Accurately Determining Esterase Activity via the Isosbestic Point of p-Nitrophenol

Peng, Yangyang; Fu, Shiyu; Líu, Hao; Lucia, Lucian A.

doi:10.15376/biores.11.4.10099-10111

Cited by 36 publications

(22 citation statements)

References 33 publications

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“…In the case of biopolymer materials that undergo swelling in aqueous media, dye-based adsorption offers an alternative approach for estimating the adsorbent SA. The measurements were conducted at the isosbestic point of PNP to avoid errors due to changes in solution pH [ 59 ]. Herein, a dye-based method was employed, where p -nitrophenol (PNP) served as the dye probe for estimating the bead SA (cf.…”

Section: Resultsmentioning

confidence: 99%

Modular Chitosan-Based Adsorbents for Tunable Uptake of Sulfate from Water

Steiger

Wilson

2020

IJMS

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The context of this study responds to the need for sorbent technology development to address the controlled removal of inorganic sulfate (SO42−) from saline water and the promising potential of chitosan as a carrier system for organosulfates in pharmaceutical and nutraceutical applications. This study aims to address the controlled removal of sulfate using chitosan as a sustainable biopolymer platform, where a modular synthetic approach was used for chitosan bead preparation that displays tunable sulfate uptake. The beads were prepared via phase-inversion synthesis, followed by cross-linking with glutaraldehyde, and impregnation of Ca2+ ions. The sulfate adsorption properties of the beads were studied at pH 5 and variable sulfate levels (50–1000 ppm), where beads with low cross-linking showed moderate sulfate uptake (35 mg/g), while cross-linked beads imbibed with Ca2+ had greater sulfate adsorption (140 mg/g). Bead stability, adsorption properties, and the point-of-zero charge (PZC) from 6.5 to 6.8 were found to depend on the cross-linking ratio and the presence of Ca2+. The beads were regenerated over multiple adsorption-desorption cycles to demonstrate the favorable uptake properties and bead stability. This study contributes to the development of chitosan-based adsorbent technology via a modular materials design strategy for the controlled removal of sulfate. The results of this study are relevant to diverse pharmaceutical and nutraceutical applications that range from the controlled removal of dextran sulfate from water to the controlled release of chondroitin sulfate.

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Section: Resultsmentioning

confidence: 99%

Modular Chitosan-Based Adsorbents for Tunable Uptake of Sulfate from Water

Steiger

Wilson

2020

IJMS

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“…Lipolytic activity, referred to only when activity was confirmed, was tested utilizing the degradation of para-nitrophenyl ( p NP) bound fatty acid substrates [51–53]. The test was carried out by mixing 83.3 μl of a testing buffer with 16.7 μl protein solution and the absorption measured at 410 nm over a period of 45 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Diversity and function of microbial lipases within the mammalian gut

Hitch

Masson

Streidl

et al. 2020

Preprint

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BackgroundCurrent estimates suggest the majority of microbial proteins within the mammalian gut lack meaningful annotation. One such functional group are microbial lipases (EC:3.1.1.3), which can alter host access and utilisation of dietary fat. In this paper, we describe the diversity of lipolytic bacteria, including in vitro characterisation of a new lipase.ResultsMetagenomic sequence-based network analysis identified that the majority of microbial lipases in the gut of three host species (human, mouse, pig) belong to two unique clusters. These clusters were characterized by the presence of two novel motifs, AHSKGG and TTxxTPH, which may play a key functional role due to co-localisation in the active site, as identified by structural modelling. Analysis of metagenomic assembled genomes (MAGs) indicated that the majority of lipase-positive species belong to the phylum Firmicutes, although all dominant phyla within the human gut were represented by positive species. Metabolic analysis of these genomes identified a high prevalence of glycerol rather than fatty acid metabolism. The occurrence of microbial lipases determined across ~800 metagenomic gut samples depended on dietary fat consumption, with lipase expression increased in lard fed compared to palm oil fed mice. A representative lipase encoded within the genome of the species Clostridium symbiosum was cloned and its characterization confirmed the in silico prediction and provided detailed annotation to 373 proteins.ConclusionsMicrobial lipases within the gut represent a conserved group characterized by unique amino acid sequence motifs. While an increase in microbial lipase occurrence was positively associated with dietary fat intake, lipase-producing species seemed unable to metabolise the released fatty acids. In this paper, we provide a global analysis of the functional importance and diversity of microbial lipases within the intestine of mammals, which will improve the resolution of future sequence-based studies and open avenues for mechanistic experiments based on isolates.

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“…2a). Since pNP is deprotonated under alkaline conditions to form p-nitrophenolate, with a yellow color (Martin et al, 2012;Peng et al, 2016), the enzyme activity can easily be monitored. Consistent with the structural resemblance between TW9814 and PhnP, the addition of TW9814 made the bpNPP solution yellow, indicating the production of p-nitrophenolate.…”

Section: Identification Of the Function Of Tw9814mentioning

confidence: 99%

Structural and functional identification of the uncharacterized metallo-β-lactamase superfamily protein TW9814 as a phosphodiesterase with unique metal coordination

Heo¹,

Park²,

Jeon³

et al. 2022

Acta Cryst Sect D Struct Biol

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Metallo-β-lactamase (MBL) superfamily proteins have a common αβ/βα sandwich fold and perform a variety of functions through metal-mediated catalysis. However, because of the enormous scale of this superfamily, only a small percentage of the proteins belonging to the superfamily have been annotated structurally or functionally to date. Therefore, much remains unknown about the MBL superfamily proteins. Here, TW9814, a hypothetical MBL superfamily protein, was structurally and functionally investigated. Guided by the crystal structure of dimeric TW9814, it was demonstrated that TW9814 functions as a phosphodiesterase (PDE) in the presence of divalent metal ions such as manganese(II) or nickel(II). A docking model between TW9814 and the substrate bis(p-nitrophenyl)phosphate (bpNPP) showed the importance of the dimerization of TW9814 for its bpNPP-hydrolyzing activity and for the interaction between the enzyme and the substrate. TW9814 showed outstanding catalytic efficiency (k cat/K m) under alkaline conditions compared with other PDEs. The activity of TW9814 appears to be regulated through a disulfide bond, which is a feature that is not present in other MBL superfamily members. This study provides a platform for the functional characterization of other hypothetical proteins of the MBL or other superfamilies.

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Accurately Determining Esterase Activity via the Isosbestic Point of p-Nitrophenol

Cited by 36 publications

References 33 publications

Modular Chitosan-Based Adsorbents for Tunable Uptake of Sulfate from Water

Modular Chitosan-Based Adsorbents for Tunable Uptake of Sulfate from Water

Diversity and function of microbial lipases within the mammalian gut

Structural and functional identification of the uncharacterized metallo-β-lactamase superfamily protein TW9814 as a phosphodiesterase with unique metal coordination

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