A sensitive pH indicator-based spectrophotometric assay for PHB depolymerase activity on microtiter plates

Camacho-Ruiz, Maria Angeles; Müller-Santos, Marcelo; Hernández-Mancillas, Xitlalli Desideria; Armenta‐Perez, Vicente Paul; Zamora-González, Edgar Oswaldo; Rodríguez, Jorge A.

doi:10.1039/d0ay00840k

Cited by 2 publications

(2 citation statements)

References 42 publications

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“…ω-Hydroxy acid production was monitored as shown in Figure S4. 48 Briefly, 200 μL of a reaction mixture containing 20 mM 1,5-PD, 1 mM NAD + , 0.15 mM FAD + , and 0.1 mM Cresol Red in 2.5 mM sodium phosphate buffer at pH 8 was incubated with 10 μL of a suspension of HBs (properly diluted) at 30 °C. The decrease in the absorbance at 580 nm due to the decrease in the pH was recorded.…”

Section: Rra (%)mentioning

confidence: 99%

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids

Santiago-Arcos,

Velasco-Lozano,

Diamanti

et al. 2024

ACS Sustainable Chem. Eng.

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Cell-free biocatalysis is gaining momentum in producing value-added chemicals, particularly in stepwise reaction cascades. However, the stability of enzyme cascades in industrial settings is often compromised when free enzymes are involved. In this study, we have developed a stable multifunctional heterogeneous biocatalyst coimmobilizing five enzymes on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved the operational efficiency and stability of the heterogeneous biocatalyst by fine-tuning the enzyme loading and spatial organization. Stability issues are overcome through postimmobilization polymer coating. The general applicability of this heterogeneous biocatalyst is demonstrated by its scale-up in both batch and packed bed reactors, allowing a product yield of >80%. The continuous process is fed with H 2 O 2 as the oxygen source, reaching a space-time yield (STY) of 0.76 g•L −1 •h −1 , maintained for the first 12 h. Finally, this flow system is telescoped with a second plug-flow reactor packed with a different heterogeneous biocatalyst integrating an additional transaminase. As a result, this 6-enzyme 2-reactor system sequentially transforms 1,ω-diols into 1,ω-amino acids while in situ recycling NAD + , depleting H 2 O 2 , and generating O 2 .

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Section: Rra (%)mentioning

confidence: 99%

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids

Santiago-Arcos,

Velasco-Lozano,

Diamanti

et al. 2024

ACS Sustainable Chem. Eng.

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show abstract

“…ω-Hydroxy acid production: ω-Hydroxy acid production was monitored as shown in Figure S4. 32 Brie y, 200 µL of a reaction mixture containing 20 mM 1,5-pentanediol, 1 mM NAD + , 0.15 mM FAD + , and 0.1 mM Cresol Red in 2.5 mM sodium phosphate buffer at pH 8 was incubated with 10 µL of suspension of HBs (properly diluted) at 30 ºC. The decrease in the absorbance at 580 nm due to the decrease in the pH was recorded.…”

Section: Preparation Of Hb14mentioning

confidence: 99%

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids

Santiago-Arcos,

Velasco-Lozano,

Diamanti

et al. 2023

Preprint

View full text Add to dashboard Cite

Cell-free biocatalysis is gaining momentum in the production of value-added chemicals, particularly in stepwise reaction cascades. However, the stability of enzyme cascades in industrial settings is often compromised when using free enzymes. In this study, we have developed a stable multifunctional heterogeneous biocatalyst co-immobilizing five enzymes on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved the operational efficiency and stability of the heterogeneous biocatalyst by fine-tuning enzyme loading and spatial organization. Stability issues are overcome through post-immoblization polymer coating. The general applicability of this heterogeneous biocatalyst is demonstrated by its scale-up in both batch and packed bed reactors, allowing a product yield > 80%. The continuous process is fed with H2O2 as oxygen source, reaching a Space-Time Yield (STY) of 0.76 g·L− 1·h− 1, maintained for the first 12 hours. Finally, this flow system is telescoped with a second plug-flow reactor packed with a different heterogeneous biocatalyst. As a result, this 6-enzyme 2-reactor system sequentially transforms 1,ω-diols into 1,ω-aminoacids while in-situ recycling NAD+, depleting H2O2 and generating O2.

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A sensitive pH indicator-based spectrophotometric assay for PHB depolymerase activity on microtiter plates

Abstract: A continuous spectrophotometric assay for the screening of PHB depolymerase activity in microtiter plates was developed. We evaluated crystalline PHB in suspension and coated with the addition of pH indicator...

Cited by 2 publications

References 42 publications

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids

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