Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli

Nakazawa, Hikaru; Okada, Katsunori; Kobayashi, Ryota; Kubota, Tetsuya; Onodera, Tomoko; Ochiai, Nobuhiro; Omata, Naoki; Ogasawara, Wataru; Okada, Hirofumi; Morikawa, Yasushi

doi:10.1007/s00253-008-1667-z

Cited by 82 publications

(58 citation statements)

References 32 publications

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“…For example, the GH3 CdxA (cellodextrinase A) from Prevotella bryantii B 1 4 is highly active on cellohexaose, but it can also degrade xylohexaose to some extent (32). The GH7 endoglucanase I of Trichoderma reesei can degrade birch wood xylan, in addition to cellulosic substrates (33). Substrate promiscuity is rarely observed for xylanases, particularly for crystalline cellulose.…”

Section: Discussionmentioning

confidence: 99%

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

Xue

Wang

et al. 2015

Appl Environ Microbiol

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bThe genome of the thermophilic bacterium Caldicellulosiruptor bescii encodes three multimodular enzymes with identical Cterminal domain organizations containing two consecutive CBM3b modules and one glycoside hydrolase (GH) family 48 (GH48) catalytic module. However, the three proteins differ much in their N termini. Among these proteins, CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) with a GH9/CBM3c binary partner in the N terminus has been shown to use a novel strategy to degrade crystalline cellulose, which leads to its outstanding cellulose-cleaving activity. Here we show that C. bescii Xyn10C (CbXyn10C), the N-terminal GH10 domain from CbXyn10C/Cel48B, can also degrade crystalline cellulose, in addition to heterogeneous xylans and barley ␤-glucan. The data from substrate competition assays, mutational studies, molecular modeling, and docking point analyses point to the existence of only one catalytic center in the bifunctional xylanase/␤-glucanase. The specific activities of the recombinant CbXyn10C on Avicel and filter paper were comparable to those of GH9/CBM3c of the robust CelA expressed in Escherichia coli. Appending one or two cellulose-binding CBM3bs enhanced the activities of CbXyn10C in degrading crystalline celluloses, which were again comparable to those of the GH9/CBM3c-CBM3b-CBM3b truncation mutant of CelA. Since CbXyn10C/Cel48B and CelA have similar domain organizations and high sequence homology, the endocellulase activity observed in CbXyn10C leads us to speculate that CbXyn10C/Cel48B may use the same strategy that CelA uses to hydrolyze crystalline cellulose, thus helping the excellent crystalline cellulose degrader C. bescii acquire energy from the environment. In addition, we also demonstrate that CbXyn10C may be an interesting candidate enzyme for biotechnology due to its versatility in hydrolyzing multiple substrates with different glycosidic linkages. P lant cell wall polysaccharides (PCWPs), composed mainly of cellulose and hemicellulose, are a promising rich resource for renewable biofuel development (1). The complete deconstruction of PCWPs into fermentable, simple mono-or oligosaccharides requires the concerted action of a complex array of glycoside hydrolases (GHs), including cellulases and hemicellulases (2, 3). The genomes of Gram-positive bacteria of the genus Caldicellulosiruptor encode an arsenal of thermophilic plant cell wall polysaccharide-degrading enzymes (3-5), which are appealing candidates in the design of novel, robust enzyme cocktails for PCWP deconstruction.In the genome of Caldicellulosiruptor bescii, there is a gene cluster containing three genes which encode proteins harboring two tandemly linked CBM3b modules and a GH family 48 (GH48) cellobiohydrolase in the C terminus. These CBM3b and GH48 modules, as well as their linker sequences, are extremely similar at the level of the amino acid sequence (see Fig. S1 in the supplemental material). Notably, however, the three multimodular enzymes differ much in their N termini: CelA (or C. bescii Cel9A [CbCel9A]/Cel48A) ha...

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Section: Discussionmentioning

confidence: 99%

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

Xue

Wang

et al. 2015

Appl Environ Microbiol

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“…Additionally, the structure reveals four disulfide bonds, in direct contrast with a previous report suggesting the absence of such elements. 21 While an attempt to engineer Hj_Cel5A for optimum catalytic efficiency at a particular pH has met with some success, this effort relied on a highly inaccurate homology model built from Ta_Cel5A coordinates. 22 The information presented here may better inform future efforts to rationally engineer Hj_Cel5A for various needs, as well as understand the wildtype activity of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…The deeper cleft contains a hydrophobic patch (F14, V27, Y28, Y40, F34, W292, A294, F297, Y301) surrounded by the b1-a1 loop (residues [15][16][17][18][19][20][21][22], the sidechain of W185, residues 104-107, residues 146-150, and the b6-a6 loop (residues 225-229) [ Fig. 1(C)].…”

Section: Active Site Architecturementioning

confidence: 99%

A structural study of Hypocrea jecorina Cel5A

et al. 2011

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Interest in generating lignocellulosic biofuels through enzymatic hydrolysis continues to rise as nonrenewable fossil fuels are depleted. The high cost of producing cellulases, hydrolytic enzymes that cleave cellulose into fermentable sugars, currently hinders economically viable biofuel production. Here, we report the crystal structure of a prevalent endoglucanase in the biofuels industry, Cel5A from the filamentous fungus Hypocrea jecorina. The structure reveals a general fold resembling that of the closest homolog with a high-resolution structure, Cel5A from Thermoascus aurantiacus. Consistent with previously described endoglucanase structures, the H. jecorina Cel5A active site contains a primarily hydrophobic substrate binding groove and a series of hydrogen bond networks surrounding two catalytic glutamates. The reported structure, however, demonstrates stark differences between side-chain identity, loop regions, and the number of disulfides. Such structural information may aid efforts to improve the stability of this protein for industrial use while maintaining enzymatic activity through revealing nonessential and immutable regions.

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“…Cellulose is a polymer of glucose linked by β-1,4 glucosidase bond with a crystalline structure that is stabilized by intermolecular and intramolecular hydrogen bonds [9]. Cellulase enzymes can degrade the crystalline structure into glucose that can be used as raw material for bioethanol.…”

Section: Introductionmentioning

confidence: 99%

Isolation, Cellulase Activity Test and Molecular Identification of Selected Cellulolytic Bacteria Indigenous Rice Bran

et al. 2018

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Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli

Cited by 82 publications

References 32 publications

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

The N-Terminal GH10 Domain of a Multimodular Protein from Caldicellulosiruptor bescii Is a Versatile Xylanase/β-Glucanase That Can Degrade Crystalline Cellulose

A structural study of Hypocrea jecorina Cel5A

Isolation, Cellulase Activity Test and Molecular Identification of Selected Cellulolytic Bacteria Indigenous Rice Bran

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