Efficient extracellular secretion of an endoglucanase and a β-glucosidase in E. coli

Gupta, Sapna; Adlakha, Nidhi; Yazdani, Syed Shams

doi:10.1016/j.pep.2012.11.006

Cited by 25 publications

(19 citation statements)

References 24 publications

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“…Yan with coworkers [10] reported that β glucosidase gene from Paecilomyces thermophila was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as active extracellular enzyme. Gupta with coauthors [11] found efficient extracellular secretion of a β glucosidase fused with the hyperosmotically induc ible periplasmic protein, OsmY, in E. coli and exhibited 2.2 U/mL of β glucosidase activity in the extracellular fraction at 16 h post induction. Chen et al [12] reported expression of a secretory β glucosidase from Trichoderma reesei in P. pastoris that the maximum recombinant β glucosidase activity achieved 60 U/mL.…”

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confidence: 98%

“…Mendoza Aguayo with coworkers [13] revealed extra cellular expression of β glucosidase of Cellulomonas fla vigena PN 120 in the supernatant of a rich medium showing 582 U/L. On the other hand, there are a few reports on expression and extracellular secretion of β glucosidase from the thermophilic bacteria in E. coli [9][10][11][12][13]. The advantages of thermostable enzymes in industrial processes include reduced risk of contamina tion, increased substrate solubility, higher stable of enzymes against denaturing agents and proteolytic enzymes, and reduced cost of external cooling [14].…”

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confidence: 99%

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Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

Xue

Hou

et al. 2015

Appl Biochem Microbiol

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The β glucosidase Tm bglA gene from hyperthermophile Thermotoga maritima was cloned into expression vectors pET 28a, producing two proteins of 55 kDa and 52 kDa in cell, which could be referred to Tm BglA with and without 23 amino acids. The results showed that fusion of 23 amino acids to Tm BglA improved its soluble expression and product tolerance, resulting over 60% yield of recombinant Tm BglA secreted into the growth medium in Escherichia coli JM109 (DE3). Tm BglA with 23 amino acids had higher k cat and K M values for p nitrophenyl β D glycopyranoside and much stronger product inhibition than Tm BglA without 23 amino acids. Subsequently, the Tm BglA was immobilized on chitin efficiently by genetically fusing the chitin binding domain of chitinase A1 from Thermoanaerobacterium thermosaccharolyticum DSM571. The immobilized Tm BglA had higher optimal temperature (95°C) and was more thermostable at the range from 75 to 100°C than its free form. This immobilized protein exhibited high stability and the con version yield exceeding 90%. The high level soluble expression, combined simultaneous purification and immobilization of the enzyme on chitin offers a novel approach for the low cost production of β glucosidase to produce lactose free fresh dairy products.

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confidence: 98%

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confidence: 99%

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

Xue

Hou

et al. 2015

Appl Biochem Microbiol

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“…). Moreover, the extracellular endoglucanase activity obtained was 2‐fold higher compared with a recent study that employs OsmY as the carrier protein …”

Section: Discussionmentioning

confidence: 59%

“…In our case, the point of optimal induction was determined as OD 600 = 0.45 with 50 µmol L −1 IPTG (Fig. ), which was in the lower range of IPTG inductions typically performed at 0.1–1 mmol L −1 , but similar to the earlier examples of secretory production in E. coli , with endoglucanase induction optimized at 20 µmol L −1 IPTG and bacteriocin release protein induction optimized at 50 µmol L −1 IPTG . Supplementation of the culture medium with Mg 2+ ion was also tested, at 0.4 and 0.8 mmol L −1 final concentrations.…”

Section: Discussionmentioning

confidence: 71%

“…Moreover, the extracellular endoglucanase activity obtained was 2-fold higher compared with a recent study that employs OsmY as the carrier protein. 28 Extracellular production can also be enhanced by increasing the permeability of the outer membrane by mechanical (ultrasound), chemical (addition of magnesium, calcium, EDTA, glycine, Tween-80 and Triton X-100), or enzymatic (lysozyme) treatments. 10,29 Moreover, process conditions such as temperature, inducer concentration, induction time and medium pH have been shown to significantly affect the extracellular production of recombinant proteins.…”

Section: Discussionmentioning

confidence: 99%

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Periplasmic and extracellular production of cellulase from recombinant Escherichia coli cells

Yıldırım

Çelik

2016

J of Chemical Tech & Biotech

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BACKGROUND Secretory production of recombinant enzymes in E. coli has significant advantages in bioprocess development, such as simplicity of purification, preventing proteolytic degradation, and providing greater access to substrates such as cellulose that opens the path to consolidated bioprocessing. RESULTS In this study, an endoglucanase (Cel5A) from Fusarium graminearum was investigated for extracellular production in E. coli as a fusion to the YebF carrier protein, and compared with periplasmic production enabled by fusing the DsbA signal peptide to the N‐terminus of Cel5A. Yields of extracellular YebF‐Cel5A (175 mg L−1) were nearly 2‐fold higher than yields of periplasmic Cel5A. Periplasmic but not extracellular production of Cel5A increased ∼1.5‐fold following the addition of 0.4 mmol L−1 MgSO4 to the growth medium. While most of the divalent cations had an inhibitory effect on the periplasmic Cel5A activity, all cations tested enhanced the extracellular endoglucanase activity, with Co2+ having the highest activation (4.3‐fold increase). Moreover, YebF‐Cel5A had 20% higher residual activity than Cel5A following 1 h heat treatment. CONCLUSION The differences demonstrated for periplasmic and extracellular production of cellulases in E. coli provide the basis of bioprocess design parameters, paving the way to consolidated bioprocessing. © 2016 Society of Chemical Industry

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Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement

Wells

Robinson

2016

Biotechnology Journal

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Recombinant proteins offer many therapeutic advantages unavailable in traditional small molecule drugs, but the need for cellular versus chemical synthesis complicates production. Avenues for producing therapeutic biologics are continuously expanding, and developments in biochemistry, cell biology, and bioengineering fuel new discoveries that promise safer, more efficient, and cheaper drugs for consumers. Numerous approaches to express recombinant proteins exist, but Escherichia coli, Saccharomyces cerevisiae, and mammalian systems (e.g. Chinese hamster ovary cells, CHO) are the most widely utilized. Improvements to production in these hosts have focused on novel expression cassettes, cell line modifications, engineering secretion pathways, and media design. Here, we describe recent developments for improving protein production in E. coli, S. cerevisiae, and CHO systems and compare recent advancements to previous knowledge in the field. With the expanding importance and prevalence of protein therapeutics, these improvements will serve as the framework for future discoveries.

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Efficient extracellular secretion of an endoglucanase and a β-glucosidase in E. coli

Cited by 25 publications

References 24 publications

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

Enhanced soluble expression of a thermostble β-glucosidase from Thermotoga maritima in Escherichia coli and its applicaton in immobilization

Periplasmic and extracellular production of cellulase from recombinant Escherichia coli cells

Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement

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