Identification and molecular modeling of a family 5 endocellulase from Thermus caldophilus GK24, a cellulolytic strain of Thermus thermophilus

Kim, Doo-Il; Park, Bo Hyun; Jung, Bo-Won; Kim, Mi-Kyung; Hong, Seok-In; Lee, Dae-Sil

doi:10.3390/i7120571

Cited by 11 publications

(3 citation statements)

References 56 publications

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“…To the best of our knowledge, hydrogen bonds are also crucial in substrate recognition and binding [ 38 , 39 ]. Therefore, we also investigated the hydrogen bonds between the enzyme and substrate.…”

Section: Discussionmentioning

confidence: 99%

Enhancing the catalytic activity of a novel GH5 cellulase GtCel5 from Gloeophyllum trabeum CBS 900.73 by site-directed mutagenesis on loop 6

Zheng

Wang

et al. 2018

Biotechnol Biofuels

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BackgroundCellulases of glycosyl hydrolase (GH) family 5 share a (β/α)8 TIM-barrel fold structure with eight βα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions. However, the role of these flexible loops in catalysis of GH5 cellulase remains to be explored.ResultsIn the present study, an acidic, mesophilic GH5 cellulase (with optimal activity at pH 4.0 and 70 °C), GtCel5, was identified in Gloeophyllum trabeum CBS 900.73. The specific activities of GtCel5 toward CMC-Na, barley β-glucan, and lichenan were 1117 ± 43, 6257 ± 26 and 5318 ± 54 U/mg, respectively. Multiple sequence alignment indicates that one amino acid residue at position 233 on the loop 6 shows semi-conservativeness and might contribute to the great catalytic performance. Saturation mutagenesis at position 233 was then conducted to reveal the vital roles of this position in enzyme properties. In comparison to the wild type, variants N233A and N233G showed decreased optimal temperature (− 10 °C) but increased activities (27 and 70%) and catalytic efficiencies (kcat/Km; 45 and 52%), respectively. The similar roles of position 233 in catalytic performance were also verified in the other two GH5 homologs, TeEgl5A and PoCel5, by reverse mutation. Further molecular dynamics simulations suggested that the substitution of asparagine with alanine or glycine may introduce more hydrogen bonds, increase the flexibility of loop 6, enhance the interactions between enzyme and substrate, and thus improve the substrate affinity and catalytic efficiency.ConclusionThis study proposed a novel cellulase with potentials for industrial application. A specific position was identified to play key roles in cellulase–substrate interactions and enzyme catalysis. It is of great importance for understanding the binding mechanism of GH5 cellulases, and provides an effective strategy to improve the catalytic performance of cellulases.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1080-5) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Enhancing the catalytic activity of a novel GH5 cellulase GtCel5 from Gloeophyllum trabeum CBS 900.73 by site-directed mutagenesis on loop 6

Zheng

Wang

et al. 2018

Biotechnol Biofuels

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“…The isolation of fungi was carried out using serial dilution method (Reddy et al 2014). The primary screening was performed on the basis of the zone of cellulose hydrolysis (Kim et al 2006), while secondary screening was performed via submerged fermentation. The strain showed highest cellulolytic potential was identified both morphologically and molecularly.…”

Section: Isolation Of Cellulolytic Fungimentioning

confidence: 99%

Process optimization for enhanced production of cellulases form locally isolated fungal strain by submerged fermentation

et al. 2021

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Cellulase has myriad applications in various sectors like pharmaceuticals, textile, detergents, animal feed and bioethanol production, etc. The current study focuses on the isolation, screening and optimization of fungal strain through one factor at a time technique for enhanced cellulase production. In current study sixteen different fungal cultures were isolated and the culture which quantitatively exhibits higher titers of cellulase activity was identified both morphologically and molecularly by 18S rDNA and designated as Aspergillus niger ABT11. Different parameters like fermentation medium, volume, temperature, pH and nutritional components were optimized. The highest CMCase and FPase activities was achieved in 100ml of M5 medium in the presence of 1% lactose and sodium nitrate at 30 oC, pH5 after 72 hours. The result revealed A. niger can be a potential candidate for scale up studies.

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“…Although Thermus spp. produce different hydrolytic enzymes like proteases or lipases, only few glycoside hydrolases have been characterized so far (Dion et al, 1999 ; Fridjonsson et al, 1999 ; Pantazaki et al, 2002 ; Kim et al, 2006 ; Nam et al, 2010 ; Blank et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with Î²-Glucosidase Activity

Schröder

Blank

Antranikian

2015

Front. Bioeng. Biotechnol.

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Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2.

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Identification and molecular modeling of a family 5 endocellulase from Thermus caldophilus GK24, a cellulolytic strain of Thermus thermophilus

Cited by 11 publications

References 56 publications

Enhancing the catalytic activity of a novel GH5 cellulase GtCel5 from Gloeophyllum trabeum CBS 900.73 by site-directed mutagenesis on loop 6

Enhancing the catalytic activity of a novel GH5 cellulase GtCel5 from Gloeophyllum trabeum CBS 900.73 by site-directed mutagenesis on loop 6

Process optimization for enhanced production of cellulases form locally isolated fungal strain by submerged fermentation

First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with Î²-Glucosidase Activity

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