Appl Microbiol Biotechnol (1990) 34:26-30

Applied Microbiology Biotechnology © Springer-Verlag 1990

Quantification and identification of the main components of the Trichoderma cellulase complex with monoclonal antibodies using an enzyme-linked immunosorbent assay (ELISA) Judith Kolbe*, and Christian P. Kubicek Abteilung far Mikrobielle Biochemie, Institut for Biochemische Technologic und Mikrobiologie, TU Wien, Getreidemarkt 9, A-1060 Vienna, Austria Received 23 March 1990/Accepted 17 May 1990

Summary. An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies has been developed to measure the concentration of three main cellulase components from Trichoderma reesei, cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and endoglucanase I (EG I), in both commercial enzyme preparations as well as in samples from laboratory fermentations. The sensitivity of the assay is 1-10 ng protein, depending on the type of cellulase. The coefficient of variability is between 10% and 20%. By a combination of two different domain-specific monoclonals against CBH I or II it is also possible to quantify the concentration of intact and truncated forms of these two enzymes, respectively. The use of the ELISA to quantify the formation of the three cellulase components under different cultivation conditions is described.

Introduction The complexity of the multi-component cellulolytic system secreted by microorganisms such as Trichoderma sp., is the subject of current investigation and speculation (Enari and Niku-Paavola 1987; Knowles et al. 1987). It basically consists of two different types of enzymes, exo-cellobiohydrolases (CBH, EC 3.2.1.91) and endo-fl-l,4-glucanases (EG, EC 3.2.1.4), each occurring in at least two genetically distinct isoenzyme forms, i.e. CBH I, CBH II, E G I and E G III (Teeri et al. 1983, 1986; Chen et al. 1987; Shoemaker et al. 1983; Saloheimo et al. 1988; Pentill~i et al. 1986; van Arsdell et al. 1987). However, the exact number o f isoenzymes present in culture fluids of Trichoderma sp. is difficult to assess because of a lack of suitable methods for separating and measuring the individual enzymes: while * Present address: Zentrum for Ultrastrukturforschung, Universit~it for Bodenkultur, Gregor Mendel Strasse 33, A-1180 Vienna, Austria Offprint requests to: C. P. Kubicek

various staining methods have been described to detect cellulases after electrophoresis (Bartley et al. 1984; Biely et al. 1985; van Tilbeurgh and Claeyssens 1985; Biely and Markovics 1988; Nummi et al. 1980), separation under conditions preserving the enzyme activity is often poor and subject to pitfalls (Sprey 1987; Sprey and Lambert 1983). Alternatively, distinguishing cellulase isoenzymes by chromogenic oligosaccharides is possible but laborious (Tomme et al. 1988). Immunological techniques have only been introduced infrequently in the analysis of cellulase components (Nummi et al. 1980; Fagerstam and Petterson 1979; Szakacs-Dobozi and Halasz 1986). We have recently prepared monoclonal antibodies against CBH I, CBH II (Mischak et al. 1989) and E G I (Luderer et al., unpublished results) from T. reesei. Here, we present a sensitive enzyme-linked immunosorbent assay (ELISA) for the quantification of CBH I, CBH II and E G I in both industrial and laboratory-produced Trichoderma cellulase preparations. Due to the availability of different domain specific monoclonal antibodies against CBH I and CBH II it can also be used to selectively quantify intact and degraded forms of CBH I and II.

Materials and methods Instruments

Absorbances were read with a Multiscan Plus microtitre-plate spectrophotometer (Titertek, Flow lab, McLean, Va, USA). Plates with 96 wells (Costar, Cambridge, MA), and a 12-channel pipette, 50-250 ~xl(Labsystems, Helsinki, Finland), were used for ELISA. Ultrafiltrations were carried out with Amicon Diaflo PM 10 ultrafiltration membranes (Danvers, Mass, USA). For electrophoresis, a Mightly-Small unit (Hoefer Instruments, San Francisco, Calif, USA) was used.

Reagents

All standard antigens (CBH I, CBH II and EG I from T. reesei) were prepared from a commercial cellulase preparation (Cellu-

27 clast, Novo Industries, Bagsvaerd, Denmark). Other commercial cellulases investigated were obtained from the suppliers indicated with the results in Table 2. Monoclonal antibodies were prepared as described previously (Mischak et al. 1989; Luderer et al., unpublished results). Bovine serum albumin (BSA) was from Serva (Heidelberg, FRG). Atkaline-phosphatase-linked, anti-mouse goat antibodies were obtained from Accurate Chemicals (New York, NY, USA). PBE 94 polybuffer exchanger, polybuffer 74 and PD 10 prefilled desalting columns were purchased from Pharmacia (Uppsala, Sweden). All other reagents and buffers were of analytical or biochemical grade and obtained from local suppliers.

pH 6.8, for elution of CBH I. Samples of approximately l mg CBH I protein, previously equilibrated on PD 10 columns with 10 m M sodium phosphate buffer, pH 6.8, were applied for a single run. CBH II and EG I were purified to electrophoretic homogeneity by preparative SDS-PAGE (Spanos and Ht~bscher 1983). Evidence for the purity of these three cellulase proteins is given in Fig. 1.

Optimized ELISA for CBH L CBH H and EG I Step 1:plate coating. Purified standard antigens and samples to be

Isolation of standard antigens Celluclast (10 ml, containing 280-350 mg protein depending on the charge) was diluted fourfold with 25 mM imidazole-HC1 buffer, pH 7.9, and passed over a column (25 x 800 mm) of Biogel P-30, previously equilibrated with the same buffer. Fractions of 10 ml were collected and assayed for the presence of CBH I, CBH II and EG I by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting and immunostaining using the respective monoclonal antibodies (see below). All three enzymes eluted in the same peak fractions. The collected fractions were then subjected to chromatofocusing on a column (16 x 250 mm) of PBE 94, previously equilibrated in the same buffer (see above). When no more protein was eluted with the starting buffer, a pH gradient was started by applying 500 ml Polybuffer 74, diluted to a tenth with distilled water and adjusted to pH 3.5 with HC1. Fractions of 4 ml were collected and assayed for CBH I, CBH II and EG I immunologically as described above. Separation of all three cellulases from each other was accomplished by this step, but they were still contaminated with other proteins. For CBH I, final purification to homogeneity was achieved by subjecting the peak fractions from chromatofocusing to anion exchange fast liquid chromatography on Mono Q (Pharmacia, Uppsala, Sweden), equilibrated with 10 m M sodium phosphate buffer, pH 6.8, using a gradient of 0.01-1.0 M sodium phosphate,

asssayed were diluted to a concentration of about 10-100 ~tg protein/ml with "coating buffer" (0.1 M sodium carbonate, pH 9.6). To each well of a 96-well plate 0.2 ml of these individual solutions was added. After overnight incubation at 4 ° C, 0.2 ml of 0.25% (w/v) BSA-phosphate-buffered saline (PBS) (containing 0.05%, w/v, Tween 20 and 0.1%, w/v, sodium azide) were added and the plates incubated for 1 h at room temperature.

Step 2: antibody-antigen binding. Preparation of all monoclonal antibodies used has already been described (Mischak et al. 1989; Luderer et al., unpublished results). They were used after ammonium sulphate precipitation and dialysis. Individual monoclonal antibodies were diluted 1 : 10 in 0.25% (w/v) BSA-PBS, and pipetted in aliquots of 100 Ixl into the wells. The plates were then incubated at 37 ° C for 45 min. Thereafter the antibody solutions were removed, and the plates washed four times with 0.05% (w/v) Tween 20-PBS.

Step 3: antibody-antibody binding. Following these washings, a 1:5000 dilution of the alkaline-phosphatase-conjugated, antimouse immunoglobulin G (IgG) goat antibody was pipetted into the wells, and incubated at 37 ° C for 45 min. Thereafter the plates were again washed 4-times as described at step 2.

Step 4: enzyme reaction. Substrate solution (100 ~1 of 2.7 m M pnitrophenyl phosphate in 50 m M sodium carbonate buffer, pH 9.6; 0.5 m M MgC12 and 0.02%, w/v, sodium azide) were pipetted into each well, and the plates incubated for 20 min at room temperature. Thereafter the reaction was stopped, and the microtitre plates were read on a microtitre-plate spectrophotometer at 405 nm. The absorbances measured for the standard proteins were plotted against the respective concentrations of the proteins on semilogarithmic paper. Unknown samples were quantified by using absorbances from the linear portion of the graph only.

Other methods Laboratory cultivations of Trichoderma reesei QM 9414 on cellulose, lactose or glucose were carried out as previously described (Labudova and Farkas 1983). Induction of cellulase formation by sophorose (Serva) in a resting-cell medium was performed essentially as described by Kubicek (1987). Filter-paper units of commercial cellulase preparations were determined as described by Wood and Bhat (1988). Protein concentrations were determined by the dye binding procedure (Bradford !976); SDS-PAGE/Western blotting/immunostaining was carried out as described previously (Kubicek et al. 1987).

Results Fig. 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDS-PAGE/protein stain of cellobiohydrolase (CBH) I, CBH II and endoglucanase (EG) I after purification as described here. MP indicates marker proteins (relative molecular mass in daltons x 10-3). Staining was carried out with Coomassie Brilliant Blue

Characterization of the enzyme immunoassay and preparation of samples Both optimal coating of the microtitre plate and optimal antiserum dilution were determined by binding dif-

28

A~os

A~o5

1.25

CH-6 •

.......~..~,• I: 10

/

1.0

~,,-'-'~ •

/o//o

o

o

~ I: 20

•

CE-16 • E-I t3 E5-3 ~"

1,00

//~/L/~/~"

0.75 O25 1:80

0.50

Q25 025

~,~ I

I

10

100

I

1000

CBH I [/ug/t] Fig. 2. Effect of monoclonal antibody CH-6 dilution on the standard curve of absorbance at 405 nm (A4o5). Data are shown from a single experiment only, but are consistent with several further experiments

Table 1. Properties of monoclonal antibodies (Mab) used during this study Mab

Recognized enzyme (epitope)

Type and concentration of protein (mg/ml)

CH-6 H-7 CE-16 E-1 EG-3

CBH CBH CBH CBH EG I

IgG (3.8) IgG (7.8) IgG (7.9) IgG (7.8) IgG (3.1)

I - core I - AB II - core II - ABB' - core

IgG, immunoglobulin G; CBH, cellobiohydrolase; EG, endoglucanase; for domain nomenclature of cellulase enzymes, see Biely et al. (1985)

ferent dilutions of the antibodies (1/10 to 1/80) to plates coated with several concentrations of purified enzymes (3-1000 ng/ml). Figure 2 shows the optimization of antibody concentration for C B H I, using CH-6. This figure shows that the optimal antibody dilution was 1 : 10, which corresponds to a final concentration of anti-CBH I - I g G of 0.13 m g / m l . Optimal concentrations for C B H I detected were 10-100 lxg/l, which is sufficiently sensitive to detect C B H I in undiluted samples from culture broths of T. reesei under various conditions. Typical standard curves obtained for each class of antibody investigated (cf. Table 1 for properties of the monoclonals used in this study) are shown in Fig. 3: CH-6 had the highest sensitivity, followed by H-7. Also the anti-CBH II core antibody was somewhat more sensitive than the anti-CBH II tail antibody. Anti E G I was least sensitive but was, however, able to recognize E G I in samples containing between 0.1 and 1 rag/1 E G I.

~

I 10

100

1000

Antigen conc. [/ug/t] Fig. 3. Typical standard curves for CBH I, CBH 1I and EG I determination, using different monoclonal antibodies (diluted 1: 10). Each standard curve represents a single experiment, but consistent data have been obtained in several further experiments. The antigen for CH-6 and H7 was CBH I, for CE-16 and E-I CBH II, and for EG-3 EG I

Again this sensitivity appears sufficient to quantify E G I in T. reesei culture fluids from both high and low producer strains. Since the antibodies had been prepared against denatured (SDS-treated) cellulases, it was of interest whether the E L I S A might be able to detect non-SDSdenatured cellulases as well. By comparing different dilutions of both native as well as previously SDS-treated and boiled samples, no differences in the values beyond the statistical deviation (+_13%) were observed.

Quantification of cellulases in some commercial preparations It has previously been shown that a typical T. reesei cellulase mixture contains about 60%-70% C B H I, 10%15% E G I and 10%-20% C B H II (Enari and Niku-Paavola 1987; Knowles et al. 1987). Also, we have recently demonstrated that some commercial samples already contain significant amounts of degradation products of all three enzymes (Mischak et al. 1989). Having the E L I S A method for quantification of cellulases in hand, we have now analysed some commercially available T. reesei cellulases with respect to their content of both intact as well as degraded individual cellulases (Table 2). It is clearly shown that the amount of C B H I and C B H II found in individual preparations roughly confirms the p r e d o m i n a n c e of CBH I over C B H II and E G I. However, some variations are apparent. A compari-

29 Table 2. Concentration of CBH I and II and their degradation products in some commer-

cial cellulase preparations from Trichoderma reesei Sample

1 2 3 4 5 6 7 8

CBH I (gg/mg)

CBH II (I.tg/mg)

Intact

Truncated

Intact

Truncated

2.0 (+ 0.6) 110.6 (+18.3) 4.3 (+ 0.9) 4.2 (+ 0.8) 62.4 (+ 0 . 4 5 ) 40.8 (+12.4) 27.5 (+ 4 . 3 ) 0.46 (+ 0.075)

ND 38.3 (+8.1) ND ND 24.9(+4.8) 1.5(+0.6) 14.9(+2.3) ND

0.17 (+0.05) 31.0 (+6.4) 0.30 (+0.04) 0.34 (+0.06) 0.40 (+0.15) 5.60 (+0.83) 5.30 (+0.70) 0.052 (+0.007)

ND 4.2 (+1.3) 0.13 (+0.04) 0.07 (+0.02) 0.86 (+0.17) 2.43 (+0.50) 1.35 (+0.20) 0.018 (+0.002)

Sample numbers indicate the following enzymes (filter-paper units per mg solids and supplier in brackets); 1, cellulase TC (0.06, Serva, Heidelberg, FRG); 2, cellulase DK-85 (0.25, Pilot Plant, Bratislava, CCSR); 3, Onozuka R-10 (0.09, Serva); 4, Cellulase Type V (0.07, Sigma, St. Louis, Mo, USA); 5, Cellulase, Type VI (0.08, Sigma); 6, Cellulase V 44-MCG 77 (0.14, Pilot Plant, Graz, Austria); 7, Celluclast (0.19, Novo Industries, Bagsvaerd, Denmark); 8, Cellulysin R (0.10, Calbiochem, San Diego, CA, USA). Values in brackets indicate standard deviations from at least five independent determinations. Concentrations for truncated cellulase enzymes were determined by calculating the differences measured with antibodies CH-6 and H7, and CE-16 and El, for CBH I and II, respectively. The validity of the calibration curve was checked by adding known quantities of standard cellulase proteins to the individual samples

Table 3. Concentrations of CBH I, CBH II and EG I in the culture fluid upon cultivation of T. reesei under different conditions

Conditions

Glucose Cellulose Lactose Sophorose

Cellulase concentration (mg/l) CBH I

CBH II

EG I

< 5 (5) 960 (78) 231 (38) 140 (22)

12 (6) 270 (45) 69 (22) 25 (7)

< 5 (5) 250 (42) 87 (25) 15 (6)

The conditions for cultivation are indicated in Materials and methods. Samples from cultivation on glucose were harvested after 18 h, and for lactose and cellulose after 42 h growth; the sample from replacement-induction by sophorose was harvested after 8 h incubation. Data given are means of at least four independent measurements (SD given in brackets). The validity of the calibration curve was checked by adding known quantities of cellulase standard proteins to the individual samples

son of the values obtained by antibodies recognizing the core and the AB region of the same enzyme (i.e. CH-6 and H7, and CE-16 and E-l, respectively) also allows quantification of the content of truncated cellulases. The quantitative values thereby obtained fit well with the qualitative pattern seen in immunostained Western blots of these samples (A. Gsur and C. P. Kubicek, unpublished data). Some of up to 25 commercial preparations tested by us, showed only a weak response and hence disproportionally low values (unpublished results). These preparations appeared to contain substances interfering with the E L I S A ; when the proteins from these samples, however, were precipitated with a twofold vol. of ethanol, reasonable values were obtained. We have not yet identified the substances responsible for this effect.

Quantification of cellulases from laboratory cultivations of T. reesei Individual cellulases have been reported to be subject to the same regulatory circuits, and therefore their proportions remain fairly constant under different cultivation conditions (Knowles et al. 1987; Enari and NikuPaavola 1987). Because of the lack of appropriate methods to quantify the main cellulase components, these observations have always been based at best on semi-quantitative assays. We have therefore investigated the individual concentrations of these enzymes during cultivation on cellulose (natural inducer), glucose (repressing carbon source), lactose (derepressing carbon source) and in a resting cell system on sophorose (the best known in-vitro inducer) by ELISA, The data obtained are compared in Table 3. It is evident that all three enzymes from the cellulase complex were synthesized in approximately constant proportions, irrespective of whether cellulose, lactose or sophorose were used. It is also clearly indicated that lactose led to lower enzyme yields than cellulose. Most interesting, however, is that T. reesei secreted a low level of CBH II also during growth on glucose. This is the first demonstration of a constitutively secreted cellulase from T. reesei, which is therefore currently being further investigated in our laboratory.

Discussion

We describe here an immunoassay that is fast and sensitive for three major cellulase enzymes from T. reesei; as little as 5 ng enzyme can be detected on the microtitre plate for EG I, and the detection limit for CBH I is even a tenth of that. Hence, the assay developed should

30 b e a p p l i c a b l e n o t o n l y to c o m m e r c i a l or l a b o r a t o r y cellulase p r e p a r a t i o n s , b u t also for c h e c k i n g c r o s s - c o n t a m i n a t i o n o f p u r i f i e d cellulase p r o t e i n s , a p r e r e q u i s i t e for studies o n s y n e r g i s m o r s u b s t r a t e specificity. M o r e over, the sensitivity a p p e a r s to b e s a t i s f a c t o r y f o r detecting c e l l u l a s e s also f r o m i n t r a c e l l u l a r c o m p a r t m e n t s , as s t u d i e d q u a l i t a t i v e l y b y G l e n n et al. (1985), a n d t h u s m a y b e o f use in s t u d y i n g the T. reesei s e c r e t o r y p a t h way. F o r b o t h p u r p o s e s , the E L I S A a p p e a r s to b e sup e r i o r o v e r v a r i o u s o t h e r a n a l y t i c a l m e t h o d s u s e d to q u a n t i f y c e l l u l a s e s (cf. W o o d a n d Bhat 1988) b e c a u s e it can b e p e r f o r m e d on inactive p r o t e i n s as well, a n d because, in the case o f C B H I a n d C B H II, it c a n d i s c r i m inate b e t w e e n i n t a c t a n d p r o t e o l y t i c a l l y t r u n c a t e d p r o teins. Note. The antibodies described in this paper may be obtained from the senior author. Conditions for this will be sent on request. Acknowledgements. This work was supported by IJsterreichisches

Bundesministerium far Wissenschaft und Forschung. The authors are also grateful fo Prof. Dr. H. Esterbauer, Graz, Austria, and Dr. V. Farkas, Bratislava, CSSR, for supplying samples of pilot plant cellulase productions, and to Prof. Dr. H.-W. Lubitz, University of Vienna, Austria, for using the ELISA reader. The help of Marion Luderer and Karl Hagspiel in the preparation of antigens is gratefully acknowledged.

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