Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-β-endoglucanase

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.

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confidence: 79%

Section: Figmentioning

confidence: 99%

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confidence: 99%

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

Vermaas

et al. 2019

Appl Environ Microbiol

“…The effect of enzyme extract concentration on enzyme stability may come from different facts: if the enzyme is multimeric and the first step of the inactivation is enzyme dissociation (but in this case the enzymes are described to be monomeric), if there are some enzyme–enzyme interactions with effect on enzyme stability (e.g., favoring inactivation by aggregation, or stabilizing the enzymes by forming some enzyme complexes), or if some component of the extract may have some effect on enzyme stability (some stabilizing reagent). To evaluate the effect of enzyme concentration on inactivation, two solutions with 1% (0.05 mg mL −1 of protein) and 10% (0.5 mg mL −1 of protein) of enzyme preparation were incubated at pH 10 at 4°C for PE, PG, and PL, at 25°C for PME and 50°C for CE.…”

Section: Resultsmentioning

confidence: 99%

“…The effect of enzyme extract concentration on enzyme stability may come from different facts: if the enzyme is multimeric and the first step of the inactivation is enzyme dissociation (but in this case the enzymes are described to be monomeric), [83][84][85][86][87] if there are some enzyme-enzyme interactions with effect on enzyme stability third sample for each enzyme was prepared with 1% enzyme preparation plus 9% inactivated enzyme solution after boiling. We cannot find any significant difference on the inactivation courses of all these preparations ( Figure 5).…”

Section: Influence Of the Enzyme Concentration On The Stability Of mentioning

confidence: 99%

Stability/activity features of the main enzyme components of rohapect 10L

Magro

Kornecki

Klein

et al. 2019

Biotechnology Progress

Rohapect 10L is an enzyme cocktail commercialized for juice clarification. Here, we characterized the activity and stability of five enzymatic activities present in this cocktail: total pectinase (PE), polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), and total cellulase (CE) activities. All these enzyme activities have the maximum activity and stability at pH 4, conditions near those found in most fruit juices. However, if the enzymes need to be handled under different conditions (e.g., to immobilize them), their stability becomes extremely low in some cases, just at pH values slightly higher than the optimal one. For example, at pH 10 only CE was reasonably stable at 25°C, while many other enzyme activities were rapidly almost inactivated, even at 4°C. For these cases, different additives were evaluated, and we found that polyethylene glycol was positive or very positive for all enzyme stabilities, allowing keeping reasonable activities after several hours at pH 10 and 25°C. Another additive, that is, dextran, has a small positive effect for PE, PG, and CE, and a very positive effect for PL, albeit significantly destabilizing PME. Thus, the handling and use of this extract requires some care when is performed out of optimal conditions.

Biotech and App Biochem

Characterization of an endo‐beta‐1,4 glucanase gene from paper‐degrading and denim bio‐stoning cellulase producing Aspergillus isolates

Ahmed

Asma‐Ul‐Taslim

Raihan

et al. 2022

Cellulases are used in textile, pulp and paper, brewery and wine, sugars, and ethanol industries. Four fungal isolates obtained from organic municipal solid wastes (OMSW) were selected based on their cellulolytic activity on carboxymethyl cellulose (CMC) agar medium. Based on the internal transcribed spacer (ITS) sequence of the ribosomal DNA, the four cellulolytic isolates were identified as Aspergillus fumigatus AKAL1, Aspergillus oryzae AKAL4, Aspergillus flavus AKAL8, and Aspergillus flavus AKAL9. After 9 days of fermentation at 30°C and pH 6.5 under 110 rpm agitation, these isolates produced the maximum amount of cellulase. The cellulase showed optimum activity at temperature 35–40°C and pH 6.0–7.0 and was stable for 1 h at 25–45°C and pH 5.0–7.0. The Mg2+ and Zn2+ significantly increased but Hg2+, K+, and Ca2+ severely repressed the cellulase activity. Degradation of filter papers and bio‐stoning of denim was successfully done with the crude cellulase. An endo‐β‐1,4‐glucanase was isolated and characterized from Aspergillus isolates. Genome‐wide analysis revealed that the genomes of A. oryzae, A. fumigatus, and A. flavus, the pertinent species of the fungal isolates, had 23, 25, and 22 cellulase genes, respectively. Phylogenetic analysis revealed that the cellulases in these fungal species were divided into three major groups, and the isolated endo‐β‐1,4‐glucanase clustered to Group II. Ten different motifs are present in cellulases of the three species. Results herein provide a valuable resource for understanding cellulase genes in Aspergillus species and potential application of cellulase in textile and fermentable sugars production industries.