Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

Conference Dates

September 15-19, 2019


D-Tagatose is a rare ketohexose sugar with sweetness similar to that of sucrose. However, its glycemic index and caloric value is much lower because of low bioavailability, making it an attractive GRAS (generally regarded as safe) sugar substitute. Recent studies have also demonstrated that it is anti-hyperglycemic and prebiotic, which promotes gut health. Thus, there exists a high demand in food industry for the economical production of rare sugars, like tagatose.

The enzyme L-arabinose isomerase (LAI) that responsible for the reversible isomerization of the pentose L-arabinose to L-ribulose can also isomerize the hexose D-galactose to D-tagatose. LAI has thus been the enzyme of choice to produce tagatose, although, to date, few commercial bioprocesses exist. A variety of LAIs from different microorganisms have been isolated and have reported optimal activity at a range of temperatures and pH. Some of the limitations of tagatose biosynthesis using LAI that may be hindering commercial viability are, 1) unfavorable enzymatic kinetics since galactose is not the native substrate of LAI, 2) low enzyme stability, particularly in the absence of divalent metal ions, and 3) low equilibrium constant for galactose to tagatose isomerization.

Few previous reports have been successful at engineering enzymatic properties of LAI for industrial application; often addressing only one of the bottlenecks to productivity. To address the kinetic issue, several groups have used enzyme engineering methods to enhance catalytic efficiency of LAI toward galactose and have shown moderate increases in productivity. To counter low-stability issues, many groups have tested the utility of thermophilic enzymes. However, most thermophilic enzymes rely on divalent metal ions (Mn2+, Co2+, Fe2+) for stability, and high reaction temperatures (≥ 80 °C) result in significant caramelization, which are all undesirable and must be removed from product, adding to processing costs. Surface-display or encapsulation in particles or whole-cells can stabilize enzymes. Finally, the thermodynamic limitations of isomerization of galactose to tagatose are severe and, arguably, the most recalcitrant issue since ΔG°rxn ≈ +1.2 kcal/mol, which indicates theoretical maximum equilibrium conversion ~ 14 % at room temperature. Several approaches have been used to overcome this limitation. Thermophilic enzymes can achieve higher conversions than mesophilic enzymes since the equilibrium shifts toward tagatose at higher temperatures. Whole-cell biocatalysts with GRAS organisms (e.g. lactic acid bacteria (LAB) and E. coli) that disproportionately partition substrate and product across their membrane has also been shown to partially circumvent this thermodynamic limitation while simultaneously enhancing enzyme stability; albeit at a kinetic penalty imposed by substrate transport limitations. Recently, cell permeabilization and sugar transport overexpression were demonstrated as methods to overcome the kinetic penalty imposed by cellular encapsulation.

There have currently been no studies that look to systematically analyze all three limitations – kinetic, thermodynamic, and enzyme stability – of the enzymatic isomerization of galactose to tagatose. This work clearly demonstrates the presence of these three limitations and provides a novel approach to balance their advantages and limitations. We use the food-safe engineered probiotic bacterium Lactobacillus plantarum as the expression host due to its increasing relevance to biochemical and biomedical research. This approach enabled ~ 50 % conversion of galactose to tagatose in 4 h (productivity of ~ 38 mmol tagatose L-1 h-1) ultimately reaching ~ 85 % conversion after 48 h at high galactose loading (300 mM) in batch culture. This is among the highest conversions and productivities reported to date for tagatose production using a mesophilic enzyme. Such an approach is expected to be applicable to other biocatalytic systems where similar trade-offs between kinetics, thermodynamics, and/or stability pose hurdles to process development.

This work is currently under consideration for publication: Bober & Nair (2019) Nature Communications (in revision)


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