Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Islam, Mohammad S.; Harnett, C. K.

doi:10.1002/elsc.201900059

Cited by 5 publications

(4 citation statements)

References 31 publications

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“…The m-cresol and its s isomers o-cresol and p-cresol can be enzymatically oxidized but with different efficiencies owing to favorable position of ring substituents in their structures 21,22 . We chose m-cresol for catalytic reaction due to high substrate specificity at room temperature (RT: 20 °C).…”

Section: Detection Of M-cresolmentioning

confidence: 99%

Gallic acid nanoflower immobilized membrane with peroxidase-like activity for m-cresol detection

Dadı

Çelik

Öçsoy

2020

Sci Rep

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We report fabrication of new generation nanoflowers (NFs) using gallic acid (GA) and copper (II) ions (Cu2+) acted as an organic and inorganic component, respectively with effective peroxidase mimic activities in solution and on filter membrane. Unlike the typical protein NFs synthesis mechanism, gallic acid NFs (GA-NFs) was formed via coordination reaction between carboxyl groups of GA and Cu2+. The different morphologies of the GA-NFs were acquired based upon whether the carboxyl groups in gallic acid are active or not. The peroxidase mimic activity of the GA-NFs relied on the Fenton reaction in the presence of hydrogen peroxide (H2O2) was tested towards m-cresol as a function of concentration of the GA-NFs, m-cresol, H2O2 and reaction time. Under the optimized conditions, the oxidative coupling of m-cresol with 4-aminoantipyrine (4-AAP) was catalyzed by the GA-NFs dispersed in solution and adsorbed on filter paper to form an antipyrine dye and it was visually and spectrophotometrically recorded. The m-cresol with range of 0.05–0.5 mM was detected in 10 min and 15 min by using the GA-NFs in solution and on filter paper, respectively. We demonstrated that the NFs can be produced from non-protein molecules and GA-NFs can be used as a promising nanocatalyst for a variety of applications.

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Section: Detection Of M-cresolmentioning

confidence: 99%

Gallic acid nanoflower immobilized membrane with peroxidase-like activity for m-cresol detection

Dadı

Çelik

Öçsoy

2020

Sci Rep

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“…The crude enzyme of all CtLac V243 mutants from the site-directed mutation was reacted with 0.5 mM 2,6-DMP in 50 mM citrate phosphate buffer containing 1 mM CuCl 2 (pH 8.0) at 25 °C to compare laccase activities among the mutants. The concentration of the oxidized product of 2,6-DMP was calculated according to the Beer-Lambert law by considering the absorbance at 468 nm, the molar extinction coefficient (ε = 49.6 mM −1 cm −1 ), and path length (Islam and Harnett 2019 ). The results were used to select the mutant with the highest laccase activity, which was compared with wild-type CtLac using the purified protein.…”

Section: Methodsmentioning

confidence: 99%

Improvement of thermoalkaliphilic laccase (CtLac) by a directed evolution and application to lignin degradation

Yang

Ghatge

Hur

2022

Appl Microbiol Biotechnol

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Thermoalkaliphilic laccase (CtLac) from the Caldalkalibacillus thermarum strain TA2.A1 has advantageous properties with potential industrial applications, such as high enzyme activity and stability at 70 °C and pH 8.0. In the present study, a directed evolution approach using a combination of random and site-directed mutagenesis was adopted to enhance the laccase activity of CtLac. Spectrophotometric assay and real-time oxygen measurement techniques were employed to compare and evaluate the enzyme activity among mutants. V243 was targeted for site-directed mutagenesis based on library screening. V243D showed a 25–35% higher laccase activity than wild-type CtLac in the spectrophotometric assay and oxygen consumption measurement results. V243D also showed higher catalytic efficiency than wild-type CtLac with decreased Km and increased kcat values. In addition, V243D enhanced oxidative degradation of the lignin model compound, guaiacylglycerol-β-guaiacyl ether (GGGE), by 10% and produced a 5–30% increase in high-value aldehydes than wild-type CtLac under optimal enzymatic conditions (i.e., 70 °C and pH 8.0). Considering the lack of protein structural information, we used the directed evolution approach to predict Val at the 243 position of CtLac as one of the critical amino acids contributing to the catalytic efficiency of the enzyme. Moreover, it found that the real-time oxygen measurement technique could overcome the limitations of the spectrophotometric assay, and apply to evaluate oxidase activity in mutagenesis research. Key points • CtLac was engineered for enhanced laccase activity through directed evolution approach • V243D showed higher catalytic efficiency (kcat/Km) than wild-type CtLac • V243D produced higher amounts of high-value aldehydes from rice straw than wild-type CtLac

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“…The lysate of all CtLac V243 mutants from the site-directed mutation was reacted with 0.5 mM 2,6-DMP in 50 mM citrate phosphate buffer containing 1 mM CuCl 2 (pH 8.0) at 25℃ to compare laccase activities among the mutants. The concentration of the oxidized product of 2,6-DMP was calculated according to Beer-Lambert law by considering the absorbance at 468 nm, the molar extinction coe cient (ε=49.6 mM -1 cm -1 ), and path length [40]. The results selected the mutant with the highest laccase activity, and the laccase activity was compared with wild-type CtLac using the puri ed protein.…”

Section: Spectrophotometric Assaymentioning

confidence: 99%

Enhanced Enzyme Activity of Thermoalkaliphilic Laccase (CtLac) of Caldalkalibacillus thermarum Strain TA2.A1 by Random and Site-directed Mutagenesis and Its Application to Lignin Degradation

Yang

Ghatge

Hur

2022

Preprint

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Background: Thermoalkaliphilic laccase, CtLac, has advantageous properties such as high enzyme activity and stability at 70℃ and pH 8.0 in the presence of organic solvents and surfactants, which confers a potential industrial application. Moreover, CtLac depolymerized lignin and produced high-value benzaldehyde chemicals such as vanillin. In the present study, the directed evolution approach using the combination of random and site-directed mutagenesis was performed to enhance the laccase activity of CtLac. Two methods of spectrophotometric assay and real-time oxygen measurement were employed for comparing and evaluating laccase activity among mutants. Results: Random mutagenesis was carried out by error-prone PCR to introduce 6-8 mutations per 1000 bps of CtLac coding gene. Among the 1,300 tested transformants, RM484 with higher laccase activity was selected, and V243 was targeted for site-directed mutagenesis. V243D showed 35% and 25% higher laccase activity than wild-type CtLac from the spectrophotometric assay and the oxygen consumption measurement, respectively. Besides, V243D showed higher catalytic efficiency with the decreased Km and the increased kcat value than wild-type CtLac. Furthermore, V243D showed the enhanced oxidative degradation of lignin under the optimal enzymatic conditions, 70℃ and pH 8.0, with 10% higher GGGE oxidation and 50-95% increased high-value aldehyde production than wild-type CtLac.Conclusions: CtLac was engineered for improved laccase activity through the directed evolution approach involving random and site-directed mutagenesis in the lack of protein structural information. Considering the limitation of the spectrophotometric assay, the real-time measurement of oxygen consumption could be a more accurate method to evaluate oxidation of laccase activity in mutagenesis research.

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Miniaturized systems for evaluating enzyme activity in polymeric membrane bioreactors

Cited by 5 publications

References 31 publications

Gallic acid nanoflower immobilized membrane with peroxidase-like activity for m-cresol detection

Gallic acid nanoflower immobilized membrane with peroxidase-like activity for m-cresol detection

Improvement of thermoalkaliphilic laccase (CtLac) by a directed evolution and application to lignin degradation

Enhanced Enzyme Activity of Thermoalkaliphilic Laccase (CtLac) of Caldalkalibacillus thermarum Strain TA2.A1 by Random and Site-directed Mutagenesis and Its Application to Lignin Degradation

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