期刊
CHEMICAL ENGINEERING JOURNAL
卷 427, 期 -, 页码 -出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.cej.2021.131439
关键词
Multi-enzyme cascade; Monolithic microreactors; Trehalose; NDP-glucose; UDP-glucose; Modular production system
资金
- National Centre for Research and Development (NCBiR, Poland) [ERA-NET-IB/NO-P/11/2016]
- Saxonian Government [100263899]
- Federal Ministry for Innovation, Science and Research of North Rhine-Westphalia [PtJ-TRI/1411ng006]
- ERANET on Industrial Biotechnology [ERA-IB-15-110]
In this study, a two-step continuous flow enzymatic synthesis process was demonstrated using basic sugars as substrates in monolithic microreactors. Immobilisation of the enzymes on functionalised silica supports was found to be most effective through covalent bonding by amino groups using a glutaraldehyde linker. The process allows for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides, with high space-time-yield values achieved for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions.
Here, we present a two-step continuous flow enzymatic synthesis process in monolithic microreactors using basic sugars as substrates. In the first step UDP-glucose pyrophosphorylase (TaGalU) catalyses the synthesis of uridine-diphosphate-glucose (UDP-Glc) using uridine triphosphate (UTP) and glucose-1-phosphate (Glc-1-P). This is followed by the trehalose transferase (mCherry-TuTreT) catalysed reaction of UDP-Glc and Glc, to obtain trehalose. First, procedures for immobilisation of both enzymes on functionalised silica supports were studied and it was found that covalent bonding by amino groups using a glutaraldehyde linker gives highly active biocatalysts. Due to a drastic difference in temperature range of activity and stability of the immobilised enzymes a bi-reactor cascade was rationally the best solution. Depending on the applied flow rate and hence reaction (residence) time (1.5-10 min) the space-time-yield values varied, respectively, from 1.9 to 14.4 and 8.3 to 49.6 g(product).L-1.h(-1).mg(protein)(-1), for UDP-glucose pyrophosphorylase and trehalose transferase catalysed reactions. Prolonged (100 h) continuous flow operation showed that the system is operationally stable, but owing to neutral pH, it is prone to microbiological infections. They can be eliminated applying an antibacterial/antifungal therapy or preventive actions by storing and washing the reactors with a NaN3 solution. The presented process paves the way for the continuous in flow synthesis of natural and non-natural trehalose analogues and disaccharides.
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