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

Abstract

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.

Biochemical characterization of a novel thermo‐halo‐tolerant GH5 endoglucanase from a thermal spring metagenome

“…The endoglucanase from Talaromyces emersonii exhibits an optimum temperature of 90°C (Wang et al, 2014). It is considered as the most thermostable GH5 endoglucanase reported so far (Zheng et al, 2019). Some other GH5 thermotolerant endoglucanases, exhibiting the optimum temperature of 70°C, had been reported from C. bogoriensis (Li et al, 2020), T. alba (Ohta et al, 2019), and Thermoanaerobacterium sp.…”

“…Nevertheless, the enzyme investigated in the present study (Cel M ) is one of the most thermotolerant GH5 endoglucanases. The enzyme with high thermal tolerance and stability is crucial for elevated temperature catalytic operations, and thus critical in the economic viability of an industrial process (Zheng et al, 2019).…”

“…Nevertheless, the enzyme investigated in the present study (Cel M ) is one of the most thermotolerant GH5 endoglucanases. The enzyme with high thermal tolerance and stability is crucial for elevated temperature catalytic F I G U R E 2 Sequence alignment of Cel M (MW039807) with homologous endoglucanases, showing the conserved residues [Color figure can be viewed at wileyonlinelibrary.com] operations, and thus critical in the economic viability of an industrial process (Zheng et al, 2019).…”

An engineered cellobiohydrolase I for sustainable degradation of lignocellulosic biomass

“…Irrespective of the conditions used either high temperatures or seawater or ILs, or DES, it is challenging to find stable enzyme or design enzyme that can function efficiently at the industrially required operating conditions (Lehmann et al, 2014; Nakabayashi et al, 2019; Wallraf et al, 2018). Protein engineering showed great success in improving the thermal tolerance of various endoglucanases (Barruetabeña et al, 2019; Zheng et al, 2019), for example, Cel5A (A. S. Dotsenko et al, 2019) and EGL7 (Vianna Bernardi et al, 2019). Besides, protein engineering was previously employed to tailor endoglucanases CelA2 tolerance in ILs, DES, seawater (Lehmann et al, 2012, 2014).…”

Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor

“…A positive solution to this problem is referred to structure‐guided recombination, which utilizes structural and evolutionary information to design highly mutated and, yet still natively folded, chimeric proteins and protein libraries (Chang et al, ; Otey et al, ; Zheng et al, ). By taking this approach, the creation of chimeric proteins with higher biological activity or catalytic properties was achieved (Kang et al, ; Pardo, Vicente, Mate, Alcalde, & Camarero, ), indicating its effectiveness in protein engineering with unknown crystalline structures.…”