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Transaminase mutant, immobilized transaminase and application of immobilized transaminase to preparation of sitagliptin

A technology for immobilizing transaminase and sitagliptin, applied in the preparation of sitagliptin or its intermediates, in the field of preparation of sitagliptin, can solve the problem of high conversion rate, non-recyclable use and low activity of transaminase , Enzyme activity is not high, etc., to achieve the effect of increased reusability, simple operation, and good stability

Pending Publication Date: 2021-07-02
ABIOCHEM BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The technical problem to be solved by the present invention is to overcome the low enzymatic activity of transaminase in the prior art, low enzymatic activity and poor stability when it is prepared as an immobilized enzyme, and then used to catalyze the ketoamide substrate to produce sitagliptin When the conversion rate of its intermediate is high and it cannot be reused, a kind of transaminase mutant, immobilized transaminase and its use for preparing sitagliptin or its intermediate are provided

Method used

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  • Transaminase mutant, immobilized transaminase and application of immobilized transaminase to preparation of sitagliptin
  • Transaminase mutant, immobilized transaminase and application of immobilized transaminase to preparation of sitagliptin
  • Transaminase mutant, immobilized transaminase and application of immobilized transaminase to preparation of sitagliptin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1 A kind of preparation of transaminase mutant enzyme liquid

[0080] The genes shown in the nucleotide sequences of SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8 were synthesized, and the gene preparation company was Suzhou Jinweizhi Biotechnology Co., Ltd. (Suzhou Industrial Park Building C3, Bio-Nano Technology Park, No. 218 Xinghu Street). The above-mentioned genes respectively code for SEQ ID NO.1 in the sequence list (ie, SEQ ID NO:110 in US8293507), SEQ ID NO.3, SEQ ID NO.5 (ie, SEQ ID NO:130 in US9617573), SEQ ID NO .7 Transaminases indicated.

[0081] table 3

[0082]

[0083]

[0084] Then synthesize gene enzyme-linked pET28a, restriction site NdeI&HindIII, and enzyme-linked vector to transform host Escherichia coli BL21 competent cells. Inoculate the constructed strain into TB culture based on 37°C, 200rpm shaker, induce overnight with IPTG concentration of 0.1mM, collect the strains, and obtain engineering strains containing tran...

Embodiment 2

[0102] Embodiment 2 Preparation of immobilized transaminase mutant

[0103] Take 12g of transaminase mutant sludge, add 120mL of 100mM phosphate buffer, mix evenly under high pressure and homogeneously break the cells, centrifuge to collect the supernatant enzyme solution, add 22gK 2 HPO 4 ·3H 2 O, 2.2gKH 2 PO 4 and 0.024g of PLP, stir to dissolve, add 10g of resin (see Table 5 for details of resin type, all purchased from Suzhou Huitong Chromatography Separation and Purification Co., Ltd.) Immobilized enzyme was obtained by suction filtration. The enzyme activity detection of the immobilized enzyme was carried out with morpholinodione and sitadione as substrates respectively, and the enzyme activity detection method was as follows:

[0104] Weigh 1.2g of morpholinodione or sitadione substrate into a 50mL Erlenmeyer flask, add 19mL of isopropanol, 0.6mL of 16mg / mL PLP solution and 0.4mL of isopropylamine, shake on a shaker at 45°C until it is completely dissolved, and coo...

Embodiment 3

[0109] Immobilized enzyme (resin: EC HFA) for the catalysis of morpholinodione

[0110] In the conical flask, add 25mL isopropanol, 2.5g morpholino, 2mL water, 32mgPLP, 1.135mL isopropylamine, 10g immobilized enzyme prepared in Example 2, and react on a shaking table at 45°C and 200rpm. After 24 hours of reaction, samples were taken to detect the conversion rate. Filter the reaction liquid to obtain immobilized enzyme, continue to add 25mL of isopropanol, 2.5g of morpholino, 2mL of water, 32mg of PLP, 1.135mL of isopropylamine, 45 ℃, 200rpm shaker reaction, react for 24 hours and take samples to detect the conversion rate. The immobilized enzyme was repeated according to the above method. The results are shown in Table 6, which shows that the conversion rate of Enz.1-M122Q-P233T and Enz.2-M122F remained above 85% and the ee value was >99.9%. Enzase is stable; Enz.1 and Enz.2 (the effect is not good after applying mechanically for 3 times) have low conversion rate and are n...

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Abstract

The invention provides application of immobilized transaminase in preparation of sitagliptin and / or (R)-3-amino-1-morpholine-4-(2,4,5-trifluorophenyl)-1-butanone. The immobilized transaminase comprises resin and a transaminase mutant, wherein the amino acid sequence of the transaminase mutant is shown as SEQ ID NO. 3 or SEQ ID NO. 7. The invention also provides the immobilized transaminase, a transaminase mutant and a preparation method and application of the immobilized transaminase and the transaminase mutant. The transaminase mutant is very high in enzyme activity when catalyzing a ketoamide substrate; the enzyme activity is still very high after the transaminase mutant is made into immobilized transaminase; and when the transaminase mutant is used for catalyzing a ketoamide substrate to produce sitagliptin or an intermediate thereof, a screened solvent reaction system is combined, and the immobilized transaminase is high in conversion rate, good in stereoselectivity, good in stability, higher in reusability and simpler to operate, so production cost is reduced, and industrial production is facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a transaminase mutant, an immobilized transaminase, and the application of the immobilized transaminase in the preparation of sitagliptin or its intermediate, and the invention also relates to a preparation of sitagliptin method. Background technique [0002] Diabetes mellitus is a metabolic disease that occurs due to changes in insulin secretion, leading to insulin deficiency and weakened action, or decreased insulin activity, or under the combined influence of the two. It is characterized by high blood sugar and accompanied by protein, sugar and fat metabolism. disorder. Diabetes and its complications are the third most harmful to human health after cardiovascular diseases and tumors, becoming an important disease that endangers human health. Among the four types of diabetes, type II diabetes accounts for more than 90%, and is more common in middle-aged and elderly peo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N11/089C12N15/70C12N1/21C12P17/18C12P17/14C12R1/19
CPCC12N9/1096C12Y206/01C12N11/08C12N15/70C12P17/182C12P17/14C12Q1/52
Inventor 田振华程占冰丁少南焦琦郝聚喜冀勇良
Owner ABIOCHEM BIOTECH CO LTD
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