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Immobilized transaminases and process for making and using immobilized transaminase

A technology for immobilizing transaminase and transaminase is applied in the fields of immobilized transaminase and production and use of immobilized transaminase, and can solve the problems of inability to reuse catalyst, discarding, lack of stability, etc.

Inactive Publication Date: 2014-02-26
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although advances in the generation of chiral amines using transaminases have been of high interest, there are still certain drawbacks to the enzymatic approach
Currently, enzymatic methods can only be performed in aqueous solvent systems due to the instability of transaminases in 100% organic solvents
Furthermore, during the separation of the product amines, the transaminase catalyst is deactivated and discarded, making it impossible to reuse the catalyst
[0008] Thus, although attempts have been made to immobilize transaminases, they have never been successful in overcoming their lack of stability, more specifically their lack of stability in organic solvents

Method used

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  • Immobilized transaminases and process for making and using immobilized transaminase
  • Immobilized transaminases and process for making and using immobilized transaminase
  • Immobilized transaminases and process for making and using immobilized transaminase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-5

[0187] Example 1-5: Immobilization of Transaminase

[0188] Five different resins as shown in Table 2 were evaluated.

[0189] Table 2

[0190] Example Resin combination 1 SEPABEADS EC-EP Polymethacrylate / epoxide 2 SEPABEADS EC-HFA / S Polymethacrylate / amino epoxide 3 SEPABEADS EXA252 Styrene / DVB copolymer 4 SEPABEADS EXE119 Polymethacrylate / epoxide 5 SEPABEADS EXE120 Polymethacrylate / octadecyl

[0191] A 25 g / L solution of SEQ ID NO: 110 with 1 g / L PLP (pyrodoxial-5-phosphate) in 100 mM potassium phosphate buffer (pH 7.5) was prepared. Incubate 1 g of each resin with 5 mL of enzyme solution in a shaker at room temperature overnight. The resin was filtered from the transaminase solution and rinsed 5 times with 5 mL of 100 mM potassium phosphate buffer (pH 7.5). The resin performance in the following transaminations was then evaluated compared to the freeze-dried enzyme preparation:

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[0193] The immobilized transaminase preparation exhibits superior specific activity co...

Embodiment 6

[0194] Example 6: Evaluation of the drying method of SEPABEADS EXE120 resin and confirmation of the activity of the immobilized enzyme in a 100% organic solvent system

[0195] The immobilized enzyme (SEQ ID NO: 110 / SEPABEADS EXE120 resin) was dried under vacuum and purged with nitrogen to remove water from the outer surface of the immobilized enzyme resin. The immobilized preparation is stirred while drying to allow a uniform moisture content throughout the immobilized enzyme bed, and to prevent any part of the immobilized enzyme preparation from over-drying or insufficiently drying. 100 mg of ketoamide substrate was dissolved in 1 mL of water-saturated IPAc (isopropyl acetate). Add 40 uL IPM (isopropylamine) to the reaction solution. 100 mg of dried immobilized transaminase (SEQ ID NO: 110 / SEPABEADS EXE120 resin) was added to the reaction. The reaction was stirred at 1000 rpm in a Thermomixer at 50°C. After 70 hours, a sample was taken and the conversion rate and ee were det...

Embodiment 7

[0196] Example 7: Plug flow reaction (PFR) method for the production of sitagliptin in IPAc

[0197] Prepare 10 mL of ketonamide substrate solution (100g / L-400g / L ketone, 40uL / mL-160uL / mL isopropylamine in IPAc). 0.75 g of immobilized transaminase (SEQ ID NO: 110 / SEPABEADS EXE120 resin) slurry was packed in the column using IPAc under gravity. The substrate solution was added to the column via a syringe pump, which was layered at 50°C. Set the flow rate to 0.1 mL / h.

[0198] In steady state, sitagliptin amine was observed at the exit of the column (> 99% ee) 85-90% conversion rate.

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Abstract

The invention is directed to immobilized transaminases and methods of making and using them.

Description

field of invention [0001] The present invention relates to immobilized transaminases and methods of producing and using them. Background of the invention [0002] Enzymes are protein molecules used to speed up chemical reactions (usually by several orders of magnitude) in living cells. In the absence of enzymes, most biochemical reactions would be so slow that even life processes would not be possible. Enzymes display enormous specificity and are not permanently modified by their participation in reactions. Since they do not change during the reaction, enzymes can be used cost-effectively as catalysts for desired chemical transformations. [0003] Transaminases are a specific class of enzymes that catalyze the direct amination of ketones to chiral amines. Enantiomerically pure chiral amines are key intermediates in many pharmaceutical compounds with a wide range of biological activities. Many efforts are currently underway to utilize biocatalysts to develop efficient cat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12Q1/52
CPCC12N9/1096C12P17/182C12N11/08C12N11/082C12N11/087C12P13/001Y02P20/52
Inventor M.D.特拉波J.M.珍妮G.休斯
Owner MERCK & CO INC
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