Components of Trichoderma reesei Cellulase Complex on Crystalline

Chapter 11

Components of Trichoderma reesei Cellulase Complex on Crystalline Cellulose Three-Dimensional Visualization with Colloidal Gold Rafael

A.

1

2

Nieves , Roberta J. Todd , Robert and Michael E. Himmel

2

P. Ellis ,

Downloaded by MONASH UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: October 7, 1994 | doi: 10.1021/bk-1994-0566.ch011

1

1Applied Biological Sciences Branch, National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, CO 80401-3393 Department of Microbiology, Colorado State University, Fort Collins, CO 80523 2

Bacterial cellulose and pretreated natural aspen cellulose were used as substrates for observation of cellulose-bound cellulases. Specific monoclonal antibodies which had previously been adsorbed to 10 nm and 15 nm gold spheres were used to detect bound endoglucanase and cellobiohydrolase via transmission electron microscopy. Three -dimensional electron micrographs demonstrated individually bound cellulases as well as clusters of bound enzymes. Significant changes in the interpretations of the micrographs were seen when these were observed in a three dimensional format as opposed to a two -dimensional view. The three dimensional electron micrographs indicated the sensitivity of this technique for these studies by revealing individual enzymes bound to individual cellulose microfibril(s).

M i c r o b i a l cellulases have been of interest in the last few decades because of their potential for industrial applications. The most widely studied cellulases are those produced by the soft-rot fungus, Trichoderma reesei. These enzymes work synergistically to degrade crystalline cellulose to simple carbohydrates via hydrolysis. The glucose produced from this mechanism can be utilized as a primary feedstock for many fermentation processes. Hydrolysis of the cellulose is thought to be initiated by the 1,4-B-D-glucan glucanohydrolases (endoglucanase I and endoglucanase II) which attack randomly along the cellulose chain exposing reducing and non-reducing ends. These hydrolyzed regions can then be attacked by the 1,4-B-D-glucan cellobiohydrolases ( C B H I and C B H II) which proceed with the subsequent degradation of cellulose. The cellobiose and small oligosaccharides produced from this synergistic effect are then converted to glucose by B-D-glucosidase (1-3). Electron microscopy studies of the action of cellulases on cellulosic substrates have been reported previously (4-6). These studies have relied on indirect visualization of hydrolysis as opposed to direct visualization and localization of the 0097-6156/94/0566-0236S08.00/0 © 1994 American Chemical Society

In Enzymatic Conversion of Biomass for Fuels Production; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

11.

NIEVES E T A L .

237

Components of T r i c h o d e r m a reesei Cellulase

enzymes (4,6). Another study demonstrated binding of cellobiohydrolases indirectly using gold colloids (5), but no studies have shown distinguishable, simultaneous binding of endoglucanases and exoglucanases. W e have recendy demonstrated the apparent association of endoglucanase and cellobiohydrolase on natural aspen cellulose by immunogold labelling and negative staining (7). Specific monoclonal antibodies ( M o A b ) directed against different cellulases were used to indirectly observe the enzymes bound to the solid substrate. Here we present further studies of cellulase binding using immunogold labels in three dimensional electron micrographs. Interpretation of these images becomes clearer as different planes of view are distinguished.


Materials and Methods P r e p a r a t i o n o f B a c t e r i a l a n d A s p e n Cellulose. Lyophilized cultures of Acetobacter xylinum were initially cultured at 30°C in 5 m L of Hestrin-Schramm medium until a visible pellicle at the air-liquid interface could be observed (8). The 5 m l culture was used to inoculate 100 m L of the medium and this was incubated for 3 days i n stationary culture at 30°C. U s i n g a sterile glass rod, the pellicle was transferred to 100 m L of cold 50 m M phosphate buffer p H 7.0. The pellicle was washed 3 X i n this manner and sliced into small squares. The squares were suspended i n 25 m L of phosphate buffer, agitated for 2 m, and filtered through cheesecloth. The resulting cell suspension was washed 2 X with phosphate buffer by centrifugation. Dilute sulfuric acid-pretreated aspen meal was prepared as described previously (7). Ascites P r o d u c t i o n a n d Purification of M o n o c l o n a l Antibodies. M i c e used for ascites production were injected with 0.5 m l of pristane (Sigma Chemical Co.) 1 to 4 weeks prior to injection of hybridoma cells. For each mouse, 5 x 10 cells of the specific hybridoma cell line was injected intraperitoneally. Ascites fluid was collected 5 days later and allowed to coagulate overnight at 4 ° C . The supernatant was clarified by centrifugation. Antibodies were purified using rProtein G Minicolumns (Gibco, Gaithersburg, M D ) . Ascites was diluted 1:10 with starting buffer (0.1 M sodium phosphate, p H 7.0, 0.15 M sodium chloride) and applied to columns which had been equilibrated with 10 bed volumes of starting buffer at a flow rate of 0.5 m L / m i n . The column was washed and the antibodies were eluted with 0.1 M glycine hydrochloride, p H 3.0. Thirty microliter aliquots of 1 M Tris buffer p H 10 were added to the fraction tubes prior to collection. Tubes containing the antibodies were pooled and analyzed for purity. Purified antibodies were bound to gold spheres as described previously (7). Purified C B H I antibodies were adsorbed to 10 n m gold colloids and E G I antibodies were adsorbed to 15 nm gold colloids. 6

I m m u n o l a b e l l i n g o f Cellobiohydrolase I a n d Endoglucanase I B o u n d to Cellulose. E M grids were prepared by placing 200-mesh copper grids on floating films of 0.5% formvar in ethylene dichloride. The grids were carbon coated, air dried, and placed on top of drops of bacterial or aspen cellulose suspensions. The grids were stream washed with distilled water, blot dried, and stored until used. E M grids to which aspen or bacterial cellulose had previously been adsorbed were incubated for 45 m with 1.3% milk-phosphate buffer p H 6.5. The grids were


238

ENZYMATIC CONVERSION OF BIOMASS FOR FUELS PRODUCTION


washed with distilled water and blot dried. The grids were placed face down on a drop of Genencor 150L cellulase (Genencor International, San Francisco, C A . ) which contained T. reesei cellobiohydrolase I and endoglucanase I. Total protein concentration of the commercial cellulase was 1 mg/mL. The grids were washed, blot dried and then placed face down on a drop of an antibody mixture solution which contained a n t i - C B H I and/or anti-EG I M o A b s adsorbed on colloidal gold particles. M o A b s were diluted 1:10 with 1 part milk block and 9 parts immunolabel. After 30 m , the grids were stream washed, blot dried, and immediately stained with \ % phosphotungstic acid for 5 m. The grids were washed, blot dried, and stored for observation. E l e c t r o n M i c r o s c o p y . Electron microscopy was performed on a Philips 400T transmission electron microscope at an accelerating voltage of 80 K v . F o r three dimensional electron micrographs, fields of interest were initially photographed at a 0 ° angle on the specimen stage. A point of reference was identified, and the stage rotated approximately 4 ° C . The point of reference was then aligned to its original position on the screen and the second image was then photographed. The three dimensional effect could be observed by pairing stereo images and viewing with a stereoscope (Abrams Instrument Corp., Lansing, M I ) . Results Figure 1 demonstrated minimal background labelling of a negative control. Negative controls were performed by omitting the exposure of the grids to the commercial cellulase solution. This low magnification electron micrograph showed a single A. xylinum bacterial cell with protruding microfibrillar cellulose. A l l negative controls showed minimal background labelling as shown here, and previously (7). Figure 2 is a high magnification photomicrograph of bacterial cellulose. The width of the bacterial cellulose fibers ranged from 2.5 to 12.5 nm. When the bacterial cellulose was exposed to the commercial cellulase solution and then labelled with either a n t i - C B H I immunogold label (10 nm) or anti-EG I label (15 nm), sporadic labelling along the long axis of the fiber was observed (data not shown). Aggregation of immunolabel was often seen on bacterial and pretreated aspen cellulosic substrates. Aggregation of the gold label had previously been observed i n two dimensions and was not found to be an artifact (7). Figure 3 is a three dimensional photomicrograph of one of these aggregates on bacterial cellulose. C B H I was bound to the fiber but the majority of the enzyme appeared to be bound to non-ribbon cellulose (9). This was often seen on both cellulose substrates. W i t h i n the apparent non-ribbon cellulose there were also visible individual crystalline fibers to which the enzymes were bound at various planes on the viewing field. W h e n pretreated aspen cellulose was used, the conserved "wood" structure that is normally associated with cellulose of higher plants was observed (Figure 4). This electron micrograph demonstrated binding of both C B H I and E G I as seen by immunogold size differential. The convex deformation of the formvar (in the middle) demonstrated the depth perception that was obtained with this technique. When viewed i n stereo, one wood fiber overlapped the other. A t that junction some apparent amorphous cellulose was observed. The C B H molecules located at one end



11.

NIEVES ETAL.


239

F i g u r e 1. A negative control electron micrograph of a n t i - C B H I immunogold and bacterial cellulose. The adsorbed bacteria were reacted with the immunogold in the absence of cellulase. M i n i m a l background labelling was observed on all negative controls. Bar = 1 micron.

F i g u r e 2. A high magnification photo electron micrograph of bacterial cellulose. The width of the bacterial microfibers ranged from 2.5 nm to 12.5 nm. Note the spiral formation and resolution of the fibers. Bar = 0.1 micron.



240


F i g u r e 3. A stereo electron micrograph showing aggregated binding of C B H I to bacterial cellulose fibers. This aggregated binding may actually be to cellulose II which has been displaced. Bar = 0.1 micron.

F i g u r e 4. This stereo electron micrograph shows E G I (15 nm) and C B H I (10 nm) bound to intact aspen cellulose. The "woody" nature of the cellulose is still conserved. Bar = 0.1 micron.



11.

NIEVES E T A L .


241

of the cellulose fiber may indicate the terminal end containing the parallel nonreducing ends of the cellulose chains. This observation has been noted before in previous studies (4,7). The advantage of obtaining a three dimensional representation as opposed to a two dimensional view was illustrated i n the following figures. W h e n Figure 5 was initially examined i n two dimensions, it appeared as i f an endoglucanase was present adjacent to eleven cellobiohydrolases bound to the terminal end of the aspen fibers. After careful observation in stereo it was revealed that the larger immunogold ( E G I) was adjacent to eight cellobiohydrolases but these cellobiohydrolases were bound at different planes in the field of view. The three cellobiohydrolases bound to the terminal end of the microfiber were shown to be located at the plane closest to the grid surface. L o g i c suggested that the large immunogold label was not floating in space (a physical impossibility), but was actually bound to the terminal end of a bundle of fibers or to amorphous cellulose that projected towards the viewer. This was confirmed by noticing a single fiber(s) extending up from the surface to one of the 10 nm a n t i - C B H I immunogold spheres (arrow). Figure 6 illustrated the importance of correct interpretation of electron photomicrographs. Initially, when this field was not observed i n stereo, it appeared as i f an endoglucanase were closely followed by two cellobiohydrolases along the axis of microfibers. W h e n observed i n stereo all three enzymes were at different planes. W h e n this occurred, it was difficult to determine which label was actually bound to an endoglucanase or a cellobiohydrolase, because the difference i n distance from one label to the other may have been significant enough to interpret a 10 nm sphere as a 15 nm sphere. A g a i n the sensitivity of these images was revealed as the apparently larger immunogold label is actually bound to an upright fiber(s). Discussion Electron microscopy studies of biological materials has proven to be an invaluable tool. Indisputably, electron micrographs can sometimes be misinterpreted without careful examination. Three dimensional electron micrographs are useful for obtaining a more definitive interpretation of the observed field. The observations mentioned i n this article are examples indicating the need for more useful visual information such as three-dimensional electron micrographs. The aggregation of enzyme at a specific site, as observed on the bacterial cellulose was unique. In a previous report, overall binding of C B H I was observed along the long axis of the bacterial cellulose (5). The bacterial cellulose used i n the present study was not pretreated and therefore may not have had as many non-reducing ends exposed as had the extracted cellulose used by Chanzy. The production of cellulose II by this particular A. xylinum strain was observed i n other micrographs (data not shown). C B H I bound very w e l l to this particular substrate in aggregates. C B H I binds so well to cellulose II (the amorphous substrate), that were it present at some point along the axis of the fiber, aggregated binding of the enzyme to this cellulose would be expected. Observations made of immunogold labelling at different planes on aspen cellulose was not unexpected when working with natural cellulosic substrates. The rigidity and insolubility of cellulose could cause this type of observed binding. The fibers were



242


F i g u r e 5. This figure demonstrates the appearance of immunogold in separate planes. O n the lowest plane C B H I can be seen bound to the terminal ends of aspen cellulose. O n a higher plane several cellobiohydrolases and one endoglucanase are seen. Close observation of this micrograph reveals a cellulose fiber extending from the lower plane of vision to one of the smaller gold particles (arrow). Bar = 0.1 micron.

F i g u r e 6. A stereo electron micrograph demonstrating a similar observation as that seen in figure 5. The arrow points to a single cellulose fiber extending from the grid, up to a large gold colloid (arrow). B a r = 0.1 micron.


11.

NIEVES ET AL.


243

extending out i n all directions. Observations of the kind shown i n Figures 5 and 6 were seen quite readily. The point must be made that an "apparent" endoglucanase was observed in Figure 6 since the difference i n distance from the surface of the grid to the large immunogold may have been significant enough to have made a 10 n m gold sphere appear as a 15 nm sphere. The sensitivity of the immunolabels and this technique were demonstrated by Figures 5 and 6. The ability to be able to localize individual enzymes on the termini of individual fiber(s) was noteworthy. Without the use of three dimensionality, such interpretations could not have been made. U n t i l three dimensional micrographs are utilized more readily for studies such as these, clarification of electron micrographs w i l l not be complete.


Acknowledgments This work was funded by the Biofuels Program Office at the National Renewable Energy Laboratory by the Biochemical Conversion Program at the Department of Energy Biofuels and Municipal Waste Technology Division. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

Coughlan, M.P. Biotechnol. Genet. Eng. Rev. 1985, 31, 39-109. Wood, T . M . ; McRae, S.I. Adv. Chem. Ser. 1979, 181, 181-209. Goyal, A . ; Ghosh, B.; Eveleigh, D. Bioresource Technol. 1991, 36, 37-50. Chanzy, H . ; Henrissat, B. FEBS Lett. 1985, 184, 285-288. Chanzy, H . ; Henrissat, B.; Vuong, R. FEBS. Lett. 1985, 172, 193-197. White, A.R.; Brown, R . M . Proc. Natl. Acad. Sci. USA 1981, 78, 1047-1051. Nieves, R.A.; Ellis, R.P.; Todd, R.J.; Johnson, T.J.A.; Grohmann, K . ; Himmel, M . E . Applied and Environ. Microbiol. 1991, 57, 3163-3170. Hestrin, S.; Schramm, M . Biochem. J. 1954, 58, 345-352. Roberts, E . M . ; Saxena, I.M.; Brown, R . M . In Cellulose and Wood Chemistry Technology Proceedings of the Tenth Cellulose Conference, Schuerch, C., Ed.; John Wiley & Sons: New York, N Y , 1984; 689-704.

RECEIVED M a r c h 17, 1994


Components of Trichoderma reesei Cellulase Complex on Crystalline

Recommend Documents