Biachimica et Biophysica Acta, 1076 (1991) 215-220 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)0167-4838/91/$03.50 ADONIS 016748389100057(:

BBAPRO 33820

Purification and charactcrisation of a fl-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

Saswati Sengupta, Anil K. Ghosh and Subhabrata Sengupta Department of Applied Biochemislry, Indian Institute of Chemical Biology. Calcutta (India)

(Received 25 June 1990) (Revised manuscript received 10 September 1990)

215

Key words: /~Glucosidase; Enzymepurificalion; (Mushroom)

A ~-ghimsidase with c e l ~ activity was porifh~d to homogeneity from the culture filtrate of the Te~tomyces d.rpeatat The enzyme had optimum activity at pH $.0 and temperature 6S°C and was stable up to 60°C and within pH 2-10. Among the subs/rates tested, p-nitrophenyl.B.D-ghieopyranoside and ¢ellobiose were hydrolysed best by the enzyme. K . and g. values for these substrates were 0.5, 1.25 mM and 95, 91 pmol/min per m ~ respectively. The enzyme had low activity towards gentiobiose, salicin and O-metbyl-~glucoside. Glucose and cellebiese inhibited the fl-D-glucosidase ( P ~ ) activity competilivdy with K I of 1.7 and 19 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate pelyacrylamide gel electrophoresls indicated a molecular mass of II0 kDa. The high molecular weight enzyme protein was pr--,~mt both intracellularly and extracellulady from the very early g r o ~ phase. The enzyme had a pi of 4.5 and appeared to be a glycoproteln.

Introduction

Cellulose hydrolysis primarily depends on at least three enzymes. These include several endo- and exo-cell- ulases and ~-v-glucosidase or eellobiase. The former two enzymes can degrade native cellulose synergistically to generate cellobiose which is a product inhibitor for these enzymes. Ceilobiase (~-l>-glucoside glucohydro- lase, EC 3.2.1.21) plays an important role of scavenging the end product cellobiose by reaving the/3, 1--, 4 linkage to generate D-glucose and also in the regulation of exo- and endo fl(1 ~ 4) glucanase synthesis [1,2]. Cellobiases purified from different sources, were re- ported to have molecular weights ranging from 40000 to 300000 or more with broad substrate specificity [1,3]. Presence of isoenzymes have also been indicated [4].

Most of the macrofungi can survive utilising lignocellulosic materials among which mushroom is considered to be one. Consequently, it is supposed to be very rich in several polysaccharide hydrolysing enzymes. In this context, we have already reported isolation, purification and characterisation of a number of carbohydrase enzymes e.g., two xylanases, mannanase and inulinase produced extracellularly during sub- merged propagation of mushroom mycelia [5-8]. Dur- ing the course of this investigation, constitutive produc- tion of cellobiase by the mushroom, Termifomyces clypeatus was characterised and the presence of casein hydrolysate in the medium was found to have a signifi- cant role in the excretion of the enzyme [9]. The present paper reports physicochemical properties of the ~-glu- cosidase (cellobiase) purified from the culture filtrate of the mushroom grown under cellulolytic conditions.

Abbreviatioas: PMSF, phenylmethyl sulfonyl fluoride; PNPG, p- nitrophenyl-O-D-glucopyranoside; PNP, p-nitrophenol; CMC, c.arboxymethyl cellulose, sodium salt (low viscosity); PCMB, p- chloromercuribenmate, sodium salt; AUFS, absorbance unit full scale; PAGE, po|yacrylamide gel electrophoresis: SDS, sodium dodecyl sulphate.

Correspondence: S. Sengupta, Indian Institut© of Chemical Biology, 4 Raja S C Muliick Road, Calcutta 700032, lndia~

Materials and Methods

Materials. Cellulose (Sigmacell); carboxym~thyl c~;l- lulose - Na salt; xylan (larchwood); a-lactose;maltose, Grade I; isomaltose; a-~+)melibiose; D(+)trehalose, dihydrate; methyl-fl-v-galactopyranoside; D(+)meliTk rose, gentiobiose; iodoacetic acid, Na salt; sodium azide; phenylmethylsulphonyl fluoride; tris(hydroxymethyl)a-

216

minomethane; and ammonium persulphate were purchased from Sigma (U.S.A.). BioGel P-200 (100-200 mesh), acrylamide, N',N'-tetramethylethylenediamine were products of Bio-Rad Laboratories (U.S.A.). DEAE-Sephadex A-25, Sepharose 4B, standard marker protein kit and pl calibration kit were purchased from Pharmacia (Uppsala, Sweden).

Enzyme production. T. clypeatus was grown in shake flasks at 30 + 2°C for 9 days in a medium reported earlier [5] replacing dextrin by cellulose (Sigmaceil, type 100) and casein hydrolysate 1~ (w/v) each. The culture filtrate was taken for the purification of cellobiase.

Enzyme assay. Cellobiase assay mixture (0.4 ml) in 0.1 M acetate buffer (pH 5.0) contained 1.6 #tool ceb lobiose and enzyme sotution (0.6 #g). The mixture was incubated at 40°C for 15 rain and the reaction was stopped by placing the reaction mixture in a boiling water bath for 5 rain. Glucose liberated was measured by glucose oxidase-peroxidase method [10]./]-D-Gluco- sidase assay mixture (2.0 ml) contained 2/~mol PNPG and enzyme solution (0.2 pg) in above mentioned assay buffer and incubated at 40°C for 15 nun. Reaction was terminated with the addition of 1 ml of Na2CO3 solu- tion (1 M) and the amount of PNP liberated was measured at 400 nm [11].

CMCase assay mixture (0.4 ml) contained 2 mg of CMC-Na salt and enzyme solution (0.6/~g) in the same buffer and was incubated at 40 o C for 30 min. Reaction was terminated by the addition of alkaline copper re- agem and the free reducing group liberated was esti- mated according to Nelson [12] and Somogyi 113]. Unit (U) of the above enzyme activities were expressed, respectively, as/Lmol of glucose or PlqP or free reducing group (glucose equivalent) liberated per rain under the assay condition. Specific activity was expressed as U per mg of enzyme protein.

Properties of the enz)~e. (i) Optimum temperature for cellobiase activity was determined by incubating the assay mixture at various temperatures (10-80 ° C) for 15 min. (ii) Optimum pH for cellobiase activity was de- termined by incubating the assay mixture at 40°C for 15 min using the following buffer systems: 0.1 M glycine-HCl (pH 2.5, 3.5), 0.1 M sodium acetate (pH 4.0, 4.5, 5.0, 5.5), 0.1 M Tris-HCl (pH 6.86) and 0.1 M Tris-glycine (pH 9.0). (iii) Stability of cellobiase activity was determined by preincubating the enzyme solution at 40°C for 1 h at different pH values (pH 2-10) or at pH 5.0 for 30 rain at different temperatures (30-80 ° C). Activity of these preincubated samples were de- termined. (iv) Lineweaver Burk plot: enzymic activity was measured using various amount of cellobiose (0.1- 4.0 raM) or PNPG (0.05-1.0 raM) as described earlier. Inhibitory effect of glucose or ceilobiose on Michaelis- Menten kinetics of //-n-glucosidase activity was de- termined by coincubating PNPG (0.05-1.0 mM) with glucose (2.5 and 5.0 mM) or PNPG (0.05-1.0 mM)

cellobiose (0.625 and 1.25 mM), respectively. All in- cubations were done for 15 min at 40°C and pH 5.0 following measurement of PNP or glucose liberated. The plot was obtained using regression analysis to de- termine the slope of the best fitting line. (v) Effect of some metal iol~s and inlfibitors on/~-glucosidas¢ activity was determined by coincubating enzyme solution, PNPG (4 mM) and respective inhibitors at pH 5 and 40°C for 15 win. Residual enzymic activity was de- termined as stated cartier (-,4) Activity towards different substmtes was determined by incubating enzyme solu- tion and respective substrates (4 mM) at 40 ° C, pH 5 for 15 rain following measurement of either PNP [11] or glucose [10] or reducing sugar [12,13].

Protein and carbohydrate determination~ Protein was determined according to Lowry et al. [14] using bovine serum albumin as standard or by measuring absorbance at 260 nm during column chromatography. Carbo- hydrate content of the enzyme protein was determined according to Dubois et al. [15].

Enzyme purification. Buffer A: 10 mM acetate buffer (pH 5.0) containing 50/tM PMSF, Buffer B; 100 mM acetate buffer (pH 5.0) containing 50 #M PMSF.

Each step of pudfieaton was carried out at 0-4°C unless otherwise specified. About 850 ml of culture filtrate containing 243 unit CMCase and 366 unit cel- Iobiase activity was concentrated by ultrafiltration un- der nitrogen pressure using a Diaflo PM-10 membrane. The enzyme concentrate (86 ml) was then subjected to ammonium sulphate fractionation. The protein fraction precipitating in between 0-60~ ammonium sulphate saturation was discarded. The superuatant was further made 100~ saturated with ammonium sulphate and protein precipitated was collected by centrifugation at 100000 × g for 30 rain. The precipitated pellet was then dissolved in buffer A and subsequently dialysed against the same buffer. The enzyme solution (10 ml) was then applied to DEAE-Sephadex (A-25) column (2.5 X 30.2 cm) which was pre-equilibrated with buffer A, The column was washed with a 4 bed volume of buffer A and the enzyme was dated with a linear gradient of NaCI from 0-1 M in the buffer A (total volume, 300 ml). Fractions of 4.6 ntl were collected at a flow rate of 27.6 ml/h and active fractions were pooled together, dialysed against buffer B and concentrated to 2 ml. It was then applied to a BioGel P-200 column (1.5 x 56 cm) pre-equilibrated with buffer B and was eluted at a flow rate of 5 ml/h. Fractions of 2.5 ml were collected and active fractions were pooled together, dialysed against buffer B and concentrated to 1.5 ml. It was then applied to a Sepharose 413 column (1.5 × 35 cm) pre- equilibrated with buffer B. The enzyme was finally eluted with buffer B at a flow rate of 2.8 ml/h with 1.4 ml of each fraction. Active fractions were pooled, con- centrated and dialysed against buffer B. The final volume of the enzyme solution was 3 mL

217

lntracellular enzyme, lntramycelial enzyme was pre- pared according to Dekker [16] by disrupting 3-day- grown mycelia with sand in a chilled mortar-pestle (checked under microscope). The clear supernatant ob- tained by centrifugatiun of the homogenate at 12000 × g was dialysed against buffer B prior to HPLC

Electrophoresis. PAGE was done st pH 83 qzsiug 12% acrylamide slab (13 × 13 cm and 1.0 mm thick) at a constant current of 20 mA [17]. The protein band in the polyacrytamide gel was detected either by Coomassie brilliant blue staining, carbohydrate staining [18] or by activity staining for ~-glucosidase [11].

Minimum molecular weight of ~-glucosidase was de- termined by comparing relative mobility of the enzyme to those of protein standards using SDS-PAGE. Pro- teins were dissolved in 1.7_5~ SDS buffer, heated on a boiling water bath for 5 rain and electrophoresed using 12% acrylamide slab gel [19].

Isoelectric focusing. To determine the isoelectric point of the enzyme, analytical isoelectric focusing was car- tied out in 0.5 mm thick 5% (w/v) PAGE containing 13% (w/v) sucrose, 0.002% (w/v) silver nitrate and 7% (v/v) Pharmalyte (pH 2.5-5.0), using a distance of 10 cm between electrodes at a constant current of 30 mA and at 10°C for 3½ h. 1 M H3PO 4 and 0.4 M Hepes were used as anode and cathode solutions, respectively. After the focusing, the gel was fixed for 1 h in aqueous solution of 10~ (w/v) trichloroacetic acid - 5% (w/v) sulfosalicylic acid and then equilibrated overnight in an aqueous solution of 25% (v/v) methanol - 5% (v/v) acetic acid. The gel was stained for 10-20 rain Ja 0.1% Coomassie blue G-250 in an aqueous solution of 25% (v/v) methanol - 5% (v/v) acetic acid and destained in the equilibrating solution. Approx. 7 ~tg protein of the purified enzyme was loaded on the gel. Low pl calibra- tion kit of pH 2.5-6.5 (Pharmacia) containing pepsino- gen (2.8), amyloglucosidase (3.5), glucose oxidase (4.15), soybean trypsin inhibitor (4.55),/~-lactoglobulin A (5.2) and bovine carbonic anhydrase B (6.55) were used as the marker proteins.

HPLC of ~.glucosidase. Purified enzyn~e as well as crude intra and extracellular enzyme were chromato- graphed on a gel fdtratiun column, protein Pak 300 SW (0.75 x 30 cm) with constant monitoring of absorbance at 280 nm (481 LC spectrophotometer) using 0.2 AUFS and equipped with a 745 B data module (Waters). Fractions of 0.25 ml were collected and cellobiase activ- ity was measured using 0.05 ml of these protein frac- tions. Mobile phase used was buffer B at a flow rate of 0.5 ml/min. Protein ~tandards (Phatmacia); ribo- naclease A (13700), ovalbumin (67000), aldolas¢ (158000), catalase (232000), fertitin (440000) and thyroglobulin (669000) were dissolved in mobile phase and chromatographed separately for column calibra- tion. Molecular weight was approximated from a plot of log molecular weight vs. K,~ v~ues.

Results and Discussion

Enzyme purification The enzyme was purified 122-fold with 30% recovery

ft,llowing the steps mentioned in Table L About 3-fold purification and 18~ increase in enzyme activity was noticed after ultrafiltration of the culture fdtrate. This might be due to removal of low-molecular-weight com- pounds inhibitory to the enzyme. During ammonium sulphate fractionation, 70% of the CMCase activity (170 U) with only 5~ f glucosidase activity of the culture filtrate was retained in protein precipitadng up to 60% saturation and so discarded. Majority of the/l-gluco- sidase activity was present in 60-100% saturated frac- tion containing negligible CMCase activity. After this step of purification about 60% of total enzyme activity with ll-fold purification was obtained. Enzyme pooled from DEAE-Sephadex (A-25) chromatography did not have any CMCase activity and yielded 495 recovery with 38-fold purification. Next step of gel permeation chromatography (BioGel P-200) resulted in the recovery of 34~ of total activity with 102-fold purification. Al- though the enzyme during BioGel P-200 chromatog- raphy was eluted in void volume (Fig. 2a), this step was found to be very important for subsequent purification to eliminate low-molecular-weight contaminating pro- teins. Finally the enzyme was purified using a second gel filtration column (Fig. 2b) where about 30~ of the total activity was recovered with 122-fold purification. After this step of purification, the enzyme was found to be electrophoretically homogeneous either by Coomas- sic brilliant blue staining or by carbohydrate staining or by activity staining. The enzyme contained about 24% carbohydrate.

TABLE 1

Purification of cellobiase

Enzyme sample Protein Total Specific (rag) activity activity

(U) (U/ms) 1 Culture filtrate (850 ml) 779 366 0.47

2 Ultrafillrat[on, 104 mol.wt. cut off (86 ml) 298 431 1,45

3 Ammonium sulfate, 60-100~ saturation (10 ml) 42 217 5.15

4 DEAE-Sephadex (A-25) fractions 221-246 (2.0 m]) 10 177 I7.73

5 BioGel P-200 fractions 11-16 (1.5 ml) 2.64 126 47,60

6 Sepharos: 4B fractions 41-49 (3.0 rnl) 1.90 109 57.47

218

05

Poolecl i i

r

i i

" (,,, r , . : \

J " - ' " " ' ; "'-- --I

I I

s-!y .... i i:

,: ii i! !L,.,

20 40 6() 210 230 250 270 Frac t i on n o (4 6 m l iTube)

J

50 i O

25 05

Fig- l. DEAE-Sephadex A-25 column chromatography (Step 4). Pro- cedures are described in Materials and Methods. ( . . . . . . ), protein as absorbance at 280 nm.( ), cellobiase activity measured as glucose equivalent in terms of absorbance at 420 nm per nil of enzyme

solution. ( . . . . . ), NaCI concentration.

Physicochemical properties ~-Glucosidase, purified from T. clypeatus, had opti-

mum activity at pH 5.0, 65°C and was stable up to temperature 0 0 ° C and between p H 2 to 10 at least for half an hour. Thus the enzyme from mushroom was similar to those of other fungi, e.g., Alternaria alternata [20], Aspergillus niger [21], Trichoderma reesei [22] and Pyricularia oryzae [23] with respect to thermal stability. In this context, this enzyme is more thermostable than those obtained from S. thermophile [24], S. cerevisiae, S. frag#is and C. gu#liermondii [25]. The enzyme was

0 4

poole,4

ii ' "O

03 ',,, ii, I o ~5o I l ,

' [ " i ° 11 i -

g 11 ° • ~'"~'o ~ ,oo i t l Frochon nO ( I . 4 m l / t u b e ]

' i//i , i ~ ucl

I i o.,l Ili', ' l l i t I /

5 25 45 65 85 Fraction nol2.Sml/tube)

Fig. 2. BioGel P-200 gel filtration (a) (Step 5). Sepharose 4-B gel filtration (b) (Step 6). 1~'ocedu~ arc described in Materials and Methods. ( . . . . . . ), protein and ( ), cellobiase activity repre-

sented are similar to Fig. 1.

capable of deaving glucosidic linkage present in each of PNPG, cellobiose, gentiobiose, salicin or methyl p-D- glucopyranoside (Table II). It has no hydrolytic activity towards CMC, starch (soluble), xylan (larchwood), a - D-lactose, a-D-maltose, a-D-isomaltose, a-D-melibiose, D-trehaiose, methyl ~-D-xylopyranoside, methyl /~-D- galactopyranoside, methyl a-D-glucopyranoside and D ( + )melizitose.

Among the substratcs tested, c¢llobiose and PNPG appeared to be the best substrates having K m 1.25 rnM, 0.5 mM and V m 91, 95 F m / m i n per rag, respectively as determined from the Lineweaver-Burk plot. Competi-

~ 0 2 E

1:1_ ®

>_.

0

0 o

i I

I0 20 i /S(mM) I

~ t / /

t 1

0 IC 20 L/S (ram)"

Fig. 3. Line,~eaver-Burk plots of ~glucosidase activity showing the effects of {a) cellobiose and (b) D-glucose. p-glucosidasc activity was measured using PNPG (0.05-1.0 raM) in the absence (12 12) and presence of cellobiose (t~ a) 0.625 mM, (o o) 1.25 raM and glucose ( A ~ A ) 2.5 mM, ( O ~ O ) 5.0 raM, respectively. Incubation was carried out for 15 min followed by measurement of PNP as described

earlier. The plot was obtained using regression analysis to dc:.e,,"mine the slope of best fitting line.

219

TABLE II

Activity towards di.[ferent substrates

Cellobiase activity was determined according to the methods de- scribed in Materials and Methods. Values represent mean+standard error of two sets of duplicates.

Substrate Specific activity (4 raM) (U/rag protein) PNPG 92.3 + 1.88 D( + )Cellobiose 85.5 + 1.93 Salicin 125:1:0.15 Gentiobiose 11.3 + 0.05 ~Methyl-D-glucopyranoside 3.4 + 0.20

tive inhibition of the enzymic activity by cdlobiose (K i 1.9 mM), using PNPG as a substrate, indicated the involvement of the same active site for these two sub- strates (Fig. 3a). This is unlike the enzyme from Asper- gillus foetidus [261. End product, glucose, also appeared to be a competitive inhibitor (Ki 1.7 raM) using PNPG as a substrate (Fig. 3b), as reported elsewhere [4,11,27]. In view of these observations, formation of a binary complex containing either enzyme molecule and PNPG or cellobiose or glucose was suggested. Among various enzyme inhibitors tested, N-bromosueeinimide, PCMB and HgCI2 appeared to be most effective, causing 93, 69 and 50% inhibition at 0.5, 0.5 and 2 mM concentra- tions, respectively (Table III). Probable involvemem of -SH group was suggested similar tot hat reported for the enzyme from Clostridium stercorarium [28]. However, none of the components like iodoaeetie acid, Na salt; EDTA, disodium salt, 2-mercaptoethanol, MnCI 2, NaN 3, CaCI2 and MgSO4 at 20 mM concentration were found to have any effect on fl-glucosidase activity.

SDS-PAGE indicated minimum molecular weight of this homogeneous enzyme preparation to be around 110 kDa. However, during gel-permeation chromatography (HPLC), molecular weight of this enzyme was ap-

TABLE Ill

Effect of some metal ions and inhibitors on fl-D-glucosidase activity

Incubation was made for 15 mill according to the methods described in Materials and Methods. Values represent mean+standard error of two sets of duplicates.

Chemicals Concen- Residual tration activity (raM) (%)

N-Bromosuccinimide 0.5 7.3 ± 1.17 PCMB 0.5 30.7 +0.45 HgCI 2 2.0 50.32 :t: 2.35 SDS 34.7 70.6 +0.16 CuSO4 2.0 79,8:1:2,52 FeSO 4 2.0 81.1:1:0.16 N-Acetylimidazole 0.5 92.1 + I.SI Urea 1000 92_8 +3.08

proximated to be 450 kDa (retention time, 12 rain), Isoelectric pH of the enzyme was found to be 4.5.

fl-Glucosidases may be divided into two groups. One is high-molecular weight (>> 110 kDa) glycoprotein and may contain subunit and has isoelectric pH around 4.5 [3]. Other group is relatively low-molecular-weight pro- tein or glycoprotein ( ~ 40 kDa) containing no subunit and has isoelectric pl-I around 8.5 [11,27]. Former group can specifically cleave fl(1 ~ 4) glucosidic linkage, while the latter cae cleave fl(l --, 2) and fl(1 ~ 6) glucosidie linkages in addition to fl(1 ~ 4) linkages [3,11,27]. Con- sidering the substrate specificity, isodectric point and molecular weight, fl-gtucosidase purifial horn T. clypeatus may be placed in the former group. However, unlike the former group, the enzyme could hydrolyse fl(1 --, 6) glucosidic linkage slowly although activity of the enzyme on fl(1 --, 2) linkage as present in sophorose is not known.

Regarding the molecular weight of the purified en- zyme it may be referred that the enzyme was a glyco- protein and glycoproteins bemve anomalously during gel filtration apparently because of their greater hydra- tion compared to globular proteins [29]. The molecular weights reported here were calculated relative to globu- lar protein markers and, therefore, expected to be higher than the true v~ues. Thus the subunit structure of the molecule was not definite. However, excretion of such a large molecule into the culture filtrate was very puz- zling. Consequently both intracellular and extracdlular enzyme levels from a 3-day-old culture were analysed by HPLC gel-permeation chromatography. Surprisingly all samples showed cellobiase emerging at 12 rain, with in the case of mycelial extracts and culture filtrates, additional protein duting at 20-25 min or 20-30 rain (major) and 28-32 min (minor), respectively. Therefore, presence of enzyme with a molecular mass of 450 kDa analogous to purified enzyme preparation (Step 6, Ta- ble I) was present in both of these preparations. The enzyme is really a large molecule and may be a part of the cell wall which was released by wall lytic enzymes as suggested for invertase and trehalase in Neurospora [301. However, the distribution of fl-glucosidase may be further complicated by the existence of small and large molecular forms of enzyme as well as the presence of isoenzymes as reported by other laboratories [!,3,4]. So it was not definite whether the enzyme was purified in aggregated from or contained similar subunits at pre- sent.

R e f e r e n c e s

I Shewale, J.G. (1982) Int. J. Biochem. 14, 435-443. 2 Gong, C.S. a'~d Tsao, G.T. (1979) in Annual Reports on Fermen- tation Processes (Perlman, D., ed.), Vol. 3, pp. 111-l,t0, Academic Press, New York.

3 McHale, A. and Coughlan, M.P. (1982) J. Gen. Microbiol, 128, 2327-2331.

220

4 Woe..], T.M. and M.-Cra~ S. (1982) J. Gen. Mcirobiol. 128, 2973-2982.

5 Ghosh, A.K., Banerjee PC, and Sengupta, S. (1980) Biochim. Biophys. Acta 612.143 - 152.

6 Mub, J'.e~ee.. M. and Sengupta, S. (1985) J. Gen. Microbiol. 131. 1881-1885.

7 Khowah, S. and Scngupta, $. 0985) Arch. Biochem. Biophys. 241. 533-539.

8 Mukherjee, K.C. and Sengupta, $. 0985) Can. J. Microbiol. 31. 773-777.

9 Sengupta, Saswati and ,Y~:ngupta. S. (1990) Enzyme Micro. Tech- nol. 12, 309-314.

10 Bergmeyer, H.U~ and Beret, IL (1974) Methods of Enzymatic Analysis, 2nd. Edn., VoL 3, pp. 1205-1215, Academic Pscss, New Yo~k.

11 Dekker, ILF.H. (1981) J. Gen. Microbiol. 127, 177-184. 12 Nelson. N. (I944) J. Biol. Chem. 153, 375-380. 13 Somogyi, M. (1952) J. Biol. Chem. 195, 19-23. 14 Lowry, O.H., Rosebrough, N.J., Fat-t, A.L. and Randall, RJ.

(1951) J. Biol. Chem. 193, 265-275. 15 Dubois, M., Gilles, K~ Hamilton, J.K., Rcbers, PAr and Smith. F.

(1956) Analyt. Chem. 28, 350-356. 16 Dekker, R.F.H. (1980) J. Gen. Microbiol. 120, 309-316. 17 Gabriel, O. (1971) MEthods Enzymol. 22, 565-578.

18 Fairbanks, G., Steck, T.L and Wallach, D.L.H. (1971) Biochem- istry 10, 2606-2613.

19 Wcher, K., Pringlc, J.R. and Osbom, M. (I972) MEthods Enzymol. 26. 3-27.

20 Martinez, MJ~ Vazquez, C, Gaillen, F. and Reyes, F. (1988) FEMS Microbiol. Le:L 55, 263-267.

21 Dekker, R.F.H. (1986) Biotechnol. Bioeng. 28, 1438-1442. 22 Gong, C.$., Ladi~h, M.R. and Tsao, G,T, (1977) Biot~chnoL

Bioeng. 19, 959-981. 23 Hirayama~ T_ Nagayama. H. and Matsuda. K. (1979) J. BiochenL

85, 591-599. 24 M~er, H. and Canevascini, G. (1981) Appl. Environ. Microbioi.

41, 924--931. Woodward, J. and Wiseman, A. (1982) Enzyme Micro. Teclmol. 4, 73-79.

26 Maryanovskaya, I.G~ Rabinovich, M.L., Kulaev, I.S. and Klesov, A.A. (1986) Biokhimiya (USSR) 51, 1726-1731.

27 Berry. ILK. and Dekker, R.F.H. (1986) Carbohydr. Res. 157, 1-12.

28 Bronnenmeier, K., Staudenbauer, W.L. (1988) AppL Microbiol. Biotechnol. 28. 380-386.

29 Andrews, P. (1%5) Biochem. J. %, 595-606. 30 Chang, P.Y,L. and Trevithick, J.R. (1972) J. Gen, Microbiol. 70,

13-22.