Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution

Vermaas

et al. 2019

Appl Environ Microbiol

Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme’s stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii (TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus (SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T50) (15 and 19°C), and the melting temperature (Tm) (16.5 and 22.9°C) and extended half-lives (t1/2) (∼240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the SoCel5 and TeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent SoCel5. IMPORTANCE Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, SoCel5 and TeEgl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

Section: Figmentioning

confidence: 89%

Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents

Vermaas

et al. 2019

Appl Environ Microbiol

Proc. Natl. Acad. Sci. U.S.A.

“…In these studies, the performance of the WT TrCel7A expressed in a heterologous host was lower than that of the natively expressed TrCel7A enzyme, likely due to the differences in glycosylation pattern between the two filamentous fungi (41). Recently, the addition of an N-glycosylation site on the CD identified via directed evolution (S113N, with confirmed linkage to GlcNAc via crystal structure) was demonstrated to improve the thermal stability of TrCel7A (increasing T max by 1.5°C) (42). Combined with previous work, the results obtained here suggest a clear stabilizing function of TrCel7A N-glycans-a key attribute for cellulase stability during natural and industrial cellulose hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

Distinct roles of N- and O-glycans in cellulase activity and stability

Amore

Knott

Supekar

et al. 2017

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)-a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.

“…These methods do not need a thorough understanding of the protein structure and lean mainly in the enzyme expression and high throughput screening methods. In the case of industrially required enzymes, the thermostability of cellobiohydrolase I (Cel7A) from T. reesei cellulases was enhanced at 10.4 • C (from 62.5 • C to 72.9 • C) using ep-PCR mutagenesis followed by the QuickChange method [116]. The most Cel7A thermostable variant contains 18 mutated sites.…”

Section: Engineering Cellulases For Enhanced Thermostabilitymentioning

confidence: 99%

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Contreras

Pramanik

Рожкова

et al. 2020

IJMS

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.