A two-stage refinement approach for the enhancement of excretory production of an exoglucanase from Escherichia coli

Aims: The cell‐surface display of Cex, which encodes xylanase and exoglucanase from Cellulomonas fimi, was constructed on Escherichia coli using PgsA as the anchor protein. Characterization of the cell‐surface display of Cex was performed. Methods and Results: PgsA was fused to the N‐terminus of Cex and six histidines were utilized as spacers between the targeting and anchor proteins. Successful cell‐surface display of Cex was demonstrated by Western blot and immunofluorescence analyses on E. coli C41 (DE3). According to the time‐course analysis, the xylanase activity of Cex was achieved at 49 U g−1 dry cell weight after 12 h culture at 37°C. The optimal temperature and pH ranges of the cell‐surface displayed protein with whole‐cell were broader than the corresponding ranges of the purified form. Further determination of thermostability indicated that the half‐life of cell‐surface displayed Cex was 1·6 times longer than that of purified Cex at 60°C. Conclusions: We have successfully developed the cell‐surface display of xylanase on E. coli. The cell‐surface display can enhance the stability of xylanase against changes in temperature and has the potential of becoming a whole‐cell biocatalyst for industrial applications, such as biobleaching of paper and production of renewable energy. Significance and Impact of the Study: The results demonstrated that the cell‐surface display of xylanase embedded in the cell membrane is more stable than that of the purified enzyme. Thus, to improve the stability of heterologous proteins production, cell‐surface display using the PgsA anchor protein as a tool can be considered in E. coli.

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“…1997). Therefore, Fu et al. (2006) designed a weaker promoter to achieve maximum overexpression of secreted Cex.…”

Section: Discussionmentioning

confidence: 99%

Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli

Chen

Hwang

Lin

et al. 2012

Journal of Applied Microbiology

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“…Our development of various efficient extracellular host systems (Fu et al, 2006;Lam et al, 1997Lam et al, , 1998Wang et al, 2010bWang et al, , 2011Wong and Lam, 2000;Wong and Sutherland, 1997;Wong et al, 1988), on the one hand, may facilitate the production, thereby the understanding of the catalytic properties and substrate specialties of recombinant GH1 and GH3 Cel of C. biazotea. Our studies along this line may hopefully result in useful information on the complementary roles played by the two families of Cel enzymes in the hydrolysis of oligosaccharides.…”

Section: Discussionmentioning

confidence: 99%

“…The availability of host systems for the export of recombinant cellulases into the culture medium facilitates not only the production of the enzymes, but also the studies of their reconstitution and interaction. Although Eng and Exg have been commonly expressed as extracellular enzymes (Fu et al, 2006;Lam et al, 1997, Wang et al, 2010b, 2011Wong and Lam, 2000;Wong et al, 1988), the attainment of Cel as secreted or excreted products is not so common. The low frequency is attributable to the fact that cellulolytic microbes are commonly found to produce cell-bound Cel (Bhat and Bhat, 1997;Wakarchuk et al, 1984), thus hindering the progress of developing recombinant strains expressing extracellular Cel.…”

Section: Introductionmentioning

confidence: 99%

“…Our laboratory has been involved in research and application of microbial cellulases for a long time (Fu et al, 2005(Fu et al, , 2006Gilkes et al, 1984;Lam et al, 1997Lam et al, , 1998Lau and Wong, 2001;Skipper et al, 1985;Wang et al, 2010bWang et al, , 2011Warren et al, 1986;Wong and Lam, 2000;Wong and Sutherland, 1997;Wong et al, 1986Wong et al, , 1988. Over these years, we have cloned and expressed the cex and cenA genes from Cellulomonas fimi, which encode an Exg and an Eng, respectively, in a wide range of host systems (Fu et al, 2006;Lam et al, 1997Lam et al, , 1998Wang et al, 2010bWang et al, , 2011Wong and Lam, 2000;Wong and Sutherland, 1997;Wong et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

“…Over these years, we have cloned and expressed the cex and cenA genes from Cellulomonas fimi, which encode an Exg and an Eng, respectively, in a wide range of host systems (Fu et al, 2006;Lam et al, 1997Lam et al, , 1998Wang et al, 2010bWang et al, , 2011Wong and Lam, 2000;Wong and Sutherland, 1997;Wong et al, 1988). To search for Cel that might work co-operatively with Exg and Eng, we have put our focus on Cellulomonas biazotea due to the reasons that i) it secretes high levels of Cel activity to the culture medium (Lau and Wong, 2001;Saratale et al, 2010;, and ii) it is closely related to C. fimi (Wood and McCrae Sheila, 1979).…”

Section: Introductionmentioning

confidence: 99%

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Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea

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A revolutionary approach facilitating co-expression of authentic human epidermal growth factor and basic fibroblast growth factor in both cytoplasm and culture medium of Escherichia coli

Kwong

Wong

2013

Appl Microbiol Biotechnol

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During secretory or excretory production of heterologous proteins in Escherichia coli, peptidase processing cleaves the signal peptide off from a premature protein, which is then secreted as a mature product. Many proteins have been successfully expressed as secreted/excreted products in E. coli. However, basic fibroblast growth factor (bFGF), despite its suitability for secretory/excretory production in E. coli, has never been successfully expressed using such an approach. In this communication, we report the application of a revolutionary E. coli system to the efficient expression of not only bFGF, but also human epidermal growth factor (EGF) concurrently, as authentic products in the culture supernatant (SN). More interestingly, both polypeptides were also shown to be present at high levels as authentic products in the cell lysate (CL). The manifestation of this unusual phenomenon required a collaborative action between construct pWKW2, an efficient excretion vector engineered by our group to facilitate extracellular production of EGF, and the Sce VMA intein, which enables self-cleavage of protein sequences fused to it. Both bFGF and EGF derived from SN and CL were characterized to be bioactive. Moreover, despite employing only shake-flask cultivation, the total yields of bFGF and EGF recovered from both SN and CL were impressive, amounting to 103 and 74 mg l(-1) of culture, respectively. The novel expression approach introduced herein may prove to be practically useful for the production of a wide range of proteins in the future.

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A two-stage refinement approach for the enhancement of excretory production of an exoglucanase from Escherichia coli

Cited by 14 publications

References 23 publications

Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli

Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli

Cloning and characterization of a novel cellobiase gene, cba3, encoding the first known β-glucosidase of glycoside hydrolase family 1 of Cellulomonas biazotea

A revolutionary approach facilitating co-expression of authentic human epidermal growth factor and basic fibroblast growth factor in both cytoplasm and culture medium of Escherichia coli

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