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PanD mutant gene, encoded protein, vector, engineering bacterium and application of encoded protein

A technology of genetically engineered bacteria and mutated genes is applied in the field of preparation of β-alanine to achieve high efficiency and clear breeding goals

Active Publication Date: 2017-05-31
浙江科峰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no molecular modification of PanD by random mutation of error-prone PCR to obtain high-activity PanD enzyme for the preparation of β-alanine

Method used

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  • PanD mutant gene, encoded protein, vector, engineering bacterium and application of encoded protein
  • PanD mutant gene, encoded protein, vector, engineering bacterium and application of encoded protein
  • PanD mutant gene, encoded protein, vector, engineering bacterium and application of encoded protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Construction of Escherichia coli panD gene mutant library

[0023] (1) Acquisition of the pET28b(+)-panD plasmid containing the E. coli panD gene: Escherichia coli BL21(DE3) / pET28b(+)-panD is the parent strain A (preservation number CCTCC NO: M206114, which has been filed in the patent application CN101210230A ) was inoculated in LB liquid medium, cultured on a shaker at 37°C and 200rpm for 12h, centrifuged at 8000rpm for 5min, collected bacterial cells, and used a plasmid extraction kit (purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. ) to extract the plasmid pET28b(+)-panD; get 2 μL of plasmid DNA to measure the concentration and purity of the sample with an ND5000 ultra-micro UV-visible spectrophotometer (Bioteke, USA); the LB liquid medium consists of: peptone 10g / L, yeast powder 5g / L, sodium chloride 5g / L, pH7.0, kanamycin 50μg / mL, water as solvent.

[0024] (2) Establishment of error-prone PCR reaction: (a) Design forward prime...

Embodiment 2

[0026] Embodiment 2: Obtaining of genetically engineered bacteria with high PanD production

[0027] The transformant and parent strain A in the mutant library constructed in Example 1 were respectively inserted into 3 mL of LB liquid medium, and lactose was added to a final concentration of 3 g / L, and cultured on a shaking table at 37° C. at 200 rpm for 1 d. After the culture, take 1.5mL of the culture solution into an EP tube, centrifuge at 8000r / min, 4°C for 10min, and collect the cells. Add 300 μL of L-Asp to make the final concentration 0.2 mol / L, mix well, and transform at 37° C., 200 rpm on a shaker for 12 hours. Take 50 μL of the transformation solution to detect the content of β-Ala and L-Asp according to the HPLC method described in the patent application CN101210230A, calculate the conversion rate, and screen out the mutant strain whose PanD enzyme activity and β-Ala production are significantly improved compared with the parent strain A. PanD enzyme activity is de...

Embodiment 3

[0034] Embodiment 3: Analysis of the panD gene sequence of the PanD high-yield mutant strain

[0035] The plasmids contained in the mutant strains 1-20, 2-30, 2-36, 3-48, 4-133, 6-83 and 6-85 screened in Example 2 were obtained by the extraction method described in Example 1, and Send to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for sequencing to determine the mutated panD gene sequence, and use DNAman software for sequence comparison; the tRNA abundance corresponding to the codon of the mutation site is obtained by querying Codon UsageDatabase, and the results are shown in Table 2. The nucleotide sequence of the mutant strain 1-20panD gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.7; the nucleotide sequence of the mutant strain 2-30panD gene is SEQ ID NO.2 As shown, the amino acid sequence of the encoded protein is shown in SEQ ID NO.8; the nucleotide sequence of the mutant strain 2-36panD gene is shown...

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Abstract

The invention relates to a panD mutant gene, encoded protein, a vector, an engineering bacterium and an application of the encoded protein. Series recombinant escherichia coli, for high-yield production of L-aspartate-[alpha]-decarboxylase (PanD), is obtained by changing codon base of the panD gene through random mutation of error-prone PCR. With the application of gene engineering bacterium for high-yield production of the PanD, the efficient transformation of the L-aspartate is achieved, so that [beta]-alanine can be prepared by virtue of a biological method, wherein PanD enzyme activity of recombinant escherichia coli numbered as 6-85 can reach 250U / L or above and [beta]-Ala yield can reach about 8.5g / L, nearly 4 times and 3 times above that of a parent strain.

Description

[0001] (1) Technical field [0002] The invention relates to the preparation of β-alanine, in particular to a series of panD mutant genes, encoded proteins, vectors, engineering bacteria and applications obtained by random mutation. [0003] (2) Background technology [0004] β-alanine, also known as β-alanine, was first discovered by Ross and Monroe in 1972 and is the only β-type amino acid in nature. β-alanine is an important chemical raw material and pharmaceutical intermediate, used in the synthesis of calcium pantothenate, carnosine, sweetener, etc., and also used as a water purification flocculant, electroplating corrosion inhibitor, etc. At present, the industrial production of β-alanine is mainly synthesized by chemical methods, which can be divided into acrylonitrile method, acrylic acid method, succinimide degradation method, β-aminopropionitrile method, etc. according to different synthetic raw materials. The high cost of chemical synthesis, strict process requireme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P13/06
CPCC12N9/88C12P13/06C12Y401/01011
Inventor 余志良裘娟萍王姗姗
Owner 浙江科峰生物技术有限公司
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