Construction of individual, fused, and co-expressed proteins of endoglucanase and β-glucosidase for hydrolyzing sugarcane bagasse

Kurniasih, Sari Dewi; Alfi, Almasul; Natalia, Dessy; Radjasa, Ocky Karna; Nurachman, Zeily

doi:10.1016/j.micres.2014.02.002

Cited by 12 publications

(6 citation statements)

References 28 publications

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“…and Co-expression of endoglucanase and beta-glucosidase were successfully carried out in E. coli [36]. Several other reports indicating fusion and co-expression of proteins belong to endoglucanase and β-glucosidase for hydrolyzing sugarcane bagasse [38]. Thus chitinase and protease can be successfully fused and expressed in E.coli without changing their catalytic activities, properties, and kinetics.…”

Section: Plos Onementioning

confidence: 99%

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Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein

et al. 2022

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The tissue of insects, pests, and fungi has a chitin layer followed by protein in the cell membrane. The complete biodegradation of chitin and protein-present in the waste requires the action of two enzymes, namely chitinase, and protease. Combining chitinase and protease in a single protein/enzyme will serve as a bifunctional enzyme that can efficiently degrade the chitin and protein-rich biomass. The present study was aimed to fuse these two enzymes to produce a single protein and study the kinetics of the recombinant fusion protein. A chitinase and alkaline protease genes were isolated, cloned, and expressed successfully as a fusion product in heterologous host Escherichia coli. The two native genes were successfully fused in E.coli by using flexible glycine–serine (G4S)2 linker (GGGGS, GS linker). The recombinant fusion protein in E.coli showed hydrolyzed chitin and protein on chitin and bovine serum albumin agar plates confirming the successful cloning and expression of chitinase and protease enzymes in a single fusion protein. The common pUC18-T7 mini vector with the ompA signal sequence helps the extracellular expression of fusion protein efficiently. The native gel electrophoresis revealed a molecular mass of purified protein as 92.0 kDa. The fusion protein’s maximal chitinase and protease activity occurred at pH 5.0 and 8.0 and 30 0C, respectively resembling the individual enzymes’. In the kinetic studies of the fusion protein, it was observed that the presence of metal ions such as Cu2+, Na2+, and Ca2+; significantly enhanced the enzyme activities while organic solvents oxidants and chemicals have drastically affected the activities of both the enzymes in the fusion protein. No such fusion protein has been produced in a heterologous host yet. The reports on fusion protein with biomass-degrading capacity are also scarce. This is probably the first report of a bifunctional chitinase/protease expressed in E. coli.

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Section: Plos Onementioning

confidence: 99%

“…The co-expression strategies have the limitation of low transformation efficiency and inconvenient screening during co-expression. Constructing a bifunctional/trifunctional gene could be an effective approach [34][35][36][37][38][39].…”

Section: Introductionmentioning

confidence: 99%

Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein

et al. 2022

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“…To avoid this problem, constructing a trifunctional cellulase with a single gene could be an effective solution. Many successful studies have been performed on developing the bifunctional cellulase with fusion expression of two different catalytic domains, including endoglucanase catalytic domain fused with β-glucosidase catalytic domain [ 14 , 22 ], exoglucanase catalytic domain fused with β-glucosidase catalytic domain [ 15 ], exoglucanase catalytic domain fused with endoglucanase catalytic domain [ 25 ]. However, it has not been reported of an active trifunctional cellulase with three different catalytic domains.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, fusion expression strategy, a biotechnology alternative to co-expression, have also been used to express multiple enzymes simultaneously with many advantages including the convenient manipulations of cloning and transforming, the high level of soluble expression, cost-effective purification, and upgraded catalytic capabilities [ 21 ]. In addition, it has been confirmed that all individual, fused, and co-expressed endoglucanases and β-glucosidases play a role in hydrolyzing sugarcane bagasse [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Construction of a trifunctional cellulase and expression in Saccharomyces cerevisiae using a fusion protein

Liu

Song

et al. 2018

BMC Biotechnol

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BackgroundCellulose is the most important component of lignocellulose, and its degradation requires three different types of enzymes to act synergistically. There have been reports of single gene duality, but no gene has been described to have more than two functions. Cloning and expression of fusion cellulases containing more than two kinds of catalytic domains has not been reported thus far.ResultsWe synthesized three different cellulase genes and linked the three catalytic domains with a (G4S)3 flexible linker. The trifunctional cellulase gene (BCE) containing three types of cellulase functions was constructed and expressed in S. cerevisiae successfully. The β-glucosidase, the exoglucanase and the endoglucanase activity of the trifunctional cellulase BCE reached 16.80 IU/mg, 2.26 IU/mg and 20.67 IU/mg, which was 46.27, 6.73 and 46.20% higher than the activities of the β-glucosidase BG, the endoglucanase CBH and the endoglucanase EG. The filter paper enzyme activity of BCE was higher than those of BG, CBH and EG, reached 2.04 IU/mg.ConclusionsThe trifunctional cellulase BCE was designed based on β-glucosidase BG, endoglucanase EG and exoglucanase CBH, and it possessed β-glucosidase activity, endoglucanase activity and exoglucanase activity simultaneously. The BCE has better filter paper activity, it means the potential practical application.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0454-x) contains supplementary material, which is available to authorized users.

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“…In addition, in order to obtain a cellulase consortium containing different cellulolytic activities, fusion expression has been applied for the synthesis of bifunctional enzymes that enable cellulose hydrolysis [ 20 , 21 ]. It has been proven that individually expressed, fused, and co-expressed endoglucanases and β-glucosidases can promote the hydrolysis of sugarcane bagasse [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Expression of β-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach

Yang

Sunkang

et al. 2023

Microorganisms

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β-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency β-glucosidase, in this study two subunits (bglA and bglB) of β-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria (Lactobacillus lactis NZ9000). The engineered strains L. lactis NZ9000/pMG36e-usp45-bglA, L. lactis NZ9000/pMG36e-usp45-bglB, and L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher (p < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K+ and Fe2+ concentrations, the Bgl enzyme activity was significantly higher (p < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn2+, Hg2+, and Tween20 concentrations, the Bgl enzyme activity was significantly lower (p < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of β-glucosidase.

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Construction of individual, fused, and co-expressed proteins of endoglucanase and β-glucosidase for hydrolyzing sugarcane bagasse

Cited by 12 publications

References 28 publications

Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein

Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein

Construction of a trifunctional cellulase and expression in Saccharomyces cerevisiae using a fusion protein

Expression of β-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach

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