Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli

Howard, RL; Masoko, Peter; Abotsi, E

doi:10.5897/ajb2003.000-1060

Cited by 12 publications

(14 citation statements)

References 17 publications

(23 reference statements)

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“…This represents significant improvement on previous cloning and expression attempts (Howard, 1997;van Rensburg et al, 1996). This paper is a continuation of our previous publication (Howard et al, 2003) with the emphasise shifting towards describing the biochemical nature of the *Corresponding author: Tel/fax +27 15 2682862, E-mail: howardr@unorth.ac.za. fused CBHI.1 protein.…”

Section: Introductionmentioning

confidence: 86%

“…coli BL21 cells transformed with pGEXcbhI.1 and pGEX plasmids used in this study were constructed by Masoko (2001) and have been previously described (Howard et al, 2003).…”

Section: Clonesmentioning

confidence: 99%

“…We previously reported in the African Journal of Biotechnology (Howard et al, 2003) on the successful cloning and expression of a functionally active Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein from Escherichia coli. The heterologously expressed glutathione S-transferase (GST) fused CBHI.1 protein exhibited hydrolytic activities against derived carboxy-methyl-cellulose (CMC) and microcrystalline Avicel without requiring in vitro chemical refolding.…”

Section: Introductionmentioning

confidence: 99%

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Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli

Howard¹,

Masoko²,

Mowa³

et al. 2004

Afr. J. Biotechnol.

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The aim of this study was to purify and analyse a Phanerochaete chrysosporium cbhI.1 gene-product expressed as an inducible, secreted, heterologous protein from an Escerichia coli pGEXcbhI.1 clone. Using glutathione Sepharose 4B affinity chromatography, the expressed protein was purified from the supernatant of an induced E. coli transformed with pGEXcbhI.1 and ran as a single band on a Sodium dodecyl sulphate-polyacrylamide gel. The glutathione S-transferase (GST) fused CBHI.1 was approximately 80 kDa in size, approximately 2.2 kDa smaller than the theoretically predicted size. The purified protein exhibited time dependent hydrolytic reaction against carboxy-methyl-cellulose (CMC) and Avicel. On CMC the highest hydrolytic reaction occurred at 120 min. whereas for Avicel it was at 150 min. Optimum pH and temperature for activity of the protein against these cellulose substrates were pH 6 and 55 o C, respectively, and the protein remained stable under these optimum conditions for 24 h.

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Section: Introductionmentioning

confidence: 86%

“…coli BL21 cells transformed with pGEXcbhI.1 and pGEX plasmids used in this study were constructed by Masoko (2001) and have been previously described (Howard et al, 2003).…”

Section: Clonesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli

Howard¹,

Masoko²,

Mowa³

et al. 2004

Afr. J. Biotechnol.

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confidence: 99%

“…Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina 3,9 , an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina 10 and Phanerochaete chrysosporium [11][12] .We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium.…”

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confidence: 99%

High Throughput Screening of Fungal Endoglucanase Activity in <em>Escherichia coli</em>

Farrow

Arnold

2011

JoVE

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Cellulase enzymes (endoglucanases, cellobiohydrolases, and β-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols 1 . The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production 2 . Of particular interest are fungal cellulases 3-8 , which are already being used industrially for foods and textiles processing.Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina 3,9 , an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina 10 and Phanerochaete chrysosporium [11][12] .We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation ("halos") at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness.Traditional bacterial whole cell CMC/CR activity assays 13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods 14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms. Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=2942 1. Generate a library of endoglucanase catalytic domain(s). This can be accomplished in many ways (e.g., site-directed or random mutagenesis, recombination, etc.) and is determined by the researcher. 2. Clone the lib...

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Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases, esterases, and lignocellulolytic enzymes

Silva

Pedezzi

Souto

2017

Appl Microbiol Biotechnol

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Fungi constitute an invaluable natural resource for scientific research, owing to their diversity; they offer a promising alternative for bioprospecting, thus contributing to biotechnological advances. For a long time, extensive information has been exploited and fungal products have been tested as a source of natural compounds. In this context, enzyme production remains a field of interest, since it offers an efficient alternative to the hazardous processes of chemical transformations. Owing to their vast biodiversity and peculiar biochemical characteristics, two fungal categories, white-rot and anaerobic Neocallimastigomycota, have gathered considerable attention for biotechnological applications. These fungi are known for their ability to depolymerize complex molecular structures and are used in degradation of lignocellulosic biomass, improvement of animal feed digestibility, biogas and bioethanol production, and various other applications. However, there are only limited reports that describe proteolytic enzymes and esterases in these fungi and their synergistic action with lignocellulolytic enzymes on degradation of complex polymers. Thus, in this minireview, we focus on the importance of these organisms in enzyme technology, their bioprospecting, possibility of integration of their enzyme repertoire, and their prospects for future biotechnological innovation.

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Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) expressed as a heterologous protein from Escherichia coli

Cited by 12 publications

References 17 publications

Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli

Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in <em>Escherichia coli</em>

Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases, esterases, and lignocellulolytic enzymes

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