The binding of cellulase variants to dislocations: a semi-quantitative analysis based on CLSM (confocal laser scanning microscopy) images

Abstract

Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.

Keywords: Cellulase binding; Confocal laser scanning microscopy; Dislocations; Fluorescence-labelled enzymes; Ratio imaging; Semi-quantitative analysis.

Fig. 1

A schematic drawing depicting the cross section of a latewood fibers (Ø = 24 μm, Ø_lumen = 8 μm) lying horizontally under the microscope and the position of ROIs (Regions of Interest, green arrows) showing dislocations. Dislocations (red, yellow areas) are here shown not to extend across the fibers, although this may also be the case. Selecting the yellow dislocation means more slices can be included for calculation. If the red dislocation is chosen, only 2 slices can be included due to emission interference from the outer and inner surface of the fiber. Grey lines slices of CLSM imaging. Fifteen slices are scanned covering a 30 μm depth, giving a 2 μm distance between slices

Fig. 2

Sensitivity of R values to changes in the absolute zero of the background level. Higher R values are more prone to shift in the absolute zero of the background level

Fig. 3

Humicola insolens EGV binding to a fiber. a PLM image showing dislocations; ROIs are shown for bulk solution (red square), normal cell wall (green square), dislocations (blue square), and lumen (orange square). b CLSM images showing binding at 4 different levels across z-axis of the microscope as shown in c. c Binding profile across z-axis within ROIs shown in a (same colour-code). SOIs (Slices of Interest; 4 slices) between 13.1 and 18.8 μm (boxed region) were selected for R calculation. Fluorescence intensity level in arbitrary unit. H. insolens EGV shows a strong binding preference for dislocations compared to the surrounding normal cell wall. The size of the images is 119.16 × 59.58 μm²

Fig. 4

Humicola insolens EGV (non CBM) binding to a fiber. a PLM image showing dislocations; ROIs are shown for bulk solution (red square), normal cell wall (green square), dislocations (blue square), and lumen (orange square). b CLSM images showing binding at 4 different levels across z-axis of the microscope as shown in c. c Binding profile across z-axis within ROIs shown in a (same colour-code). SOIs (Slices of Interest; 4 slices) between 6.2–9.3 μm (boxed region) were selected for R calculation. Fluorescence intensity level in arbitrary unit. H. insolens (non CBM) does not show increased binding to dislocations. The size of the images is 119.16 × 59.58 μm²

Fig. 5

Trichoderma reesei CBHI binding to a fiber. a PLM image showing dislocations; ROIs are shown for bulk solution (red square), normal cell wall (green square), dislocations (blue square), and lumen (orange square). b CLSM images showing binding at 4 different levels across z-axis of the microscope as shown in c. c Binding profile across z-axis within ROIs shown in a (same colour-code). SOIs (Slices of Interest; 5 slices) between 7.8 and 14 μm (boxed region) were selected for R calculation. Fluorescence intensity level in arbitrary unit. T. reesei CBHI does not show increased binding to dislocations. The size of the images is 119.16 × 59.58 μm²

Fig. 6

Trichoderma reesei CBHI (non CBM) binding to a fiber. a PLM image showing dislocations; ROIs are shown for bulk solution (red square), normal cell wall (green square), dislocations (blue square), and lumen (orange square). b CLSM images showing binding at 4 different levels across z-axis of the microscope as shown in c. c Binding profile across z-axis within ROIs shown in a (same colour-code). SOI (Slices of Interest; 4 slices) between 6.5 and 11.5 μm (boxed region) were selected for R calculation. Fluorescence intensity level in arbitrary unit. T. reesei CBHI (non CBM) does not show increased binding to the dislocations. The size of the images is 119.16 × 59.58 μm²

Fig. 7

a Plot of R values of H. insolens EGV-related enzymes/proteins. Circled data points indicate suspected outliers, b Bar graph made of data points in a after omission of suspected outliers and pooling of the unwashed and washed samples. Standard error of the mean is used to describe data variation. Enzymes/protein containing CBM from H. insolens EGV (EGV) show higher R (around 2) compared to those that do not have this binding module

Fig. 8

a Plot of R values of T. reesei CBHI, CBHI without CBM, CBHII and EGII. Note that maximum y-axis scale is different than in the equivalent graph in Fig. 7. Circled data points indicate suspected outliers, b Bar graph made of data points in a after omission of suspected outliers and pooling of the unwashed and washed samples. Standard error of the mean is used to describe data variation. Essentially the values are around 1.1–1.2 (see text) with none reaching R = 2