Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production.

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.

Transthyretin (TTR) is a homo-tetramer protein involved in the transport of thyroid hormone (thyroxine; T4) in the plasma and cerebrospinal fluid. Many pollutants have been shown to bind to TTR, which could be alarming as disruption in the thyroid hormone system can lead to several physiological problems. It is also indicated that the monomerization of tetramer and destabilization of monomer can lead to amyloidogenesis. Many compounds are identified that can bind to tetramer and stabilize the tetramer leading to the inhibition of amyloid fibril formation. Other compounds are known to bind tetramer and induce amyloid fibril formation. Among the pollutants, per- and polyfluoroalkyl substances (PFAS) are known to disrupt the thyroid hormone system. The molecular mechanisms of thyroid hormone disruption could be diverse, as some are known to bind with thyroid hormone receptors, and others can bind to membrane transporters. Binding to TTR could also be one of the important pathways to alter thyroid signaling. However, the molecular interactions that drive thyroid-disrupting effects of long-chain and short-chain PFASs are not comprehensively understood at the molecular level. In this study, using a computational approach, we show that carbon chain length and functional group in PFASs are structural determinants, in which longer carbon chains of PFASs and sulfur-containing PFASs favor stronger interactions with TTR than their shorter-chained counterparts. Interestingly, short-chain PFAS also showed strong binding capacity, and the interaction energy for some was as close to the longer-chain PFAS. This suggests that short-chain PFASs are not completely safe, and their use and build-up in the environment should be carefully regulated. Of note, TTR homologs analysis suggests that thyroid-disrupting effects of PFASs could be most likely translated to TTR-like proteins and other species.