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Highly Stable Protein Immobilization via Maleimido-Thiol Chemistry to Monitor Enzymatic Activity

Highly Stable Protein Immobilization via Maleimido-Thiol Chemistry to Monitor Enzymatic Activity

Jonas Schartner, Jörn Güldenhaupt, Sarah Katharina Gaßmeyer, Katharina Rosga, Robert Kourist, Klaus Gerwert, Carsten Kötting

Analyst. 2018 May 15;143(10):2276-2284.

PMID: 29708245

Abstract:

Immobilizing enzymes for biocatalysis offers many advantages, including easy separation of the enzyme from the product and repeated and continuous use. ATR-FTIR spectroscopy is a versatile tool to monitor immobilized enzymes and has been applied to many proteins. However, while the common and convenient immobilization via oligohistidine on mono-NTA layers is adequate for the measurement of difference spectra induced by ligand binding or photochemistry, it lacks the long term stability that is necessary for monitoring biocatalysis. Here, we report a new immobilization methodology based on maleimido-thiol chemistry. A 12-mercaptododecanoic acid NHS ester monolayer is reacted with 1-(2-aminoethyl)-maleimide to build a thiol reactive surface. Subsequently, NTA-C16-thiol is covalently attached and finally oligohistidine tagged enzymes were immobilized to this surface, which remained bound with a five times higher EC50-value compared to typical mono-NTA layers. To demonstrate the high potential of the surface we analysed decarboxylation reactions catalyzed by arylmalonate decarboxylase. With ATR-FTIR both the enzyme and its substrate conversion can be monitored label free. Correct folding of the enzyme can be evaluated based on the amide band of the immobilized enzyme. In addition, the infrared absorption spectra of educt and product are monitored in real time. We show that hybrid hard-soft multivariate curve resolution improves separation of the product and educt spectra from other effects during the experiments, leading to clean kinetic traces and reaction rates for the catalytic process. Our approach can in principle be extended to any enzyme and is ideally suited for the development of biocatalysts.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP82001534	12-Mercaptododecanoic acid		82001-53-4	Price

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