assay conditions. Protein was for cellobiose, and in this, resembled most of measured by the method of Lowry el 01. (11). the other fungi examined. Submerged growth in shake flasks was superior SnccIrnrifica/iot~s Cellulase was produced by growing T. uiricle QM9414 t o the Koji method for production of P-glucosiin 15-litre laboratory fermentors. Saccharification of dase in that the specific activity and yield per cellulose (15%) was carried out in flasks containing ceilulase in 0.05 IM citrate buffer a t p H 4.8 a n d at 50 "C gram of carbon source is greater in shake with constant rotary shaking at 200 rpnl. Substrates were flasks, e.g., 1.2 U/mg starch versus 0.2 U / m g Avicel p H 102 ( F M C Corp., American Viscose Div., bran. Furthermore, the amount of endo- 1,4-PNewark, DE), a niicrocrystalline cellulose, and Solka glucanase (CMC'ase) (EC 3.2.1.4) was inuch Floc BW 200 (Brown Co., Berlin, NH), a purified wood lower in shake-flask culture. O n the other hand, cellulose which has been ball-milled and passed through a 200-mesh screen. During hydrolysis, samples were if an economical way of producing high levels removed from the flasks, immersed in a boiling water of crude P-glucosidase was desired, Koji may be bath for 5 niiin, centrifuged, and the supernatants assayed the method of choice. i c method (3) for total sugar by the phenol - s u l f ~ ~ r acid Many cominercial enzyine preparations conand for glucose by theGlucostat method with the addition tain P-glucosidase. More than 60 crude preparaof 6-gluconolacrone to inhibit j3-glucosidase activity in tions of different enzymes, primarily fungal, were the Glucostat reagent. Some samples were analyzed by evaluated for their P-glucosidase content. Sehigh-pressure liquid chromatography using pBONDAPAK/carbohydrate column (Waters Associates, Milford, lected examples are listed in Table 2. One can MA) and a n acetonitri1e:water (80:20) solvent (15). almost predict which of these come from A . Cot~cet~/rcr/iotz ntlcl Frnc/iot~n/iotzof Etlzyt~les niger grown o n bran. They had a high ratio of Culture filtrates (12-14 days) were adjusted to p H 4.5 cellobiose activity to salicin activity; and a with citric acid and concentrated 10-fold by ultrafiltration, high 1,4-P-glucanase content. Almond emulsin with washing. T h e concentrate was adjusted to p H 3.5 showed the opposite relationship; it had low a n d kaolin added (6 g/100 ml). After 10 min of stirring, cellobiase and high aryl P-glucosidase activities. the mixture was centrifuged a n d the supernatant discarded. The precipitates were washed once with water Most of the commercial proteases and amylases and the enzyme eluted with 30 nil of 0.02 M K-PO, tested were free of P-glucosidase (not shown). (pH 7.0). This product was precipitated with 1 volume of acetone, centrifuged, redissolved in 0.02 M K-PO, buffer (pH 7.0), and added to a diethylaminoethyl (DEAE) cellulose c o l u n ~ n equilibrated with the same buffer. The c o l ~ ~ n iwas n eluted with a NaCl gradient (0-0.5 M ) a t the same p H (16, 17).
Ex-/rac/iot~o j ' E t ~ z ~ ~Jiot~l m e Myceli~rt~r Washed mycelium was ground for 1 niin in a Waring Blendor in 0.05 M citrate buffer ( p H 4.5) to break up the
B. Factors A.ffectirig the Prod~rctior7of P-Gl~~cosidose b y Black Aspergilli P-Glucosidase often remains cell-bound in microorganisms such as Sclzizopl1ylhm7 commune (22), Chuetotni~rrn tiler-r?iopliile (12), a n d Alcaligenes faecalis (6). T h e proportion of mycelial-bound t o cell-free enzyme was studied
STERNBERG E T A L
TABLE1. Organisms selected for ability to produce good yields of a-glucosidase -
-
Yield, U/ml, grown in shake flasks
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Name
QM No.
Salicinase
Cellobiase
Aspergillus niger A. phoenicis A. luchuensis Arrreobasidirmz pullula~zs Cizlot~idium7virirle Cladosporiunr resinae Fusarirn~~ moniliforme Mucor oerrrln~nsii Pe~~icilliron variable P. brefeldianurr~ P. worfir~a~nri P. parv~rn~ Pesfalotiopsis wesferclijkii
TABLE2. Commercial enzyme preparations containing P-glucosidase U/mg powder Source Cellulase concn. 9 x Cellase 1000 P-Glucosidase No. 1 Hemicellulase concn. F1329 Cellulase 19AP Snail enzymes* p-Glucosidase Cellulase P-Glucosidase Our acetone powders
Organism
Cellobiase
Salicinase
Miles Lab Wallerstein Rohm & Haas Miles R&H Miles-Seravac Onozuka Rohm
Helix Almonds T. viride A. ~ve~rtii A. niger A. piroenicis
*Snail enzyme comes as a liquid. The values shown are for Ujmg protein.
in the two aspergilli (Fig. I). After 6 days of growth tlie P-glucosidase level reached its peak with the enzynie being more or less evenly distributed between the myceliuin and the culture fluid. After this time, the mycelial level decreased while the filtrate level increased, indicating that the increased level in the filtrate was due to its release and not to further synthesis. After about 12 days most of the P-glucosidase was found in the culture filtrate. These results are similar to those noted earlier (17) for P-xylosidase (EC 3.2.1.37) production by A . 12 iger . P-Glucosidase could be induced in shakeflask cultures by the addition of P-glucosides. A s p e l g i l l ~ iplloenicis, ~ unlike A . niger, produced
appreciable quantities of P-glucosidase on a starch medium without inducer. Enzyme levels were increased in both organisms by the addition of inducer. 0-Glucosides which induced were methyl P-glucoside, amygdalin, salicin, and phenyl-P-glucoside. P-Xylosides, e.g., methyland phenyl-P-xylosides were also good inducers. Cellobiose, lactose, cellobiitol, and methyl 0cellobioside were not effective. P-Glucosidase was also produced on simple sugars, but addition of an inducer was necessary for both species and yields of tlie enzynie were less than with starch. N o inducer was required with the Koji method o r when xylan o r 1,3-P-glucan were used as carbon sources in shake-flask cultures. T h e addition of sodium oleate or Tween 80
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CAN. J. MICROBIOL. VOL. 23, 1977
Incubation
FIG.1. Location of p-glucosidase duringgrowth on starch F = filtrate; 877 = A. niger; 329 = A . plzoenicis.
+ inducer + surfactant. M = mycelium;
TABLE 3. Purification of B-glucosidase of A. phoenicis QM329 U/mg protein Fraction
p-Glucosidase*
Culture filtrate Kaolin eluate Acetone ppt. (1 vol.) DEAE fraction
1.9 9.1 13.3 57.0
Amylase 24.2 11.4 1.8 0.7
CMC'ase 0.5 2.0 0.4 0.3
1,3-p-glucanase 1.3 2.6 3.9 13.8
p-Glucosidase, yield % 100 45 8 3
*As measured against salicin.
at 1 to 2 mg/ml doubled the yields of P-glucosidase in shake-flask cultures. As the time of addition was unimportant, surfactant was usually sterilized with the medium. Nearly as effective as Tween were Carbopol 934 (B. F. Goodrich Chem. Co., Cleveland, OH) and Triton X- 100. Sucrose monopalmitate, Igepol Co630, and polyethylene glycol 1000 had no effect. Quaternary ammonium compounds were toxic. Best P-glucosidase yields (about 12 U/ml as cellobiase) were obtained with A. pl~oe/iicisin shake-flask culture containing 1 % starch, 0.2% methyl P-glucoside, and 0.2% Tween 80.
C. Purification of P-Glucosidc~seof A. phoenicis The increase in specific activity during purification of P-glucosidase is given in Table 3. T h e final product came off the DEAE-cellulose column, when the salt gradient began, as a double P-glucosidase peak which had a constant activity ratio of salicinase t o cellobiase over the range. The increase in specific activity repre-
sents a 30-fold purification. T h e final P-glucosidase preparation had 57 U/mg as salicinase and 160 U/mg as cellobiase. These values compare favorably with cellobiase values for A, crystalline P-glucosidase of A. ~ventii, 113 U/mg (1, lo), and of Alcaliget~esfaecalis, 18 U/mg (6), both of which had been purified more than 100-fold. I ,3-P-Glucanase activity paralleled the increase in P-glucosidase activity. It appeared that activity on the 1,3-P-glucan substrate was dependent on P-glucosidase in that it was strongly inhibited by nojirimycin, i.e., the ratio of inhibitor t o substrate, for 50% inhibition was 0.0005, the same for both 1,3-P-glucan a n d salicin. Exo- and endo- 1,3-P-glucanases are relatively resistant to nojirimycin inhibition (18).
D. Properties of t l ~ eP-Glucosidase of A. pkoenicis Q M329 The D E A E P-glucosidase fraction (Table 3) acts rapidly both on aryl P-glucosides and o n glucosyl P-glucosides and much less rapidly o n alkyl P-glucosides (Table 4). The high activity
STERNBERG ET AL.
TABLE4. Specificity of e-glucosidase of A. phoenicis QM329 RS mg/min per ml enzyme*
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Substrate Esculin Cellobiose Cellotriitol Cellobioside, e-methyl Amygdalin Arbutin Phenyl p-glucoside
Aryl e-glucoside 1,4-e-Diglucose Reduced cellotriose Alkyl-p-cellobioside Aryl p-gentiobioside Aryl a-glucoside
>PNOzCellobiitol Salicin Glucoustilic acid Methyl e-glucoside Phloridzin Phenyl 0-xyloside Stevioside
Reduced cellobiose Aryl p-glucoside 0-Cellobioside Alkyl p-glucoside Aryl a-glucoside Aryl-a-xyloside A a-sophoroside
*Substrate 7.5 :i lo-' M in 0.05 M citrate pH 4.5; 5 0 ° C ; RS
=
7.6 5.8 3.7 3.4 2.6 2.3 2.2 2.0 0.64 0.60 0.46 0.34 0.06 0.01 0.01
r e d u c i n ~sugar.
salicin (not shown). According to the plots, maximum velocities for cellobiose and cellotetraose were 164 and 95 pmol glucose released min-' mg-', respectively. The inhibition constant ( K , ) for nojirimycin was 0.64 x l o F 3mM with cellobiose as the substrate. Substrate inhibition occurred when cellobiose concentration exceeded 10 mM and cellotetraose concentration exceeded 4 mM. The optimum pH for activity of A. phoaiicis P-glucosidase (50 "C) is about 4.3, and between pH 3 and 6 there was more than 5 0 z of the nlaximum activity. Stability at 50 "C was optimal at pH 5.5, with 5 0 x inactivation in 22 h at pH 3.7 and 6.1. The enzyme was stable under the conditions (pH 4.8 and 50 " C ) used for the enzymatic hydrolysis of cellulose by the T. viride system, 85% of the enzyme activity being retained after 4 days. Inactivation was more rapid at higher temperatures, the half-life of 60 "C and 70 "C being 6.5 and less than 0.5 h, TABLE 5. Relative susceptibility of various gli~cose respectively. The crude P-glucosidase of A. tiiger. oligosacchar~desto e-glucosidase of A. phoer~icis was compatible with the cellulase system of T. viride. Neither system adversely affected the Substrate, 7.5 x M Rate, relative enzymic activity of the other over the 4-day period tested.
on cellobiose is the characteristic of the enzymes of the black aspergilli that makes them more applicable to our problems than the P-glucosidases of other fungi. Like typ~cal0-glucosidases (16), they act on all of the variously linked P-glucosyl glucoses, though at different rates; and less rapidly on tetramers than on diniers (Table 5). They are strongly inh~bitedby nojiri~nycin(R J / S 5 , = 0.0005). They are active on P-glucopyranosides but not on P-glucofuranosides. The P-glucosidase had no action on methyl P-xyloside, methyl P-galactoside, methyl 0thioglucoside, methyl a-glucoside, or rutinosides (hesperidin, rutin). The very low act~vity on stevioside niay ~ndicatea structural impedance. The K,, of A. phoe17icl~P-glucosidase as determined by Lineweaver-Burk plots (Fig. 2) was 0.75 mM for cellobiose, 0.36 nlM for cellotetraose, and 44 niM for the aryl glucoside,
Cellobiose p,B-Trehalose Gentiobiose Laminari biose Sophorose
1,4-p-Diglucose 1,l " 1,6 " 1,3 " " 1,2
Laminaritetraose Cellotetraose
G-3G-3G-3Gz G-4G-4G-4G4
'Reduc~ng cnd of molecule.
100 75 72 66 54 46 39
E. Effect of Addition of P-Glucosid~iseto T. uiride Cellulase 011 the Hyrl,olysis of Cell~ilose The addition of 0-glucosidase to saccharification reactions containing a fixed co~~centration of T. viride cellulase enhanced both glucose and total sugar production (Fig. 3). The T. uil.ide
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CAN. J. MICROBIOL. VOL. 23, 1977
FIG.2. Lineweaver-Burk plots of A . plroenicis P-glucosidase, and standard plots (insert). Symbols: m M nojirimycin; !J (---), cellotetraose. cellobiose; A (---), cellobiose + 0.96 x
0 (--),
cellulase preparation contained 0.5 U/ml pglucosidase (as cellobiase); for higher levels of this enzyme, Aspergillus p-glucosidase was added. By comparing values in Fig. 3 it can be seen that the time required to reach any sugar concentration with T. viride cellulase alone was reduced at least 50% when the p-glucosidase activity was increased to 2.7 U/ml of digestion mixture. Enhancement was nearly maximal at 3 U/ml (Fig. 4). The amount of stimulation by Aspergill~rs P-glucosidase was the same with either the crude or purified enzyme, from either A. niger or A. phoetzicis. In digests with T. viride cellulase alone, the concentration of cellobiose was reached and remained at about 12 mg/ml in hydrolysates of BW 200 and about 8 mg/ml in those of Avicel (Fig. 5). The addition of Aspergill~rs p-glucosidase (6 U/mI) reduced the cellobiose levels to about 2 mg/ml and 1 mg/ml in BW 200 and Avicel hydrolysates, respectively. Xylose (not shown) was present at about 8 mg/ml during the course of BW 200 hydrolysis and was either absent or nearly so in Avicel hydrolysates.
Discussion For the enzymatic conversion of cellulose to glucose on a commercial scale it is necessary to have all cellulolytic components at the optimal level. Since P-glucosidase activity in T. viride cellulase preparations is suboptimal for the saccharification process (20, 24), it is essential to find a way of supplying additional P-glucosidase to such reactions. The approach used here was to search for an alternate source of pglucosidase and to maximize its production. Aspergillus tziger QM877 and A. phoetzicis QM329 were the most active P-glucosidase producers of the organisms tested in the QM culture collection. The former organism, already recognized as a producer of p-glucosidase (9), is cultured on bran (Koji method) for commercial preparations of this enzyme. Our work has emphasized P-glucosidase production in liquid culture (shake flasks). We have obtained maximum yields (about 12 U/ml as cellobiase) with soluble starch as the carbon source, methyl P-glucoside as an inducer, and surfactants. We estimate the yields of cell-free enzymes to be
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STERNBERG E T AL.
Hwn
Hwn
FIG.3. Effect of the addition of crude a-glucosidase (as cellobiase) on the saccharification of BW 200 T. uirirle cellulase alone (FP'ase = 2.0 U/rnl, CMC'ase = and Avicel by T. uiridecellulase. Symbols: 0, 95 U/ml, !3-glucosidase = 0.5 U/ml); A, T. uiricle cellulase as above plus 1.1 U/ml A . niger a-glucosidase (total !3-glucosidase = 1.6 U/ml); 0, T. uirirle cell~llaseas above plus 2.2 U/ml A. nyer glucosidase (total a-glucosidase = 2.7 U/ml); 0 , T. uirirle cellulase as above plus 5.5 U/ml A. niger a-glucosidase (total 0-glucosidase = 6.0 U/ml; e, A. niger preparation alone (P-glucosidase = 6 U/ml).
a-
> 100-fold those obtained from Alccrligerzes (6), Schizopl~llutncornnurne (22), Chaetomi~rmthermophile (12), Botryodiplodia theobror?zae (2 l), or Aspergillrrs ~ventii(1 , 10). The specificity of the P-glucosidase of A. phoerzicis is similar to that of A. niger (16) in having good activity on all five of the P-linked dimers of glucose. The P-glucosidase of A. funzigntus (19) differs in that its activity on laminaribiose is greater than on sophorose, and there is very little action on cellobiose or on gentiobiose. All act on aryl P-glucosides. The affinity of Aspergill~rs P-glucosidase for cellobiose (K, = 0.76 mM) is somewhat greater than that reported for the Triclzodertnn enzyme ( K , = 1.5 and 1.8 mM) (2, 4, 20). But, like the P-glucosidase of Trichoderma, the K, for larger
oligosaccharides (trimer and tetramer) is lower than that for the dimer, and the enzymes are inhibited by cellobiose concentrations greater than 10 m M (3.4 mg/ml). Therefore, the Aspergillus enzyme should behave similarly to that of Trichoderma in saccharification reactions. The highest P-glucosidase activity obtained from ~richodermain this laboratory has been around 0.5 U/ml (as cellobiase). On the basis of results presented here it appears that the optimum P-glucosidase activity for the saccharification of 1 5 x cellulose is much higher, i.e., at least 3 to 4 U/ml. When the saccharification reaction contains 0.5 U/ml P-glucosidase, substantial levels of cellobiose persist throughout the course of the hydrolysis, whereas when activity is 3 U/ml or greater, cellobiose levels
146
CAN. J. MICROBIOL.
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23. 1977
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 06/06/12 For personal use only.
crystalline cellulose (Avicel) is of particular interest. T h e hydrolysis rate is markedly increased and may even exceed that of ball-milled cellulose, when the latter is saccharified without additional cellobiase.
2
4
6
2
8
4
8
6
Celloblas. U/ml
FIG. 4. Effect of the addition of (3-glucosidase (as cellobiase) o n rates of cellulose hydrolysis by T. virirle cellulase. Times required to reach 4% glucose ((I) and 5% total sugar (b) for each cellobiase concentration. Symbols: 0, 15% BW 200; A, 15% Avicel (conditions as in Fig. 3).
Acknowledgements We thank Drs. Mary Mandels, Hillel Levinson, and Gabriel Mandels for their assistance in the preparation of this manuscript. We a r e grateful to D r . Derek Ball of the Natick Laboratories for his preparation of sophorose and t o Dr. S. Inouye of Meiji Seika Co., for the gift of nojirimycin. This research was conducted while one of us (D. S.) was sponsored by the National Research Council's (U.S.A.) Research Associateship Program at the U.S. Army Natick Research a n d Development Command.
I. BAUSE,E., and G . LEGLER.1974. Isolation and amino acid sequence of a peptide from the active site of P-glucosidases A, fi-omA. i ~ ~ ~ t z Hoppe-Seyler's tii. Z. 80 Physiol. Chem. 355: 438-442. 1974. 2. BERGHEM.L . E. R.. and L . G . PETTERSSON. p-Glucosidase from Tric.lroder~t71c,~,it.itle.Eur. J . Biochem. 46: 295-305. 3. DUBOIS,M..'K. A. G I L L E SJ, . K. H A M I L T O NP.. A. RERERS, and F. S M I T H 1956. . Colorimetric method for determination of sugars and related substances. Anal. 10 20 30 40 50 10 20 30 40 50 Chem. 28: 3 5 c 3 5 6 . 4. EMERT.G. H., E. K. G U M ,J . A. LANG.T. H . L I U , and R. D. BROWN.J R . 1974. Cellulases. Irr Advances FIG. 5. Effect of the addition of (3-glucosidase (as in chemistry. Series No. 136. Etlitrtl by J . Whitaker. cellobiase) o n the accumulation of cellobiose and glucose Am. Chem. Soc.. Washington. DC. pp. 79-100. as a result of T. viride cellulase activity. Glucose and 5. H A L L I W E L G. L , 1975. Action of the componentsof the cellobiose levels during saccharification of 15% cellulose. cellulase complex. Itr Symposium on enzymatic hyglucose; A, cellobiose. (Closed symbols, Symbols: 0, drolysis of cellulose. Erlitetl hy M. Bailey, T. M. 0.5 U/ml (3-glucosidase; open symbols, 6.0 U/ml (3Enari. and M. Linko. SITRA. Helsinki, Finland. pp. glucosidase). T. viride enzyme levels same as in Fig. 3. 319-336. . P6. H A N , Y . W., and V . R. S R I N I V A S E N1969. are very low. Because cellobiose inhibits celluGlucosidase of A l t ~ r t l i ~ ~ c t r e s f i ~J~. ~Bacterioi. c ~ ~ ~ I i . ~100: . lolytic enzymes and decreases the rate of saccha1355-1363. 7. HOWELL,J . A , , and J. D. STUCK.1975. Kinetics of rification (5, 7, 23), the stimulatory effect of Solka Floc cellulose hydrolysis by Tr.icl~odertt~o ~,it.itle additional P-glucosidase is probably due to the cellulase. Biotech. Bioeng. 17: 873-893. removal of inhibitory levels of cellobiose. 8. J E R M Y NM. . A. 1961. Glycosidases. Rev. Pure Appl. For the development of a n economically Chem. 11: 92-1 15. feasible process, it is necessary t o reduce the time , and B . A. ST.ONE. 196 1. Fmc9. K R I S H N A - M U RCT.IR., tionat ion of p-glucosidases from A. tligpr.. Biochem. J. required to hydrolyze the cellulose. Addition 78: 7 15-723. o f P-glucosidase t o the T. viride system has this 10. LEGLER,G. 1968. Isolation a n d enzymatic activity of effect (Fig. 4). The time can be reduced t o onetwo P-glucosidases of Aspergillrrs n.c,t~tii. Hoppehalf by the addition of about 3 P-glucosidase Seyler'sZ. Physiol. Chem. 348: 1359-1366. A. L. FARR,and U/ml of digestion mixture. As the A . pl~oer~icis 11. LOWRY,0 . H., N. J. ROSEBROUGH. R. J. RANDALL. 1951. Protein measurement with Folin culture filtrates contain about 12 U/ml, one part phenol reagent. J . Biol. Chem. 193: 265-275. of this conveniently could be added to three 12. L.usls. A. J . , and R. R. BECKER.1973. p-Glucosidases parts of the T. viride filtrates. The effect of the ofClrnc~ror~~ir117r tl1er111o/211ile. Biochim. Biophys. Acta, 329: 5-16. addition of P-glucosidase on the digestion of l00L
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I
'
I
'
--
I
Avicel
I
I
I
I -
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV CALGARY on 06/06/12 For personal use only.
STERNBERG ET AL.
13. MANDELS, M., L . HONTZ,and J . NYSTROM.1974. Enzymatic hydrolysis of waste cellulose. Biotech. Bioeng. 16: 1471-1493. 14. NISIZAWA, K., and Y . HASHIMOTO. 1970. Glycoside hydrolases. 111 The carbohydrates, v2A. Etliretl by W. Pigman and D. Horton. Academic Press, Inc.. NY. 15. PALMER, J . 1975. Liquid chromatogl-aphyfor monitoring the conversion of cellulosic wastes to sugars. Appl. Poly. Symp. No. 28. John Wiley & Sons. NY. pp. 237-245. 16. REESE,E. T . , A. MAGUIRE. and F. W. PARRISH. 1968. Glucosidases and exo-glucanases. Can. J . Biochern. 46: 25-34. E , F. W. PARRISH. 1973. 17. REESE.E. T.. A. M A G U I R and Production of P-xylosidases by fungi. Can. J . Microbiol. 19: 1065-1074. 1971. Nojirimycin 18. REESE.E. T., and F. W. PARRISH. and gluconolactone as inhibitors of carbohydrases. Carbohydr. Res. 18: 381-388. 19. R U D I C K , MJ.. . and A. D. ELBEIN.1973. Glycoprotein
20. 21. 22. 23.
24.
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~ r t rChern. ~ s . 248: enzyme of Aspcygilllrs f i ~ ~ ~ ~ i J~. Biol. 6506-65 13. D. 1976. P-Glucosidase: its biosynthesis STERNBERG, and role in saccharification of cellulose. Appl. Enviran. Microbiol. 31: 64S654. U M E Z U R I KG. A . M. 1970. Purification and properties of P-glucosidase of Bof~yodiploitlio rllcobrotrlrrr. Biochirn. Biophys. Acta. 227: 419. WILSON,R. W., and J. J. NIEDERPRUEM. 1967. P-Glucosidases of Schi:ol~h\llirtt~ co11itl7rtt1e.Can. J. Microbiol. 13: 1009-1020. WOOD,T.. and S . I. MCCRAE.1975. Cellulase complex of Trichodrrtr~cikotlitlgii. 111 S y m p o s i ~ ~on m enzymatic hydrolysis of cellulose. Erlitttl by M. Bailey, T . M. Enari, and M. Linko. SITRA. Helsinki, Finland. pp. 23 1-254. Y A M A N A K Y., A , and C. R. WILKE.1976. Cellulose hydrolysis with a mixed enzyme system. AIChE 81st Ann. Meet. Abstr. Kansas City, MO. April 11-14, 1976.
