Direct quantification of immobilized enzymes by means of FTIR ATR spectroscopy – A process analytics tool for biotransformations applying non-porous magnetic enzyme carriers

• A cheap and easily applicable FTIR method for immobilized enzyme analysis.
• Direct quantification of immobilized enzymes instead of indirect methods.
• Results for adsorptively and covalently immobilized α-Chymotrypsin are shown.

Biotransformation using immobilized enzymes on polymer beads has become interesting in recent years. Although having advantages like higher enzyme stability, easy enzyme separation and recovery, product streams without the used enzyme, and the possibility of continuous processes, the use of immobilized enzymes in big scale process volumes concentrates on well-known enzymes like Glucose isomerase, Lipase, Lactase and Penicillin G acylase, although there is a lot more potential for immobilized enzymes in preparative and industrial applications. For a broader use of immobilized enzymes in a repetitive way it is necessary to have a reliable process analytics with regard to the immobilizates. While working on the adoption of immobilized proteases for biotransformation reactions in biotechnological and pharmaceutical industrial processes, we found no established method to measure the amount of immobilized enzymes on the carrier directly, as mostly only the activity of the immobilizates is analyzed. We propose a method to quantify the immobilized enzymes directly on non-porous polyvinyl alcohol magnetic beads using well-established Fourier Transform Infrared Spectroscopy. The method utilizes the Attenuated Total Reflection mode and a multivariate, chemometric model. Two models, one for adsorptive and one for covalent immobilization of the protease bovine α-Chymotrypsin, are developed, demonstrating the effectiveness of the method for the use in process analytics.

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