Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases

Abstract

We measured hydrolytic rates of four purified cellulases in small increments of temperature (10-50 °C) and substrate loads (0-100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q10 values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases.

Keywords: Hypocrea jecorina; Rasamsonia emersonii; carbohydrate-binding protein; cellulase; enzyme kinetics; thermoactivation; thermophile.

SCHEME 1.

Simplified reaction scheme used to characterize processive activity of Cel7A enzymes against insoluble cellulose.

FIGURE 1.

Specific enzyme activity (_pv_ss/E₀) for Hj_CBM (A), Re_CBM (B), Hj_CORE (C), and Re_CORE (D) plotted as a function of Avicel load (0–106 g/liter) between 10 and 50 °C. Symbols represent all experimental data from triplicate measurements, and lines are the best fit of Equation 3. Insets show enlargements of results at the lower temperatures, which are hard to assess on the main figures.

FIGURE 2.

Maximum specific rate (_pV_max/E₀; A), Michaelis constant (_pK_m; B), and specificity constant (_pη; C) plotted as a function of temperature for the four investigated enzymes.

FIGURE 3.

Arrhenius plots. The natural logarithm of the maximum specific rate (_pV_max; A), the Michaelis constant (_pK_m; B), and specificity constant (_pη; C) are plotted against the reciprocal of the absolute temperature.

FIGURE 4.

Effects of temperature and binding module on the activity of Cel7A from H. jecorina. The inset shows data from 30 °C of the specific rate versus substrate load. It appears that the one-domain variant was slower at low substrate but became faster than the two-domain enzyme above ∼40 g/liter. The main panel shows the location (i.e. substrate load) of this crossover as a function of temperature. The line separates the plane into regions where the CBM promoted (upper left) or reduced (lower right) enzyme activity, respectively.

FIGURE 5.

Fraction of bound Cel7A in the hydrolysis samples as function of substrate load at temperatures from 10 to 50 °C. The total enzyme concentration was 400 nm in all samples, and the measurements were made after 1-h contact time. All points are average ± S.D. (error bars) for triplicate measurements.