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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 (??)8-barrel structure, where each (??) 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 (T 50) (15 and 19°C), and the melting temperature (Tm ) (16.5 and 22.9°C) and extended half-lives (t 1/2) (?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 ?2 and ?3 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 (??) 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.

SUBMITTER: Zheng F

PROVIDER: S-EPMC6384118 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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

Zheng Fei F Vermaas Josh V JV Zheng Jie J Wang Yuan Y Tu Tao T Wang Xiaoyu X Xie Xiangming X Yao Bin B Beckham Gregg T GT Luo Huiying H

Applied and environmental microbiology 20190220 5

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)<sub>8</sub>-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 sou ...[more]

PMID: 30552196

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Project description:BackgroundEndoglucanases from thermophilic microorganisms are a valuable resource as they can be used in a wide variety of biotechnological applications including the valorisation of biomass and the production of biofuels. In the present work we analysed the metagenome from the hot spring Muiño da Veiga, located in the northwest of Spain (in the Galicia region), in search for novel thermostable endoglucanases.ResultsSequence analysis of the metagenome revealed a promising enzyme (Cel776). Predictions on protein structure and conserved amino acid sequences were conducted, as well as expression in heterologous systems with Escherichia coli and Saccharomyces cerevisiae as the host. Cel776Ec was correctly expressed and purified by taking advantage of the His-Tag system, with a yield of 0.346 U/mL in the eluted fraction. Cel776Sc was expressed extracellulary and was easily recovered from the supernatant without the need of further purification, requiring only a concentration step by ultrafiltration, with a significantly higher yield of 531.95 U/mL, revealing a much more suitable system for production of large amounts of the enzyme. Their biochemical characterization revealed biotechnologically interesting enzymes. Both Cel776Ec and Cel776Sc had an optimal temperature of 80 °C and optimal pH of 5. Cel776Ec exhibited high thermostability maintaining its activity for 24 h at 60 °C and maintained its activity longer than Cel776Sc at increasing incubation temperatures. Moreover, its substrate specificity allowed the degradation of both cellulose and xylan. Whereas Cel776Ec was more active in the presence of calcium and magnesium, manganese was found to increase Cel776Sc activity. A stronger inhibitory effect was found for Cel776Ec than Cel776Sc adding detergent SDS to the reaction mix, whereas EDTA only significantly affected Cel776Sc activity.ConclusionsOur study reports the discovery of a new promising biocatalyst for its application in processes, such as the production of biofuel and the saccharification of plant biomass, due to its bifunctional enzymatic activity as an endoglucanase and as a xylanase, as well as highlights the advantages of a yeast expression system over bacteria.

Project description:Backgroundβ-1,4-endoglucanase (EG) is one of the three types of cellulases used in cellulose saccharification during lignocellulosic biofuel/biomaterial production. GsCelA is an EG secreted by the thermophilic bacterium Geobacillus sp. 70PC53 isolated from rice straw compost in southern Taiwan. This enzyme belongs to glycoside hydrolase family 5 (GH5) with a TIM-barrel structure common among all members of this family. GsCelA exhibits excellent lignocellulolytic activity and thermostability. In the course of investigating the regulation of this enzyme, it was fortuitously discovered that GsCelA undergoes a novel self-truncation/activation process that appears to be common among GH5 enzymes.ResultsThree diverse Gram-positive bacterial GH5 EGs, but not a GH12 EG, undergo an unexpected self-truncation process by removing a part of their C-terminal region. This unique process has been studied in detail with GsCelA. The purified recombinant GsCelA was capable of removing a 53-amino-acid peptide from the C-terminus. Natural or engineered GsCelA truncated variants, with up to 60-amino-acid deletion from the C-terminus, exhibited higher specific activity and thermostability than the full-length enzyme. Interestingly, the C-terminal part that is removed in this self-truncation process is capable of binding to cellulosic substrates of EGs. The protein truncation, which is pH and temperature dependent, occurred between amino acids 315 and 316, but removal of these two amino acids did not stop the process. Furthermore, mutations of E142A and E231A, which are essential for EG activity, did not affect the protein self-truncation process. Conversely, two single amino acid substitution mutations affected the self-truncation activity without much impact on EG activities. In Geobacillus sp. 70PC53, the full-length GsCelA was first synthesized in the cell but progressively transformed into the truncated form and eventually secreted. The GsCelA self-truncation was not affected by standard protease inhibitors, but could be suppressed by EDTA and EGTA and enhanced by certain divalent ions, such as Ca2+, Mg2+, and Cu2+.ConclusionsThis study reveals novel insights into the strategy of Gram-positive bacteria for directing their GH5 EGs to the substrate, and then releasing the catalytic part for enhanced activity via a spontaneous self-truncation process.

Project description:BackgroundThe non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited.ResultsEight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1.ConclusionsOur results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.

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

Publications

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

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