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Recombinant R-type transaminase, mutant and application thereof

A transaminase and mutant technology, which is applied in the field of recombinant R-ω-transaminase and mutants, can solve the problems of harsh reaction conditions, high cost of separation and high residual rate of metal ions, and achieves the effect of high activity

Active Publication Date: 2020-03-24
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of this method are that the process steps are cumbersome, the reaction conditions are harsh, the catalyst used is expensive, the residual rate of metal ions is high, the cost of product purification and separation is too high, the optical purity of the product is low (e.e.<97%), and the environmental pollution is serious.

Method used

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  • Recombinant R-type transaminase, mutant and application thereof
  • Recombinant R-type transaminase, mutant and application thereof
  • Recombinant R-type transaminase, mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0028] Example 1: Screening of novel ω-TA, determination of stereoselectivity and precise determination of enzyme activity

[0029] 1. Enzyme source and gene synthesis

[0030] Using gene mining technology, three new enzymes were obtained from NCBI database gene mining, respectively from Mycolicibacteriumgoodii (MgTA, Genbank number WP_049743179.1), Amine transaminaseHFO (HFO, Genbank number AIN35005.1) and Mesorhizobium japonicum (MjTA, Genbank number WP_010910285 .1). Codon optimization was carried out according to the codon preference of E.coli, and the nucleotide sequences of the three enzymes were synthesized by the method of whole gene synthesis, which are respectively shown in SEQ ID NO.2, 4 and 6; the amino acid sequences of the encoded enzymes were respectively As shown in SEQ ID NO.1, 3 and 5. Add 6×his-tag tags at the end of the nucleotide sequence, add restriction sites Xho I and Nco I at both ends, clone the gene into the Xho I and NcoI sites corresponding to pE...

Embodiment 3

[0045] Example 3: Recombination and screening of enzymes

[0046] The research group applied for the high-efficiency R-selective Gibberellazeae TA mutant (hereinafter referred to as GzTA) in the patent CN201910871261.3 7 ), the activity to sitagliptin precursor ketone is 210.3U / g, can catalyze the conversion of 200mM precursor ketone into sitagliptin, and the conversion rate is 82.6%. (GzTA 7 The nucleotide sequence of the enzyme is shown in SEQ ID NO.8, and the amino acid sequence of the encoded enzyme is shown in SEQ ID NO.7). In the present invention, DNA recombination is carried out respectively with MjTA and MgTA to obtain new mutants.

[0047] 1. Clone GzTA 7 , MjTA and MgTA catalytic regions

[0048] (1) GzTA synthesized as needed 7 Design primers for the nucleotide sequence fragments, and use rapid PCR technology to recombine the vector pET28b / GzTA 7 As a template, synthesize GzTA 7 The N-terminal catalytic region (referred to as A 1 ) DNA fragment, the primers...

Embodiment 4

[0112] Example 4: Construction and screening of TAmutant-4 single point mutants

[0113] 1. Construction of mutants

[0114] The R-ω-TA screened in the mutant library was subjected to single-point mutation, and primers for site-directed mutation were designed according to the amino acid sequence of TAmutant-4 (SEQ ID NO.15), and the recombinant vector pET28b / TAmutant- 4 is a template, and a single point mutation is introduced at position 71 of the amino acid sequence of TAmutant-4, and the primers are:

[0115] Forward primer 71H: GTTCTTCCGT NNK GACGACCAC (the underline is the mutated base)

[0116] Reverse primer 71H: GTGGTCGTC MNN CGGAAGAAC (the underline is the mutant base)

[0117] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ )25μL, dNTPs10mM, forward primer 71H 2μL, reverse primer 71H 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0118] The PCR amplification conditions were 95°C for 3min; (95°C for 15s,...

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Abstract

The invention relates to recombinant R-omega-transaminase, a mutant and an application of the recombinant R-omega-transaminase and the mutant in asymmetric synthesis of sitagliptin. According to the invention, novel R-omega-transaminase is screened and recombined with the existing high-efficiency transaminase; the amino acid sequence of the obtained recombinant is shown as SEQ ID: 15, and the mutant thereof is obtained by carrying out single-site mutation or multi-site mutation on one or more of 71, 135 and 292 amino acid sequences shown in SEQ ID: 15; the optimal amino acid sequence of the R-omega-transaminase mutant strain is shown as SEQ ID: 17; and the corresponding nucleotide sequence is shown as SEQ ID: 18. The invention provides the R-omega-TA mutant with higher activity (582.4U / g) and stereoselectivity (e.e.value 99.9%). A 500mM substrate can be catalyzed to be converted into sitagliptin, and the conversion rate is as high as 99%. The mutant has an important significancefor improving the sitagliptin biocatalytic preparation technology.

Description

[0001] (1) Technical field [0002] The present invention relates to a recombinant R-ω-transaminase and its mutant, and its application in the asymmetric synthesis of sitagliptin. [0003] (2) Background technology [0004] Sitagliptin, English name sitagliptin, full name (3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo [4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one, a dipeptidyl group researched and developed by Merck and Codexis Peptidase-4 (DPP-4) inhibitors can control blood sugar levels by protecting endogenous incretins and enhancing their effects. Compared with sulfonylurea drugs and metformin drugs, it has more stable hypoglycemic function, less side effects, and better clinical effect, and can be effectively used in the treatment of type 2 diabetes, and is a therapeutic drug with great potential for type 2 diabetes. [0005] The preparation of sitagliptin mainly includes chemical synthesis and asymmetric synthesis. Chemical synthesis ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P17/18
CPCC12N9/1096C12P17/182C12Y206/01
Inventor 柳志强贾东旭郑裕国李军良彭晨徐海鹏孙晨奕
Owner ZHEJIANG UNIV OF TECH
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