Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis
Josef R. Bober & Nikhil U. Nair
Received Date: 14th February 19
There are many enzymes that are relevant for making rare and valuable chemicals that while active, are severely limited by thermodynamic, kinetic, or stability issues (e.g. isomerases, lyases, transglycosidases, etc.). In this work, we study an enzymatic reaction system − Lactobacillus sakei L-arabinose isomerase (LsLAI) for D-galactose to D-tagatose isomerization – that is limited by all three reaction parameters. The enzyme has a low catalytic efficiency for non-natural substrate galactose, has low thermal stability at temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies to overcome these limitations, we finally show that encapsulating the enzyme in a gram-positive bacterium (Lactobacillus plantarum) that is chemically permeabilized can enable reactions at high rates, high conversion, and at high temperatures. The modified whole cell system stabilizes the enzyme, differentially partitions substrate and product across the membrane to shift the equilibrium toward product formation enables rapid transport of substrate and product for fast kinetics. In a batch process, this system enables approximately 50 % conversion in 4 h starting with 300 mM galactose (average productivity of 37 mM/h), and 85 % conversion in 48 h, which are the highest reported for food-safe mesophilic tagatose synthesis. We suggest that such an approach may be invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-, and/or stability-limited.
Read in full at bioRxiv.
This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.