Steps towards the rational design of an immobilized biocatalyst with improved process stability
Integration of life sciences & engineering
Bio-transformation in the Laboratory and in Large Scale Production (T5-3)
Keywords: biocatalysis, immobilization, (process) stability, D-amino acid oxidase
The implementation of biocatalysis in large-scale industrial processes is widely seen as a major step towards a greener chemistry. Biocatalysts offer several advantages as compared to their chemical counterparts and a number of biocatalytic processes has been developed also on large scale. However, many potentially interesting enzymes fail to meet the requirements of an industrial process, especially concerning operational stability. Enzyme immobilization techniques have been used for several decades to overcome these limitations. Immobilization facilitates downstream processing and allows for the repetitive use of the enzyme or its use in continuous reactor systems. Furthermore enzymes are usually stabilized through immobilization. However, these beneficial effects are often overcompensated by the high costs of immobilization and a severe loss in specific enzyme activity. Mass transport phenomena limiting – among other effects – the activity of the biocatalyst as well as the underlying effects of stabilization have clearly been underexplored so far. Lacking mechanistic understanding, rational improvement of current immobilization techniques remains a distant prospect.
We have used D-amino acid oxidase as an example of a biocatalyst of industrial relevance to study and dissect the stabilizing effects in different immobilized enzyme forms. Immobilized D-amino acid oxidase from the yeast Trigonopsis variabilis (TvDAO) is used in a multi-ton-per-year biocatalytic process for the conversion of cephalosporin C to 7-amino cephalosporanic acid, a valuable building block for semisynthetic cephalosporin antibiotics. Although TvDAO is considered to be a comparably robust O2-dependent biocatalyst, its operational stability is not fully satisfactory and the overall economics of the above-mentioned process could significantly benefit from an increase in total turnover numbers.
Different immobilization techniques have been used in this study: covalent immobilization of the enzyme on an epoxy-activated support; encapsulation in semipermeable microcapsules [1]; and directed immobilization via an affinity tag [2]. Using a mechanistic model of the inactivation process [3, 4], we have performed a detailed kinetic analysis of the thermal inactivation of free and immobilized TvDAO forms. This comparative study was complemented by experiments in a miniaturized reactor system under operational and process-near conditions. Dissecting and understanding the various beneficial effects of immobilization on TvDAO stability will provide a mechanistic tool for rational stabilization of this and possibly other industrially important enzymes for improved process performance.
________________
[1] Nahalka J, Dib I, and Nidetzky B. 2007. Biotechnol Bioeng. submitted.
[2] Dib I, Stanzer D, Nidetzky B. 2007. Appl Environ Microbiol. 73(1):331-3.
[3] Dib I, Slavica A, Riethorst W, Nidetzky B. 2006. Biotechnol Bioeng. 94(4):645-54.
[4] Slavica A, Dib I, Nidetzky B. 2005. Appl Environ Microbiol. 71(12):8061-8.
Presented Thursday 20, 15:00 to 15:20, in session Bio-transformation in the Laboratory and in Large Scale Production (T5-3).
