ARduino‐pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in <i>Escherichia coli</i>

Hsu, Kao-Pang; Tan, Shih‐I; Chiu, Chen-Yaw; Chang, Yu-Kaung; Ng, I‐Son

doi:10.1002/btpr.2834

Cited by 18 publications

(11 citation statements)

References 41 publications

(79 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both forms of R5-SazCA demonstrated good salinity tolerance, clearly showing that they are suitable for carbon sequestration applications using seawater because its salinity is about 3.5%. SspCA also showed little loss in activity up to 6% NaCl [23], which is very similar to our results. The activities before incubation with cations were set as 100%.…”

Section: Cation and Salinity Tolerancesupporting

confidence: 92%

“…It is clear that the silica coating protects the enzyme from the inhibitory effects exerted by these metal ions. The inhibition caused by Cu 2+ and Zn 2+ has also been reported for SspCA, and the inhibition by Cu 2+ is likely due to the possible binding of Cu 2+ to the imidazole side chain of His residues in the active center [20,23].…”

Section: Cation and Salinity Tolerancementioning

confidence: 59%

See 1 more Smart Citation

Entrapment of the Fastest Known Carbonic Anhydrase with Biomimetic Silica and Its Application for CO2 Sequestration

Hsieh

Cheng²,

et al. 2021

Polymers

View full text Add to dashboard Cite

Capturing and storing CO2 is of prime importance. The rate of CO2 sequestration is often limited by the hydration of CO2, which can be greatly accelerated by using carbonic anhydrase (CA, EC 4.2.1.1) as a catalyst. In order to improve the stability and reusability of CA, a silica-condensing peptide (R5) was fused with the fastest known CA from Sulfurihydrogenibium azorense (SazCA) to form R5-SazCA; the fusion protein successfully performed in vitro silicification. The entrapment efficiency reached 100% and the silicified form (R5-SazCA-SP) showed a high activity recovery of 91%. The residual activity of R5-SazCA-SP was two-fold higher than that of the free form when stored at 25 °C for 35 days; R5-SazCA-SP still retained 86% of its activity after 10 cycles of reuse. Comparing with an uncatalyzed reaction, the time required for the onset of CaCO3 formation was shortened by 43% and 33% with the addition of R5-SazCA and R5-SazCA-SP, respectively. R5-SazCA-SP shows great potential as a robust and efficient biocatalyst for CO2 sequestration because of its high activity, high stability, and reusability.

show abstract

Section: Cation and Salinity Tolerancesupporting

confidence: 92%

Section: Cation and Salinity Tolerancementioning

confidence: 59%

Entrapment of the Fastest Known Carbonic Anhydrase with Biomimetic Silica and Its Application for CO2 Sequestration

Hsieh

Cheng²,

et al. 2021

Polymers

View full text Add to dashboard Cite

show abstract

“…The CA expressing cells were collected and washed 3 times by deionized water and finally adjusted to OD 5 for further use. The CA activity was determined by Wilbur-Anderson assay as our previous publication (Hsu et al 2019). Briefly, 9 mL ice-cold Tris-HCl (20 mM, pH 8.3) buffer and 0.2 mL enzyme were mixed and put in a 20-mL sample bottle at 0 °C under stirring.…”

Section: Determination Of Ca Activity By Wilbur-anderson Unit (Wau)mentioning

confidence: 99%

“…In our previous report, BL21(DE3) overexpressing carbonic anhydrase (CA) showed the arrested cell growth as compared to that without CA expression (Hsu et al 2019). Therefore, we considered whether W3110 strain would tolerate CA overexpression via pET32a-SyCA.…”

Section: Carbonic Anhydrase Expression and Activity In Engineered W31mentioning

confidence: 99%

Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

Ting

Tan

2020

Bioresour. Bioprocess.

Self Cite

View full text Add to dashboard Cite

Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy. Results Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively. Conclusion Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.

show abstract

“…In our previous report, BL21(DE3) overexpressing carbonic anhydrase (CA) showed the arrested cell growth as compared to that without CA expression [32]. Therefore, we considered whether W3110 strain would tolerate CA overexpression via pET32a-SyCA.…”

Section: Carbonic Anhydrase Expression and Activity In Engineered W31mentioning

confidence: 99%

Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

Ting¹,

Tan²,

Ng³

2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter were powerful tools to mediate the protein expression. Moreover, Escherichia coli W3110 strain possesses more advantages than the B strain due to more heat shock proteins and higher tolerance to chemicals. Therefore, implementation of T7-based system in W3110 strain is a conceivable strategy to develop the cell factory. Results Three novel W3110 strains with chromosome-equipped T7RNAP (i.e W3110:IL5, W3110::L5 and W3110::pI) were engineered to demonstrate the feasibility on protein expression and chemical production. At first, the LacZ and T7RNAP with IPTG induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), Cas9, carbonic anhydrase (CA) and lysine decarboxylase (CadA). All the proteins were expressed higher and enzymatic functions were better in W3110::L5 and W3110::pI. Moreover, the highest cadaverine production, lysine consumption and the yield were obtained in W3110::L5(+) strain with pET28a(+)-CadA which reached 32.2 g/L, 45 g/L and 91.7% at 24 h, while the W3110::pI(-) strain with pSU-T7-CadA achieved 36.9 g/L, 43.8 g/L and 103.4% at 12 h which is unnecessary of inducer. Conclusion Inducer and lacI/lacO regulators on chromosome and plasmid have been investigated in W3110 strains with T7RNAP. The newly engineered W3110::L5 and W3110:pI both possessed similar protein expression compared to commercial BL21(DE3). Furthermore, among all strains, W3110::pI displayed the greatest potential as cell factory in the future.

show abstract

ARduino‐pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli

Cited by 18 publications

References 41 publications

Entrapment of the Fastest Known Carbonic Anhydrase with Biomimetic Silica and Its Application for CO2 Sequestration

Entrapment of the Fastest Known Carbonic Anhydrase with Biomimetic Silica and Its Application for CO2 Sequestration

Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

Contact Info

Product

Resources

About