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Fusion protein as well as coding gene and application thereof

A fusion protein and gene technology, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of increased reaction cost

Inactive Publication Date: 2014-12-10
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the reduced coenzyme in the system needs to be consumed in the catalytic process of the enzyme, the additional addition of the reduced coenzyme will significantly increase the reaction cost

Method used

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  • Fusion protein as well as coding gene and application thereof
  • Fusion protein as well as coding gene and application thereof
  • Fusion protein as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the acquisition of fusion protein and its coding gene

[0031] In this study, alcohol dehydrogenase (ADH) from Lactobacillus brevis with high catalytic activity and strong enantioselectivity and formate dehydrogenase (FDH) from Candida boidinii) were selected according to According to the codon preference principle of Escherichia coli, the codons of the two enzyme genes were optimized separately. A rigid structural linker (EAAAK) will be added between alcohol dehydrogenase and formate dehydrogenase 2 , to obtain the fusion protein FDH-linker-ADH, the amino acid sequence of the fusion protein is sequence 2 in the sequence list. The coding gene of the fusion protein is sequence 1 in the sequence list.

[0032] Wherein the sequence 2 in the sequence listing is alcohol dehydrogenase from the 375th to 625th position of the N' end; the 365th to 374th position from the N' end is a linker; the 1st to 364th position from the N' end is a formate dehydrogenase.

...

Embodiment 2

[0034] Embodiment 2, the application of fusion protein and its coding gene

[0035] 1. Construction of dual-enzyme co-expression vector

[0036] 1. Construction of recombinant vector pETDuet-FDH-ADH

[0037] The FDH-containing vector pGH-FDH (containing the FDH sequence, the entire sequence is sequence 3) was digested with NdeI and Xhol; 3) Skeleton connection to obtain the intermediate vector pETDuet-FDH;

[0038] The ADH-containing vector pGH-ADH (containing the ADH sequence, the full sequence is sequence 4) was digested with BamHI and NotI; the obtained 768bp digested product was connected with the 6484bp intermediate vector pETDuet-FDH backbone that had undergone the same digestion to obtain a recombinant vector pETDuet-FDH-ADH (correctly identified by enzyme digestion).

[0039] 2. Construction of recombinant vector pETDuet-FDH-linker-ADH

[0040] 以pGH-FDH为模板,以FDHF:GACCATGGGCAAAATCGTTCTGGTTCTG,FLAR:TTTGGCCGCTGCTTCTTTGGCCGCTGCTTCTTTTT TGTCGTGTT为引物,得到1124bp的PCR产物;以pGH-A...

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Abstract

The invention discloses a fusion protein as well as a coding gene thereof and application thereof. The fusion protein provided by the invention is protein which is obtained by in series connecting the connecting peptides of ethanol dehydrogenase and formate dehydrogenase. The ethanol dehydrogenase is from lactobacillus brevis and the formate dehydrogenase is from candida boidinii; the connecting peptides are rigid structures Linker (EAAAK). According to the experiment in the invention, the invention provides the fusion protein which is obtained by in series connecting the connecting peptides of ethanol dehydrogenase and formate dehydrogenase, wherein enzyme activity of the formate dehydrogenase is higher than the activity for independently expressing the formate dehydrogenase after the formate dehydrogenase is expressed in escherichia coli; moreover, the stability and heat stability of the ethanol dehydrogenase in organic substrate are also improved. The construction of the fusion protein provides important reference for the improvement to the preparation method for chiral alcohol and chiral hydroxy compound.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein and its encoding gene and application. Background technique [0002] Chiral alcohols with specific functional groups play an important role in the preparation of chiral drugs, and they are important intermediates for a variety of chiral drugs that are antiviral, antifungal, antitumor, and for the treatment of nervous system, cardiovascular and other diseases. Therefore, the maturity and improvement of chiral alcohol synthesis methods have a positive effect on the synthesis of chiral drugs. [0003] Due to the high catalytic efficiency of the enzyme catalysis method, it is generally more than 100 times that of the inorganic catalyst, and the purity of the product is very high, the reaction conditions are mild, and it can generally react at room temperature. It has been widely concerned in the research on the preparation of sex alcohol. Alcohol dehydrogenase has the ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N9/02C12N15/62C12N15/53C12N15/63C12N5/10C12N1/21C12R1/24C12R1/72C12R1/19
Inventor 李玉霞陈惠鹏曲芬凌焱李炳娟岳俊杰孙芳毛艳张景海吴逊白东梅李伟东刘刚梁龙周围李北平高原
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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