Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis

Abstract

Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5–9.0 and possessed high thermal stability up to 50°C with 80% of the residual activities compared with the stability seen at the pH range of 6.5–7.0 and temperatures of up to 40°C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass together with cellulases.

When substrate inhibits and inhibitor activates: implications of β-glucosidases

“…At low substrate concentrations, the velocity increases with increasing substrate concentration but starts to decrease when substrate concentration exceeds a certain optimum concentration. The activation of BGs that show substrate inhibition (i.e., non Michaelis–Menten kinetics) by inhibitor has also reported [32, 38]. Therefore, we were curious whether the competition of inhibitor with the nonproductive binding of substrate can result in the activation of enzyme also in the presence of substrate inhibition.…”

“…This phenomenon has been often reported with the inhibition of BGs by glucose [11–36], and xylose [13, 15, 19, 21, 22, 28–31, 37], but also by other sugars [19, 28, 31]. With some of these BGs, the Michaelis–Menten saturation kinetics holds [19, 21, 26, 31], with others, it does not [32, 38]. Most common mechanistic interpretations of the activation by inhibitor include transglycosylation to inhibitor [19, 26] and binding of inhibitor to an allosteric regulatory binding site [21, 39], whereas the glucose tolerance has been explained by the deep and narrow active site architecture of GH1 BGs [40].…”

“…The most common explanation to this phenomenon is the transglycosylation to inhibitor, and, indeed, in many cases, the transglycosylation products are observed in reactions containing inhibitor [19, 26]. However, the activation by inhibitor has been reported also for BGs which do not produce transglycosylation products with inhibitors [38]. The competition of glucose with substrate for binding to the enzyme-substrate complex has recently been proposed to be responsible for the glucose activation of a mutant BG of T. reesei [38].…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Understanding the mechanism of glucose inhibition/tolerance is of crucial importance for biofuel production. Recently, replacement of two amino acids (Leu 167 and Pro 172) at the entrance of the active site of intracellular β-glucosidase of T. reesei (Bgl II) with Trp and Leu, respectively, significantly enhanced glucose tolerance e.g., Ki of 650 mM [141]. Further studies for identification of new β-glucosidase with high glucose tolerance are required.…”

How to Lengthen the Long-Term Stability of Enzyme Membranes: Trends and Strategies

“…Drobecq et al changed the cysteine residue to alanine in small ubiquitin-like modifiers to improve their thermo-stability [17]. Several other papers have also reported that site-directed mutagenesis (replacing an amino acid with another) of enzymes enhanced heat stability [21,22]. To ensure the rigid structure of enzymes, the simplest way is to introduce a disulfide-bond at an internal chain of proteins [17][18][19][20][21][22][23].…”