Characterization of a novel manganese dependent endoglucanase belongs in GH family 5 from Phanerochaete chrysosporium

Huy, Nguyễn Đức; Nguyen, Cu Le; Park, Han-Sung; Lộc, Nguyễn Hoàng; Choi, Myoung-Suk; Kim, Dae-Hyuk; Seo, Jeong-Woo; Park, Seung-Moon

doi:10.1016/j.jbiosc.2015.06.009

Cited by 13 publications

(5 citation statements)

References 23 publications

(30 reference statements)

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“…The results of the reducing sugar content highlighted the potential of switchgrass as a raw material for industrial ethanol. As Cel-5A is an endo-type enzyme, it can cleave cellulose chains of internal β-1,4-glucan linkages, thus converting biomass into shorter cellulose chains (Huy et al 2016).…”

Section: Discussionmentioning

confidence: 99%

Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds

Rakhmanova

Wang

et al. 2020

AMB Expr

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Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). The CEL-5A gene was sequenced with a full-length of 1500 bp, encoding a totally of 500 amino acids, and containing two domains: the GH5 family catalytic domain (CD) and the CBM3 family cellulose-binding domain (CBD). Recombinant endocellulase Cel-5A with a His-tag was purified of the Ni-NTA column, and SDS-PAGE results demonstrated that Cel-5A exhibited a molecular weight of 56.4 kDa. The maximum enzyme activity of Cel-5A was observed at pH 4.5 and 50 °C. Moreover, it was active over the broad temperature region of 30-60 °C, and stable within the pH range of 4.5-10.0. In addition, Co 2+ was able to increase enzyme activity, while the majority of metal ions demonstrated stable enzyme activity under low-concentration. The substrate specificity of Cel-5A exhibited a high specific activity on the β-1,3-1,4 glucan linkage from barley. The Michaelis-Menten constant and the maximum velocity of the recombinant Cel-5A for CMC-Na were determined as 14.87 mg/mL and 19.19 μmol/min/mg, respectively. When Cel-5A was applied to the switchgrass and coffee grounds, its color became lighter and the biomass was observed to loosen following hydrolyzation. The saccharification rate reached 12% of the total weight of switchgrass in 20 h. These properties highlight the potential application of Cel-5A as an endocellulase in the pretreatment of biomass, for example, in the coffee grounds/waste, and related industries.

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Section: Discussionmentioning

confidence: 99%

Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds

Rakhmanova

Wang

et al. 2020

AMB Expr

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“…These properties indicated that thermostable cellobiohydrolase CtCel6 has practical value and great commercial application potential. Generally, in most operational situations, the optimal temperature of enzymatic action is 40-60 o C, it is considered that high temperature reaction activity and thermostable enzyme is favorable (Aditya et al, 2014;Huy et al, 2016). In addition, the purified recombinant CtCel6 exhibited excellent activity in a range of pH 5 to pH 9 with the maximum hydrolysis activity at pH 5 as showen in Fig.…”

Section: Discussionmentioning

confidence: 90%

“…Generally, C. thermophilum glycoside hydrolases are thermostable and have a high optimal reaction temperature based on the previous researches. Thermostable enzymes have potential advantages in lignocelluloses conversation, on account of effectively improving hydrolysis efficiency and reducing the possible contamination at high temperature in industrial processes (Huy et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

A Novel Application Potential of GH6 Cellobiohydrolase CtCel6 from Thermophilic Chaetomium thermophilum for Gene Cloning, Heterologous Expression and Biological Characterization

Zhou¹,

Jia²,

Ji³

et al. 2017

IJAB

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Chaetomium thermophilum is a thermophilic fungus expressed a series of glycoside hydrolases. Genome sequence analysis of C. thermophilium revealed that ctcel6 gene encoded a putative cellobiohydrolase which composed of 397 amino acid residues including a predicted signal peptide sequence. Ctcel6 gene was cloned, heterologously expressed in Pichia pastoris and purified by Ni 2+ affinity chromatography. Sequence alignment indicated that CtCel6 enzyme belonged to glycoside hydrolase family 6 (GH6) and the molecular mass of purified recombinant enzyme CtCel6 was 42 kDa by SDS-PAGE analysis. Characterization of recombinant CtCel6 exhibited high hydrolysis activity and excellent thermostability. The optimum reaction temperature and pH was 70 o C and pH 5, respectively. The bivalent metallic cations Mg 2+ and Ca 2+ significantly enhanced the activity of CtCel6. The specific activity of CtCel6 enzyme was 1.27 U/mg and Km value was 0.38 mM on β-Dglucan. The substrate specificity and hydrolysis products insisted that CtCel6 was an exo-/endo-type cellobiohydrolase. The biochemical properties of recombinant CtCel6 made it potentially effective for bioconversion of biomass and had tremendous potential in industrial applications such as enzyme preparation industry and feed processing industry.

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“…This is evident in the GH3 domain family, where four GH3 enzymes from Cellulomonas fimi showed a range of activities on many xylo-and oligosaccharides (Gao and Wakarchuk 2014) even though no other catalytic domains were apparent. The GH5 domain family also displays considerable functional diversity, with recent studies highlighting specificities for substrates such as cellulose (Huy et al 2016;Valadares et al 2016;Wang et al 2016b), xylans andxyloglucans (dos Santos et al 2015;Ghatge et al 2014) mannans (Tóth et al 2016;Zang et al 2015) and glycoceramides (Han et al 2017b). Occasionally, new CAZyme domain families or rare variants of existing families are also discovered, such as a xylan degrading (Corrêa et al 2012) or multifunctional (Morrison et al 2016) GH39 enzyme, or the recently defined GH116 family, which was shown to act on glucosylceramide, N-acetylglucosaminides and xylosides (Cobucci-Ponzano et al 2010;Ferrara et al 2014).…”

Section: Cazyme Categoriesmentioning

confidence: 99%

Carbohydrate active enzyme domains from extreme thermophiles: components of a modular toolbox for lignocellulose degradation

et al. 2017

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Lignocellulosic biomass is a promising feedstock for the manufacture of biodegradable and renewable bioproducts. However, the complex lignocellulosic polymeric structure of woody tissue is difficult to access without extensive industrial pre-treatment. Enzyme processing of partly depolymerised biomass is an established technology, and there is evidence that high temperature (extremely thermophilic) lignocellulose degrading enzymes [carbohydrate active enzymes (CAZymes)] may enhance processing efficiency. However, wild-type thermophilic CAZymes will not necessarily be functionally optimal under industrial pre-treatment conditions. With recent advances in synthetic biology, it is now potentially possible to build CAZyme constructs from individual protein domains, tailored to the conditions of specific industrial processes. In this review, we identify a 'toolbox' of thermostable CAZyme domains from extremely thermophilic organisms and highlight recent advances in CAZyme engineering which will allow for the rational design of CAZymes tailored to specific aspects of lignocellulose digestion.

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Characterization of a novel manganese dependent endoglucanase belongs in GH family 5 from Phanerochaete chrysosporium

Cited by 13 publications

References 23 publications

Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds

Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds

A Novel Application Potential of GH6 Cellobiohydrolase CtCel6 from Thermophilic Chaetomium thermophilum for Gene Cloning, Heterologous Expression and Biological Characterization

Carbohydrate active enzyme domains from extreme thermophiles: components of a modular toolbox for lignocellulose degradation

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