Assembling a novel bifunctional cellulase–xylanase from Thermotoga maritima by end-to-end fusion

Hong, Su Young; Lee, Jin Suk; Cho, Kye Man; Math, Renukaradhya K.; Kim, Yong Hee; Hong, Sang Jeen; Cho, Yong Un; Kim, Hoon; Yun, Han Dae

doi:10.1007/s10529-006-9166-8

Cited by 63 publications

(37 citation statements)

References 15 publications

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“…They found that the chimeric enzyme exhibited both cellulase and xylanase activity when Cel5Z was fused downstream of XynX, but no activities were observed when Cel5Z was fused upstream of XynX. A similar result was reported by Hong et al (2006). However, the chimeric enzymes constructed in these two previous studies did not contain linker sequences between the catalytic domains.…”

Section: Hydrolytic Activity Toward Rice Strawsupporting

confidence: 60%

“…Chimeric enzymes are usually created by an end-to-end fusion technique, in which the N terminus of one catalytic domain is linked to the C terminus of the other catalytic domain (Khandeparker and Numan 2008). The sequential order of catalytic domains in chimeric enzymes may affect the enzyme specific activities Hong et al 2006). In addition, proper linker peptides between the individual catalytic domains are able to adopt the original conformation and maintain the functionality of the individual domains (Lu and Feng 2008).…”

Section: Design Of the Chimeric Xylanolytic Enzymesmentioning

confidence: 99%

“…Therefore, it is suggested that Xyn-S2-Axe has the potential to be used in a range of applications due to its efficient synergy in the degradation of xylan and its potential to save production costs. Fusion protein techniques are commonly used for purifying recombinant proteins, monitoring protein expression, displaying proteins on the cell surface, biological screening, targeting proteins, and so on (Hong et al 2006). Fusion proteins are usually created by an end-to-end fusion technique, in which the N terminus of one domain is linked to the C terminus of the other domain (Khandeparker and Numan 2008).…”

Section: Specific Activities Of the Parental And Chimeric Enzymes On mentioning

confidence: 99%

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The Construction of Bifunctional Fusion Xylanolytic Enzymes and the Prediction of Optimum Reaction Conditions for the Enzyme Activity

Pai¹,

Wang²,

Guo³

et al. 2012

BioResources

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Four chimeric xylanolytic enzymes were formed by fusion of a thermally stable xylanase XynCDBFV either to the N-terminus or C-terminus of a thermally stable acetylxylan esterase AxeS20E, with or without a Gly-rich flexible linker (S2). The three-dimensional (3D) structures of the chimeric enzymes were predicted using the I-TASSER server, and the results indicated that the structures of Axe-S2-Xyn and Xyn-S2-Axe were more similar to the native structures than were those of Axe-Xyn and Xyn-Axe. Axe-S2-Xyn and Xyn-S2-Axe were expressed in Escherichia coli and purified by means of affinity chromatography. Response surface modeling (RSM), combined with central composite design (CCD) and regression analysis, was then employed to optimize the xylanase activities of the chimeric enzymes. Under the optimal conditions, Xyn-S2-Axe had greater hydrolytic activities on natural xylans and rice straw than did the parental enzymes. These results suggested that the chimeric enzyme Xyn-S2-Axe could be effective at hydrolyzing xylan in biomass and that it has potential to be used in a range of biotechnological applications.

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Section: Hydrolytic Activity Toward Rice Strawsupporting

confidence: 60%

Section: Design Of the Chimeric Xylanolytic Enzymesmentioning

confidence: 99%

Section: Specific Activities Of the Parental And Chimeric Enzymes On mentioning

confidence: 99%

See 1 more Smart Citation

The Construction of Bifunctional Fusion Xylanolytic Enzymes and the Prediction of Optimum Reaction Conditions for the Enzyme Activity

Pai¹,

Wang²,

Guo³

et al. 2012

BioResources

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“…Single polypeptides carrying more than one cellulolytic activity that have either been isolated from nature (12,29) or constructed in the laboratory (2,11,18,19,23,24) would certainly have advantages as candidate enzymes in the cellulase consortium.…”

mentioning

confidence: 99%

Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates

Adlakha

Sawant

Anil

et al. 2012

Appl Environ Microbiol

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bIdentification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel ␤-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a ␤-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G 4 S) 3 -Gluc1C, demonstrated 3.2-and 2-fold higher molar specific activities for ␤-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.

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“…We and others have demonstrated that the creation of artificial, multifunctional, lignocellulosic hydrolases is a realistic and practical approach for the improvement of biomass conversion (5,8,9,12). These novel enzymes can be used alone, when a 1:1:1 stoichiometry is preferred, or in mixtures with small quantities of other enzymes to create an optimal enzyme cocktail when enzyme ratio FIG.…”

mentioning

confidence: 99%

Multimeric Hemicellulases Facilitate Biomass Conversion

Fan

Wagschal

Chen

et al. 2009

Appl Environ Microbiol

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Assembling a novel bifunctional cellulase–xylanase from Thermotoga maritima by end-to-end fusion

Cited by 63 publications

References 15 publications

The Construction of Bifunctional Fusion Xylanolytic Enzymes and the Prediction of Optimum Reaction Conditions for the Enzyme Activity

The Construction of Bifunctional Fusion Xylanolytic Enzymes and the Prediction of Optimum Reaction Conditions for the Enzyme Activity

Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates

Multimeric Hemicellulases Facilitate Biomass Conversion

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