Abstract
BackgroundWhole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water.ResultsTo solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA) from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP) anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC). The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution.ConclusionIn vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.
Highlights
Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses
During immobilization, enzyme activity and natural properties are lost and the reusability of the immobilized enzyme is limited. As these issues can be overcome by using whole cells, whole-cell biocatalysis has been widely adopted for the commercial synthesis of a variety of compounds, from bulk chemicals to valuable pharmaceuticals [2,3] or for bioremediation [4]
A thermostable lipase (TliA) from Pseudomonas fluorescens SIK W1 [27] was expressed on the surface of Pseudomonas putida GM730 [28] using ice-nucleation protein (INP) fusion, and the in vivo immobilized enzyme was shown to serve as a whole cell biocatalyst for three representative lipase reactions: lipid hydrolysis in a two-phase reaction, triacylglycerol synthesis without water and chiral resolution in organic solvent
Summary
To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA) from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP) anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC). Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution
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