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Genetic engineering bacteria containing L-alanine racemase genes and application of genetic engineering bacteria

A technology of alanine racemase and genetically engineered bacteria, applied in the field of bioengineering, can solve problems such as low yield, long synthetic route, and difficult separation of by-products, and achieve low dosage, short reaction time, and good industrialization foreground effect

Active Publication Date: 2014-06-04
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of these synthetic methods is high, and the use of harmful substances such as cyanide, methanol, and chloroform, and the separation of by-products are also very difficult; at the same time, it also exposes the long synthetic route, low yield, and serious "three wastes" problems, etc. defect

Method used

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  • Genetic engineering bacteria containing L-alanine racemase genes and application of genetic engineering bacteria
  • Genetic engineering bacteria containing L-alanine racemase genes and application of genetic engineering bacteria
  • Genetic engineering bacteria containing L-alanine racemase genes and application of genetic engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Extraction of Genomic DNA of Lactobacillus fermentum CGMCC2921.

[0038]According to the instruction manual provided by the manufacturer, the genomic DNA of Lactobacillus fermentum CGMCC2921 in the logarithmic growth phase was extracted with the Genomic DNA Purification Kit (Takara, Dalian), and analyzed by 1% (10 g / L) agarose gel electrophoresis. The obtained genomic DNA was tested. see results figure 1 .

Embodiment 2

[0039] Example 2: Cloning of L-alanine racemase gene (alr gene) and construction of genetically engineered bacteria.

[0040] 2.1 PCR amplification of L-alanine racemase gene (alr gene):

[0041] According to the sequence of L-alanine racemase gene from Lactobacillus fermentum reported on Genbank, use Vector NTI software to design primer 1 and primer 2, the primer sequence is:

[0042] Primer 1: 5'-G GAATTC ATGGTAAGTGCAC-3' (the underline is the EcoRI restriction site)

[0043] Primer 2: 5'-AA GCGGCCGC GCCTAGGTAGC-3' (the underline is the NotI restriction site)

[0044] Using the genomic DNA of Lactobacillus fermentum CGMCC2921 obtained in Example 1 as a template, the target gene fragment was amplified.

[0045] The amplification system of PCR (polymerase chain reaction) is: 2 μL of genomic DNA, 2 μL of primer 1 and primer 2, 4 μL of dNTP, 10 × Taq buffer (containing Mg 2+ ) 5 μL, Taq enzyme 1 μL, ddH 2 O 34 μL;

[0046] The PCR reaction program was: pre-denaturation a...

Embodiment 3

[0063] Example 3: Induced expression of L-alanine racemase.

[0064] Prepare 1 L of seed liquid, the medium is LB liquid medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, adjust the pH value to 7.0 with NaOH), and put it into several In a 500mL wide-mouth Erlenmeyer flask. Inject a ring of genetically engineered strains into the seed solution with an inoculation needle, and place it on a shaker at 37°C at a speed of 200rpm for overnight cultivation. Prepare a fermentation medium containing L-alanine 10g / L, peptone (or yeast powder) 10g / L, magnesium sulfate heptahydrate 0.25g / L, sodium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate 2g / L 1000mL, adjust the pH value to 7.5 with NaOH, and divide it into wide-mouth Erlenmeyer flasks with a capacity of 500mL, and the liquid volume of each bottle is 100mL; the above fermentation culture is sterilized based on 121°C high-pressure damp heat for 30min. After the culture medium is cooled, add 1mL of the seed solution cult...

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Abstract

The invention discloses genetic engineering bacteria containing L-alanine racemase genes and application of genetic engineering bacteria; the bacterial strain comprises a nucleotide sequence expressed by SEQ ID NO: 1. The genetic engineering bacteria disclosed by the invention can produce DL-alanine through racemizing L-alanine and is characterized by little dosage, short reaction time and multi-batch reutilization; by adoption of the genetic engineering bacteria of The invention, conversation rate in a racemizing procedure reaches to more than 99.0%; product purity reaches to more than 99.5%; and the genetic engineering bacteria disclosed by The invention has good industrialization prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a genetically engineered bacterium containing an L-alanine racemase gene and an application thereof. Background technique [0002] As the production process of L-alanine matures, the demand for high value-added derivatives from L-alanine is increasing day by day. Among them, the high-efficiency and green production process of DL-alanine is attracting the attention of the industry. DL-alanine is the structural racemate of L-alanine, its chemical name is DL-alpha-alanine (abbreviated as alanine), and its molecular formula is CH 3 CH(NH 2 )COOH, the finished product is colorless to white odorless acicular crystal or crystalline powder with strong sweet taste, easily soluble in water and non-optical. [0003] [0004] DL-Alanine has a wide range of uses. At present, it is mainly used as a nutritional enhancer and condiment in the food industry, and it is also...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12N1/21C12N15/70C12P13/06C12R1/19
Inventor 徐虹徐铮李莎许露冯小海
Owner NANJING TECH UNIV
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