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Method for producing L-norvaline through enzymatic transformation

A technology for amino acid and leucine dehydrogenase, which is applied in the field of producing L-norvaline by enzymatic transformation, can solve problems such as being unsuitable for large-scale use, high NADH price, etc., and achieves strong product specificity and conversion. Efficient effect

Inactive Publication Date: 2017-03-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, LeuDH genetically engineered bacteria from different sources have been successfully constructed to achieve high-density expression and large-scale production of LeuDH. However, the large-scale application of LeuDH requires the addition of NADH as a coenzyme. NADH is expensive and not suitable for large-scale industrial use. Therefore, building an efficient NADH regeneration system is the key to solving the large-scale application of LeuDH

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: D-amino acid oxidase, leucine dehydrogenase and formate dehydrogenase primer design

[0041] According to the daao gene sequence in the whole genome nucleic acid sequence of Trigonopsis variabilis in NCBI, PCR primers P1 and P2 of daao gene were designed.

[0042] F: 5'-GG GGTACC AAAGGAGGGAAATCATGGGATCCCAAAAGAGGGTT-3'(Kpn Ⅰ)

[0043] R: 5'-CG GAATTC TTAGTGGTGGTGGTGGTGGTGGAGCTTAGACTCGCGGGC-3' (EcoR I)

[0044] According to the leudh gene sequence in the whole genome nucleic acid sequence of Bacillus cereus in NCBI, the PCR primers P3 and P4 of leudh gene were designed.

[0045] F: 5'-ACCG GGATCC ATGACATTAGAAATCTTCG-3'(BamH Ⅰ)

[0046] R: 5'-CGC GTC GAC TTAGCGACGGCTAATAATATC-3'(Sal Ⅰ)

[0047] According to the fdh gene sequence in the whole genome nucleic acid sequence of Candida boidinii in NCBI, PCR primers P5 and P6 of fdh gene were designed.

[0048] F: 5'-ACCG GGATCC ATGAAGATCGTTTTAGTC-3'(BamH Ⅰ)

[0049] R: 5'-CGC GTC GAC TTATTTCTTATCGTGTT...

Embodiment 2

[0050] Embodiment 2: Cloning of D-amino acid oxidase gene daao

[0051] Using the total DNA of Trigonopsis variabilis as a template, use the primers provided above for PCR amplification. The amplification conditions are: 94°C pre-denaturation, 5min, one cycle; 94°C denaturation, 1min, 58°C annealing, 1min, 72°C extension, 90s , 34 cycles; final extension at 72°C for 10 min. PCR amplification system: template 1 μL, upstream and downstream primers 0.4 μL, dNTP Mix 4 μL, 10×Ex Taq Buffer 5 μL, sterilized double distilled water 37 μL, Ex Taq DNA polymerase 1 μL. The PCR product was purified and recovered using a gel recovery kit, and the concentration of the recovered product was checked by electrophoresis. The recovered product was stored in a 1.5ml centrifuge tube and stored in a -20°C refrigerator for later use. The recovered product was ligated with pMD18-T Vector, and the ligated product was transformed into E.coil JM109, and the transformed product was coated on an LB plat...

Embodiment 3

[0052] Embodiment 3: Cloning of leucine dehydrogenase gene leudh and formate dehydrogenase gene fdh

[0053] Using the total DNA of Bacillus cereus and Candida boidinii as templates, use the primers provided above for PCR amplification. The amplification conditions are: 94°C pre-denaturation, 5min, one cycle; 94°C denaturation, 1min, 58°C annealing, 1min, 72°C extension, 90s, 34 cycles; 72°C final extension 10min. PCR amplification system: template 1 μL, upstream and downstream primers 0.4 μL, dNTP Mix 4 μL, 10×Ex Taq Buffer 5 μL, sterilized double distilled water 37 μL, Ex Taq DNA polymerase 1 μL. The PCR product was purified and recovered using a gel recovery kit, and the concentration of the recovered product was checked by electrophoresis. The recovered product was stored in a 1.5ml centrifuge tube and stored in a -20°C refrigerator for later use. The recovered product was ligated with pMD18-T Vector, and the ligated product was transformed into E.coil JM109, and the tra...

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Abstract

The invention discloses a method for producing L-norvaline through enzymatic transformation. Through shuttle plasmids pXMJ19 between escherichia coli and corynebacterium crenatum, D-amino acid oxidase derived from Trigonopsis variabilis is successfully expressed in C. glutamicum ATCC13032, through construction of recombinant plasmids pET-28a(+)-leudh and pET-28a(+)-fdh, leucine dehydrogenase and formate dehydrogenase derived from Bacillus cereus and Candida boidinii are successfully expressed in E. coli BL21, and in the presence of a crude enzyme liquid of the three engineering strains as a biocatalyst, the mixed DL-norvaline as a substrate is subjected to enzymatic kinetic resolution under the optimum transformation conditions of a reaction temperature of 30 DEG C and pH of 7.5 so that L-norvaline is produced, wherein after 25h, the content of L-norvaline in the transformation liquid is 80.09g / L and the yield of L-norvaline is 98.3%. The method for producing L-norvaline has the advantages of high efficiency, environmental friendliness and high product purity.

Description

Background technique [0001] Chirality is one of the essential properties of nature, and the molecules that make up living organisms are all asymmetric chiral molecules. In the pharmaceutical industry, enantiopure compounds with pharmacological activity are called chiral drugs. When drugs of different configurations act on organisms, they play completely different roles, and there are also significant differences in drug activity, metabolic process, and toxicity. It fully demonstrates that the study of drugs in the form of single enantiomers is very necessary. [0002] L-norvaline (L-norvaline) is a non-protein branched-chain amino acid, which is widely used in the pharmaceutical industry as a precursor for the synthesis of many drugs. Perindopril, as an effective drug for treating hypertension and heart failure, is synthesized by using L-norvaline as an important intermediate. Currently, L-norvaline is mainly synthesized by chemical methods. There are many defects in the c...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/06C12N9/02C12P13/08C12R1/15C12R1/19
CPCC12N9/0024C12N9/0008C12N9/0016C12P13/08C12Y102/01002C12Y104/01009C12Y104/03003
Inventor 饶志明戚云龙杨套伟周俊平郑俊贤徐美娟张显
Owner JIANGNAN UNIV
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