A high-throughput colourimetric method for the determination of pH in crystallization screens

Kirkwood, Jobie; Wilson, Julie; O’Keefe, Simon; Hargreaves, David

doi:10.1107/s1399004714014011

Cited by 4 publications

(7 citation statements)

References 13 publications

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“…Data for any wells where neither buffer pH nor purification pH was available were removed. It has been shown that the buffering capacity of the protein itself is negligible both in vitro ( Kirkwood et al. , 2014 ) and in vivo ( Poznanski et al.…”

Section: Resultsmentioning

confidence: 99%

“…A method to accurately determine pH using spectrophotometry has been developed and is described in detail elsewhere ( Kirkwood et al. , 2014 ).…”

Section: Methodsmentioning

confidence: 99%

“…Measurement of pH using a pH meter is time consuming and can be impractical if it requires reformatting of the crystallization screen to accommodate the probe. We have shown previously that accurate measurements of pH can be obtained for crystallization solutions using spectrophotometry ( Kirkwood et al. , 2014 ).…”

Section: Introductionmentioning

confidence: 99%

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Using isoelectric point to determine the pH for initial protein crystallization trials

et al. 2015

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Motivation: The identification of suitable conditions for crystallization is a rate-limiting step in protein structure determination. The pH of an experiment is an important parameter and has the potential to be used in data-mining studies to help reduce the number of crystallization trials required. However, the pH is usually recorded as that of the buffer solution, which can be highly inaccurate.Results: Here, we show that a better estimate of the true pH can be predicted by considering not only the buffer pH but also any other chemicals in the crystallization solution. We use these more accurate pH values to investigate the disputed relationship between the pI of a protein and the pH at which it crystallizes.Availability and implementation: Data used to generate models are available as Supplementary Material.Contact: julie.wilson@york.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.

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Section: Resultsmentioning

confidence: 99%

“…A method to accurately determine pH using spectrophotometry has been developed and is described in detail elsewhere ( Kirkwood et al. , 2014 ).…”

Section: Methodsmentioning

confidence: 99%

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Using isoelectric point to determine the pH for initial protein crystallization trials

et al. 2015

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“…Some indicator compounds, such as anilines, are known to tolerate high concentrations of organic solvents, e.g., acetonitrile (49); however, they are not suitable to detect shifts from physiological pH. In this study, we observed that the indicator dye nitrazine yellow undergoes a color shift below pH 7 (29,50) and can thus be used for the determination of pH changes in the presence of different water-miscible organic solvents at concentrations of up to 50% (vol/vol). Notably, this approach is limited to testing of water-miscible organic solvents; nonpolar organic solvents form two-phase systems, which are difficult to read out with colorimetric microtiter plate (MTP) scale assays.…”

Section: Discussionmentioning

confidence: 62%

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis

Bollinger

Molitor

Thies

et al. 2020

Appl Environ Microbiol

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Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica. IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.

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“…It is well documented that estimating the pH of a crystallization solution as that of the buffer component can be inaccurate (Kirkwood et al, 2014;Bukrinsky & Poulsen, 2001). Chemical species such as PEGs and ammonium-containing compounds are known to degrade over time, thereby modifying the pH Jurnak, 1986;Mikol et al, 1989;Hampton Research, 2012).…”

Section: Analysis Of Ph and The Relationship To Pimentioning

confidence: 99%