Heterologous expression and biochemical characterization of a novel thermostable Sclerotinia sclerotiorum GH45 endoglucanase in Pichia pastoris

Chahed, Haifa; Boumaiza, Mohamed; Ezzine, Aymen; Marzouki, M. Néjib

doi:10.1016/j.ijbiomac.2017.08.062

Cited by 26 publications

(19 citation statements)

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“…The retention of the α-factor signal peptide could increase the molecular weight of the recombinant protein in 10 kDa. Hiperglycosylation is also commonly known to increase the molecular weight of recombinant proteins expressed in P. Pastoris 46 . In addition, according to the parameters for heterologous production of a recombinant enzyme, other properties, like optimal temperature and pH for enzymatic activity, can differ from the native enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, according to the parameters for heterologous production of a recombinant enzyme, other properties, like optimal temperature and pH for enzymatic activity, can differ from the native enzyme. Chahed and colleagues produced a GH45 endoglucanase from Sclerotinia sclerotiorum that shows maximum activity at pH 7.0 and 60 °C, against pH 5.0 and 60 °C for the native enzyme 46 . In another study, Akbarzadeh and colleagues presented a recombinant endoglucanase II from Trichoderma reesei expressed in P. pastoris with reduced disulfide bonds.…”

Section: Resultsmentioning

confidence: 99%

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Enzymatic activity of a recombinant β-1,4-endoglucanase from the Cotton Boll Weevil (Anthonomus grandis) aiming second generation ethanol production

Ibarra

Alves

Antonino

et al. 2019

Sci Rep

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In the last years, the production of ethanol fuel has started to change with the introduction of second-generation ethanol (2 G Ethanol) in the energy sector. However, in Brazil, the process of obtaining 2 G ethanol did not reach a basic standard to achieve relevant and economically viable results. Several studies have currently been addressed to solve these issues. A critical stage in the bioethanol production is the deployment of efficient and stable enzymes to catalyze the saccharification step into the process of biomass conversion. The present study comprises a screening for genes coding for plant biomass degradation enzymes, followed by cloning a selected gene, addressing its heterologous expression, and characterizing enzymatic activity towards cellulose derived substrates, with a view to second-generation ethanol production. A cDNA database of the Cotton Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae), an insect that feeds on cotton plant biomass, was used as a source of plant biomass degradation enzyme genes. A larva and adult midgut-specific β-1,4-Endoglucanase-coding gene (AgraGH45-1) was cloned and expressed in the yeast Pichia pastoris. Its amino acid sequence, including the two catalytic domains, shares high identity with other Coleoptera Glycosyl Hydrolases from family 45 (GH45). AgraGH45-1 activity was detected in a Carboxymethylcellulose (CMC) and Hydroxyethylcellulose (HEC) degradation assay and the optimal conditions for enzymatic activity was pH 5.0 at 50 °C. When compared to commercial cellulase from Aspergillus niger, Agra GH45-1 was 1.3-fold more efficient to degrade HEC substrate. Together, these results show that AgraGH45-1 is a valid candidate to be engineered and be tested for 2 G ethanol production.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Enzymatic activity of a recombinant β-1,4-endoglucanase from the Cotton Boll Weevil (Anthonomus grandis) aiming second generation ethanol production

Ibarra

Alves

Antonino

et al. 2019

Sci Rep

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“…When Af-EGL7 was expressed in E. coli , stability at these temperatures decreased rapidly, and residual activity was absent after 30 min at 60–90 °C [4]. Again, the better thermal stability observed in the present study was probably due to enzyme processing during its expression in P. pastoris , including post-translational modifications like glycosylation [15,16].…”

Section: Resultsmentioning

confidence: 76%

“…In our previous work, we expressed recombinant Af-EGL7 in E. coli and characterized its function [4]. Compared to the prokaryotic system, heterologous expression in the methylotrophic yeast P. pastoris offers many advantages, especially in terms of recombinant protein processing, folding, and post-translational modifications, which can influence enzyme functioning and even stability [15,16].…”

Section: Introductionmentioning

confidence: 99%

A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis

Bernardi

Yonamine

Uyemura

et al. 2019

IJMS

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In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-β-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters Km and Vmax were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0–8.0) and was highly thermostable at 55 °C for 72 h. Low Cu2+ concentrations (0.1–1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse “in natura”, sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.

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“…Otherwise, the activity is decreased at a neutral pH. Therefore, the pH shift to neutral pH or basic pH may result in the loss of hyaluronidase activity in wild-type rVesA2 [58]. However, the optimal temperature of the V. affinis venom hyaluronidase was lower than that of other venom hyaluronidases [1, 35].…”

Section: Discussionmentioning

confidence: 99%

Heterologous expression and mutagenesis of recombinant Vespa affinis hyaluronidase protein (rVesA2)

Rungsa

Janpan

Saengkun

et al. 2019

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: Crude venom of the banded tiger wasp Vespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors. Methods: The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed. Results: V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β) 7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4. Conclusion: The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.

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Heterologous expression and biochemical characterization of a novel thermostable Sclerotinia sclerotiorum GH45 endoglucanase in Pichia pastoris

Cited by 26 publications

References 50 publications

Enzymatic activity of a recombinant β-1,4-endoglucanase from the Cotton Boll Weevil (Anthonomus grandis) aiming second generation ethanol production

Enzymatic activity of a recombinant β-1,4-endoglucanase from the Cotton Boll Weevil (Anthonomus grandis) aiming second generation ethanol production

A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis

Heterologous expression and mutagenesis of recombinant Vespa affinis hyaluronidase protein (rVesA2)

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