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Gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase

A technology of genetically engineered bacteria and phosphate isomerase, applied in the field of bioengineering, can solve the problems of harmful by-products using a large amount of organic solvents, expensive reagents, low total yield, etc., achieves good industrialization prospects, saves reaction time, The effect of improving enzyme activity

Active Publication Date: 2014-02-05
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the chemical synthesis of L-ribose has been greatly improved in terms of synthesis steps and yields, there are still complex synthetic process routes, cumbersome reaction steps, expensive reagents, low overall yields, and the need to use a large amount of organic compounds. Problems such as solvents and harmful by-products are difficult to meet the requirements of industrial production

Method used

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  • Gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase
  • Gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase
  • Gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Construction of a co-expression plasmid vector containing L-arabinose isomerase gene and mannose-6-phosphate isomerase gene.

[0047] According to the L-arabinose isomerase gene and mannose-6-phosphate isomerase gene from Lactobacillus fermentum (Lactobacillus fermentum, deposit number: CGMCC2921) and Thermus thermophilus HB8 (ATCC27634) reported on Genbank The sequence of constitutive enzyme gene is used Vector NTI software to design primer, and primer sequence is as shown in table 1:

[0048] Table 1 Primer sequences

[0049]

[0050] According to the instructions provided by the manufacturer, the Genomic DNA Purification Kit (Takara, Dalian) was used to extract Lactobacillus fermentum NXTag1 (Lactobacillus fermentum, deposit number: CGMCC2921, which was disclosed in the patent document ZL200910025982. Genomic DNA of Thermus thermophilus (Thermus thermophilus, HB8, ATCC27634), and the obtained genomic DNA was detected by 1% (10g / L) agarose gel electroph...

Embodiment 2

[0069] Example 2: Induced expression of genetically engineered bacteria.

[0070] Two ways are used to induce expression of the genetically engineered bacteria obtained in Example 1:

[0071] (1) Prepare 1L of seed solution, the medium is LB liquid medium (10g / L peptone, 5g / L yeast powder, 10g / L NaCl, sterilized by high pressure at 121℃ for 30min, and put it into several 500mL wide-mouthed triangular flasks Insert a ring of genetically engineered strains in Example 1 into the seed liquid with an inoculation needle, and place 37° C. shaking table to cultivate overnight at a speed of 200 rpm. The preparation contains peptone 10 g / L, yeast powder 5 g / L, and NaCl 10 g / L. 1000mL of LB culture medium in L, divided into wide-mouthed Erlenmeyer flasks with a capacity of 500mL, and the volume of each bottle is 100mL; the above-mentioned LB culture was sterilized based on high-pressure damp heat at 121°C for 30min. After the medium was cooled, it was inserted into overnight culture 1mL...

Embodiment 3

[0073] Example 3: Effects of the type and concentration of metal ions on the enzyme activity of a genetically engineered strain co-expressing L-arabinose isomerase and mannose-6-phosphate isomerase.

[0074] Add L-arabinose to 1 mL of 100 mM sodium phosphate buffer solution (pH 6.5) to a final concentration of 100 mM, then add 12 mg of co-expressed genetically engineered bacteria, and add the final concentration of each metal ion to 1 mM or 10 mM, and bathe in water at 65 °C for 20 min , the amount of L-ribose produced was measured by HPLC, and the results are shown in Table 2 (the group without metal ions was used as the control, and the enzyme activity was set as 100%).

[0075] Table 2 Effects of different metal ions and concentrations on the enzyme activity of co-expressed genetically engineered strains

[0076]

[0077] From the results, it can be seen that when Co 2+ The concentration is 1mM, Mn 2+ When the concentration is 10mM, the enzyme activity is higher, so th...

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Abstract

The invention discloses a gene engineering bacterium for coexpressing L-arabinose isomerase gene and mannitose-6-phosphoric acid isomerase. The strain comprises nucleotide sequences disclosed as SEQ ID NO:1 and SEQ ID NO:2. The gene engineering bacterium can be used for producing L-ribose by catalyzing L-arabinose, and has the advantages of one-step conversion, simple technique and high conversion rate; by using the gene engineering bacterium, the conversion rate of L-ribose is up to 25-50%; and thus, the gene engineering bacterium has favorable industrialization prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the construction and application of a genetic engineering bacterium co-expressing L-arabinose isomerase gene and mannose-6-phosphate isomerase gene. Background technique [0002] Ribose (Ribose, C 5 h 10 o 5 ) is a typical five-carbon sugar, which is a component of various ribonucleic acid (RNA), nucleotide coenzymes, ATP, and NADP. It is closely related to biological genetics and plays an important role in regulating the physiological activities of organisms . Ribose in nature mainly exists in the form of D-ribose, which widely exists in plant and animal cells in the form of furanose. D-ribose is also a variety of vitamins, coenzymes and certain antibiotics, such as neomycin A, B and The composition of paromomycin; L-ribose is the chiral enantiomer opposite to D-ribose, which does not exist in nature and organisms, and is an extremely expensive rare sugar....

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/61C12P19/02C12R1/19
Inventor 徐虹詹伊婧徐铮李莎冯小海
Owner NANJING UNIV OF TECH
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