Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastoris

Generoso, Wesley Cardoso; Malagó-Jr, Wilson; Pereira, Nei; Henrique-Silva, Flávio

doi:10.4238/2012.may.21.11

Cited by 22 publications

(9 citation statements)

References 33 publications

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“…It is likely that Tyr7 would hold somewhat weaker interactions with the substrate due to the smaller hydrophobic contact area relative to Trp7. This is consistent with the recently reported Michaelis-Menten kinetics which yield K M ∼21.4 g/L for ThEG3 [47], suggesting that the substrate binding affinity to ThEG3 is roughly 14 times smaller than to TrEG3 ( K M ∼1.5 g/L) [48].…”

Section: Resultssupporting

confidence: 92%

X-ray Structure and Molecular Dynamics Simulations of Endoglucanase 3 from Trichoderma harzianum: Structural Organization and Substrate Recognition by Endoglucanases That Lack Cellulose Binding Module

et al. 2013

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Plant biomass holds a promise for the production of second-generation ethanol via enzymatic hydrolysis, but its utilization as a biofuel resource is currently limited to a large extent by the cost and low efficiency of the cellulolytic enzymes. Considerable efforts have been dedicated to elucidate the mechanisms of the enzymatic process. It is well known that most cellulases possess a catalytic core domain and a carbohydrate binding module (CBM), without which the enzymatic activity can be drastically reduced. However, Cel12A members of the glycosyl hydrolases family 12 (GHF12) do not bear a CBM and yet are able to hydrolyze amorphous cellulose quite efficiently. Here, we use X-ray crystallography and molecular dynamics simulations to unravel the molecular basis underlying the catalytic capability of endoglucanase 3 from Trichoderma harzianum (ThEG3), a member of the GHF12 enzymes that lacks a CBM. A comparative analysis with the Cellulomonas fimi CBM identifies important residues mediating interactions of EG3s with amorphous regions of the cellulose. For instance, three aromatic residues constitute a harboring wall of hydrophobic contacts with the substrate in both ThEG3 and CfCBM structures. Moreover, residues at the entrance of the active site cleft of ThEG3 are identified, which might hydrogen bond to the substrate. We advocate that the ThEG3 residues Asn152 and Glu201 interact with the substrate similarly to the corresponding CfCBM residues Asn81 and Arg75. Altogether, these results show that CBM motifs are incorporated within the ThEG3 catalytic domain and suggest that the enzymatic efficiency is associated with the length and position of the substrate chain, being higher when the substrate interact with the aromatic residues at the entrance of the cleft and the catalytic triad. Our results provide guidelines for rational protein engineering aiming to improve interactions of GHF12 enzymes with cellulosic substrates.

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Section: Resultssupporting

confidence: 92%

X-ray Structure and Molecular Dynamics Simulations of Endoglucanase 3 from Trichoderma harzianum: Structural Organization and Substrate Recognition by Endoglucanases That Lack Cellulose Binding Module

et al. 2013

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“…However, the specific activities of NfEG12A against barley ␤-glucan, lichenin, and CMC-Na (7,857 U/mg, 6,857 U/mg, and 2,210 U/mg, respectively) are dramatically higher than those of other known GH12 endoglucanases, including BG from Aspergillus japonicus (11), Cel12A from Chrysosporium lucknowense (12), Cel12A from Stachybotrys atra (13), EGII from Fomitopsis palustris (14), and Cel12A from T. reesei (15). Besides, NfEG12A also showed higher substrate affinity for lichenin and CMC-Na (K m values, 1.46 mg/ml and 6.54 mg/ml, respectively) than Cel12A from Trichoderma harzianum (16) and AtEglD from Aspergillus terreus (17) but similar to that of Cel12A from T. reesei (15). Furthermore, the catalytic efficiencies of NfEG12A to lichenin and CMC-Na were 3,001 ml/mg/s and 263 ml/mg/s, respectively, which are higher than those of the GH12 endoglucanases from A. terreus, T. reesei, T. harzianum, and A. niger HO (9).…”

Section: Discussionmentioning

confidence: 98%

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

Yang

Shi

Liu

et al. 2017

Appl Environ Microbiol

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Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using NfEG12A from Neosartorya fischeri P1 and EG (PDB 1KS4) from Aspergillus niger. Acidophilic and thermophilic NfEG12A had the highest catalytic efficiency (k cat /K m , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into NfEG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (k cat /K m ) of these enzymes (in descending order, NfEG12A ). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (k cat ). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.IMPORTANCE Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the Ϫ1 or ϩ1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, NfEG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (k cat ). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.KEYWORDS Neosartorya fischeri, GH12 ␤-endoglucanase, loop, catalytic efficiency, hydrogen bond I n recent decades, bioethanol as a clean energy source has become a hot spot of research and industrial application (1). The bioconversion processes of producing fermentable sugars from biomass need a few glycoside hydrolases (GH), especially the

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“…The weaker signal in the xylan zymogram indicates the presence of a ~20 kDa xylanolytic activity which is compatible with other studies of this species that describe xylanase activities around this molecular mass with the lack of cellulase activity (Rezende et al 2002; Lee et al 2009; do Vale et al 2012). The L04 cellulase activity around 20 kDa could correspond to the endoglucanase (EGIII) from T. harzianum IOC3844 characterized by Generoso et al (2012) described with a low molecular mass, lack the cellulose binding domain (CBD) and able to degrade amorphous cellulose such as CMC.…”

Section: Discussionmentioning

confidence: 99%

A novel promising Trichoderma harzianum strain for the production of a cellulolytic complex using sugarcane bagasse in natura

2013

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Brazil is a major producer of agro-industrial residues, such as sugarcane bagasse, which could be used as raw material for microbial production of cellulases as an important strategy for the development of sustainable processes of second generation ethanol production. For this purpose, this work aimed at screening for glycosyl hydrolase activities of fungal strains isolated from the Brazilian Cerrado. Among 13 isolates, a Trichoderma harzianum strain (L04) was identified as a promising candidate for cellulase production when cultured on in natura sugarcane bagasse. Strain L04 revealed a well-balanced cellulolytic complex, presenting fast kinetic production of endoglucanases, exoglucanases and β-glucosidases, achieving 4,022, U.L-1 (72 h), 1,228 U.L-1 (120 h) and 1,968 U.L-1 (48 h) as the highest activities, respectively. About 60% glucose yields were obtained from sugarcane bagasse after 18 hours hydrolysis. This new strain represents a potential candidate for on-site enzyme production using sugarcane bagasse as carbon source.

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Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastoris

Cited by 22 publications

References 33 publications

X-ray Structure and Molecular Dynamics Simulations of Endoglucanase 3 from Trichoderma harzianum: Structural Organization and Substrate Recognition by Endoglucanases That Lack Cellulose Binding Module

X-ray Structure and Molecular Dynamics Simulations of Endoglucanase 3 from Trichoderma harzianum: Structural Organization and Substrate Recognition by Endoglucanases That Lack Cellulose Binding Module

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

A novel promising Trichoderma harzianum strain for the production of a cellulolytic complex using sugarcane bagasse in natura

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