Thermostability improvement of endoglucanase Cel7B from Hypocrea pseudokoningii

Mitrović, Aleksandra; Flicker, Karlheinz; Steinkellner, Georg; Gruber, Karl; Reisinger, Christoph; Schirrmacher, Georg; Camattari, Andrea; Glieder, Anton

doi:10.1016/j.molcatb.2013.12.009

Cited by 10 publications

(3 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In particular, the presence of unique prolines and superficial ion pairs (D152-R335, D293-R264, D136-K139) an additional disulfide bridge and a more compact hydrophobic core (due to the presence of W203 and F395, for example) are all stability promoting elements [44,45]. Indeed, protein engineering studies showed that thermostability can be increased by the addition of more disulfide bridges in TrCel7B [46] and by increasing hydrophobicity of cavities in an EG from Trichoderma pseudokoningii [47].…”

Section: Structural Properties Of Recel7bmentioning

confidence: 99%

Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

et al. 2019

View full text Add to dashboard Cite

Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here, we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate‐binding module (CBM). With a temperature optimum of 80 °C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate‐binding cleft. An increased number of glycosylations were identified in ReCel7B, and we propose a stabilizing mechanism for one of the glycans. Global structure–function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding, and cellulose accessibility in GH7 endoglucanases. Database Structural data are available in RCSB Protein Data Bank database under the accession number https://www.rcsb.org/structure/6SU8. Enzymes ReCel7B, endoglucanase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/4.html) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from Rasamsonia emersonii; TrCel7B, endoglucanase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/4.html) from Trichoderma reesei; TrCel7A, cellobiohydrolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/176.html) from Trichoderma reesei.

show abstract

Section: Structural Properties Of Recel7bmentioning

confidence: 99%

Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Directed evolution provides another powerful alternative for engineering the thermostability of enzymes by random mutagenesis. The advantage of this approach mainly lies on the facts that no structure or reaction mechanism information of the protein is required (Yamada et al, 2014;Poor et al, 2014;Mitrovic et al, 2014). However, its efficiency depends on the diversity of the library and the availability of high-throughput screening methods.…”

Section: Q3mentioning

confidence: 99%

Significantly improved thermostability of a reductase CgKR1 from Candida glabrata with a key mutation at Asp 138 for enhancing bioreduction of aromatic α-keto esters

Huang

2015

Journal of Biotechnology

View full text Add to dashboard Cite

“…Thermostable multimodular cellulases provide an excellent template for modification to enhance their suitability for industrial applications. The accessory roles of non-catalytic domains (CBMs) on thermal stability and insoluble substrate degrading efficiencies have been extensively reported by removing or grafting from respective catalytic domain/s [ 17 , 18 , 19 , 20 , 21 , 22 ]. MD simulations (molecular dynamics, is used to analyze physical movements of atoms and molecules by using computational tools) have been used in several studies to evaluate the factors regulating thermostability of enzymes [ 11 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 ] and to investigate the stability of the biomolecular complex and interaction attributes [ 31 , 32 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced Thermostability and Enzymatic Activity of cel6A Variants from Thermobifida fusca by Empirical Domain Engineering

Imran

Rehman

Mirza

et al. 2020

Biology

View full text Add to dashboard Cite

Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (Cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as Cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant Cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of Cel6A.CBC which revealed thermostability of Cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of Cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.

show abstract

Thermostability improvement of endoglucanase Cel7B from Hypocrea pseudokoningii

Cited by 10 publications

References 56 publications

Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

Structural and biochemical characterization of a family 7 highly thermostable endoglucanase from the fungus Rasamsonia emersonii

Significantly improved thermostability of a reductase CgKR1 from Candida glabrata with a key mutation at Asp 138 for enhancing bioreduction of aromatic α-keto esters

Enhanced Thermostability and Enzymatic Activity of cel6A Variants from Thermobifida fusca by Empirical Domain Engineering

Contact Info

Product

Resources

About