Trehalose phosphorylase
A technology of phosphorylase and trehalose, applied in the field of phosphorylase
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Embodiment 1
[0795] Example 1: General method
[0796] Cloning of wild type TP: The trehalose phosphorylase gene from S. commune was codon optimized for expression in E. coli and synthesized from the Eurofins MWG operon. The gene was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen, Inc.). The resulting plastids were used for transformation of Escherichia coli BL21(DE3) cells.
[0797] Molecular biology methods: Mutants of TPase were generated by standard site-directed mutagenesis technology known in the art.
[0798] Expression of recombinant TP: by inoculating Medium I (4.6 g / L yeast extract, 9.3 g / L peptone, 25 mM Na 2 HPO 4 *12H 2 O, 25mM KH 2 PO 4 , 50mM NH 4 Cl 2 、Na 2 SO 4 , 5g / L glycerin, 0.5g / L glucose*1H 2 O, 2mM MgSO 4 , 50 μg / mL kanamycin) routinely expressed recombinant TP. Cultures were grown at 37°C until the optical density at 600nm was 0.6-0.8. Cultures were induced with IPTG at a final concentration of 0.1 mM. Expression was perform...
Embodiment 2
[0803] Example 2: Effect of sucrose on thermostability
[0804] Expression of recombinant TP: by inoculating Medium I (4.6 g / L yeast extract, 9.3 g / L peptone, 25 mM Na 2 HPO 4 *12H 2 O, 25mM KH 2 PO 4 , 50mM NH4 Cl 2 、Na 2 SO 4 , 5g / L glycerin, 0.5g / L glucose*1H 2 O, 2mM MgSO 4 , 50 μg / mL kanamycin) to express the wild-type enzyme SEQ ID NO:1 in shake flasks. Cultures were induced in log phase with 0.1 mM IPTG and expressed overnight at 24-25°C.
[0805] Preparation of TPase preparation: To prepare sucrose-free cell extracts, cells were harvested by centrifugation and suspended in buffer containing 50 mM potassium phosphate - buffer pH 7, 2 mM MgCl 2 , 0.5mg / mL lysozyme and 20U / mL nuclease. Cells are disrupted by sonication. Cell-free extracts containing soluble enzymes were separated from debris by centrifugation. To prepare cell extracts stabilized with sucrose, cells were harvested by centrifugation and suspended in a buffer containing 100 mM potassium phosphat...
Embodiment 3
[0807] Example 3: Residual activity of TP variants after incubation at 52°C for 15 minutes
[0808] Expression of recombinant TP: by inoculating medium I (4.6g / L yeast extract, 9.3g / L peptone, 25mM Na 2 HPO 4 *12H 2 O, 25mM KH 2 PO 4 , 50mM NH 4 Cl 2 、Na 2 SO 4 , 5g / L glycerin, 0.5g / L glucose*1H 2 O, 2mM MgSO 4 , 50 μg / mL kanamycin) and fresh overnight cultures to express recombinant TP in deep well plates. Cultures were grown at 37°C to an optical density at 600 nm of 0.6-0.8. Cultures were induced with IPTG at a final concentration of 0.1 mM. Express overnight at 24-25°C.
[0809] Preparation of TPase preparation: cells were harvested by centrifugation and suspended in 100 mM potassium phosphate buffer pH7, 2 mM MgCl 2 , 0.5mg / mL lysozyme and 20U / mL nuclease. Cells are destroyed by repeated freeze / thaw cycles. Cell-free extracts containing soluble enzymes were separated from debris by centrifugation. Dilute the cell-free extract 1:2 with 2M sucrose solution. ...
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