Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach
“…In another study, Akbarzadeh and colleagues presented a recombinant endoglucanase II from Trichoderma reesei expressed in P. pastoris with reduced disulfide bonds. The recombinant enzyme showed higher activity and thermostability than the native enzyme 47 . Such variations could even better adjust the recombinant enzyme for industrial physicochemical conditions for plant biomass conversion.Figure 4SDS-PAGE ( A ) and Western Blotting ( B ) of AgraGH45-1 expression and purification.…”
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.
“…In another study, Akbarzadeh and colleagues presented a recombinant endoglucanase II from Trichoderma reesei expressed in P. pastoris with reduced disulfide bonds. The recombinant enzyme showed higher activity and thermostability than the native enzyme 47 . Such variations could even better adjust the recombinant enzyme for industrial physicochemical conditions for plant biomass conversion.Figure 4SDS-PAGE ( A ) and Western Blotting ( B ) of AgraGH45-1 expression and purification.…”
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.
“…39 In similar cases, stabilization of the C-or N-terminal area of a GH led to an increase in thermal stability. 22,90,91 A common postulate is that there is a trade-off between activity and thermal stability in enzymes, 92 i.e., that thermodynamic stabilization produced by a reduction in the free energy (ΔG) of an enzyme leads to a decrease in enzyme flexibility. Since flexibility is needed for enzymatic activity, an increase in stability produces a reduction in the enzymatic activity at lower temperatures.…”
Understanding
the thermostability of cellulases is of high importance
for their application in lignocellulosic biomass degradation,
feedstock, and pulp and paper production. Cellulases have to withstand
high temperatures and harsh conditions in various application areas,
for instance, in bioethanol production. Engineering thermostable
cellulases increases the cellulase lifetime in processes and contributes
to more-sustainable production. Here we report the first KnowVolution
campaign toward improving the thermostability of the endo-β-1,4-glucanase PvCel5A from Penicillium
verruculosum. The C-terminal region of PvCel5A (eighth
α-helix, amino acid residues 280–314) was identified
as a key structural determinant to improve the thermostability
of PvCel5A without affecting its specific activity. The most beneficial
variant, PvCel5A-R17, harbors three substitutions (F16L/Y293F/Q289G);
its half-life at 75 °C improved 5.5-fold (from 32 to 175 min)
and the melting temperature was raised 7.7 °C (from 70.8 °C)
when compared to those of wild-type PvCel5A. Exceptionally, the thermally
improved PvCel5A-R17 variant retained its specific activity at low
temperatures (40 °C). Computational analyses revealed that the
stabilization of the C-terminal region of PvCel5A is responsible for
the improved thermostability. This knowledge will facilitate
shorter times in cellulase engineering and thereby enhance the performance
and sustainability of processes.
“…Akbarzadeh et al used site-directed mutagenesis to eliminate two disulfide bonds of Cel5A of T. reesei in order to assess if their absence correlated with increased thermostability, as expected by structural comparison with thermophilic Cel5A from Thermoascus aurantiacus . When compared to the parental Cel5A, the thermal stability of variant C99V increased 2.4-fold at 80 °C, 2.01-fold at 70 °C, and 1.8-fold at 60 °C, while, for variant C323H, it increased 2.34-fold, 1.81-fold, and 1.6-fold at 80 °C, 70 °C, and 60 °C, respectively [62]. Trudeau et al used site-directed mutagenesis for improving the thermostability of H. jecorina Cel5A by the combination of 16 previous mutations that proved to thermostabilize Cel5A.…”
Protein engineering emerged as a powerful approach to generate more robust and efficient biocatalysts for bio-based economy applications, an alternative to ecologically toxic chemistries that rely on petroleum. On the quest for environmentally friendly technologies, sustainable and low-cost resources such as lignocellulosic plant-derived biomass are being used for the production of biofuels and fine chemicals. Since most of the enzymes used in the biorefinery industry act in suboptimal conditions, modification of their catalytic properties through protein rational design and in vitro evolution techniques allows the improvement of enzymatic parameters such as specificity, activity, efficiency, secretability, and stability, leading to better yields in the production lines. This review focuses on the current application of protein engineering techniques for improving the catalytic performance of enzymes used to break down lignocellulosic polymers. We discuss the use of both classical and modern methods reported in the literature in the last five years that allowed the boosting of biocatalysts for biomass degradation.
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