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L-arabinose isomerase and application thereof in production of L-ribulose

An arabinose and isomerase technology, applied in the field of bioengineering, can solve the problem of low catalytic efficiency of L-arabinose, and achieve wide application prospects and the effect of economic value and high enzyme activity

Active Publication Date: 2016-04-13
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

L-AI from a variety of microbial sources has been discovered, and the L-AI coding gene (araA) of various L-arabinose isomerase producing bacteria such as Escherichia coli, Lactobacillus plantarum, Bacillus subtilis, and Bacillus thermophiles has been cloned and Exogenous expression, but most of the found L-AI have low catalytic efficiency to L-arabinose

Method used

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  • L-arabinose isomerase and application thereof in production of L-ribulose
  • L-arabinose isomerase and application thereof in production of L-ribulose
  • L-arabinose isomerase and application thereof in production of L-ribulose

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: Extraction of Paenibacillus polymyxa NX-1 genomic DNA.

[0052] Genomic DNA of Bacillus polymyxa NX-1 in logarithmic growth phase was extracted with GenomicDNA Purification Kit (Takara, Dalian), and the obtained bacterial genome was detected by agarose gel electrophoresis.

Embodiment 2

[0053] Example 2: Cloning of the gene encoding L-arabinose isomerase (araA) and construction of recombinant bacteria.

[0054] 2.1 PCR amplification of araA gene

[0055] According to the sequence of Bacillus polymyxa L-AI gene reported on GeneBank, primers Primer1 and Primer2 were designed using VectorNTI software. The primer sequence is:

[0056] Primer 1: 5'-CG GGATCC (BamHI) ATGTCAACAGTAAGTACAAAACAGT-3';

[0057] Primer 2: 5'-ATAAGAAT GCGGCCGC (NotI) TTATTTAATTATTACGTATTCCAGG-3';

[0058] Using the genomic DNA of Bacillus polymyxa obtained in Example 1 as a template, the gene fragment of Bacillus polymyxa was amplified.

[0059] The PCR amplification system (25 μL) is: 2 μL of genomic DNA, 1 μL of each primer 1 and primer 2, 2 μL of dNTP, 2.5 μL of 10×Tag buffer, 0.5 μL of Ex-Tag polymerase, ddH 2 O 16 μL;

[0060] The PCR reaction program was as follows: step 1: pre-denaturation at 94°C for 2 minutes; step 2: denaturation at 94°C for 2 minutes; then annealing at 55...

Embodiment 3

[0077] Example 3: Induced expression of L-arabinose isomerase.

[0078] Inoculate the recombinant Escherichia coli BL21(DE3)-AI in 5 mL of LB liquid medium supplemented with 25 μg / mL kanamycin, cultivate overnight at 37°C on a shaker; Put it into a 500mL shake flask filled with 100mL LB medium (containing 25μg / mL kanamycin), and culture it on a shaking table at 37°C for 2-3h, until the OD 600 Add IPTG at about 0.6-1.0 for induction (IPTG final concentration 1mM), or add 1g / L lactose for induction, and then continue to induce expression for 6h, and collect the bacteria by centrifugation.

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Abstract

The invention discloses L-arabinose isomerase and application thereof in production of L-ribulose. The invention further discloses a gene sequence SEQ ID NO:1 for coding the L-arabinose isomerase, a genetically engineered bacterium containing the L-arabinose isomerase and a construction method thereof and application of the L-arabinose isomerase and the genetically engineered bacterium thereof in production of the L-ribulose. The L-arabinose isomerase has higher catalytic efficiency on L-arabinose on the condition that the temperature ranges from 20 DEG C to 70 DEG C and the pH ranges from 5.0 to 10.0, and the activity and the thermal stability of the L-arabinose isomerase can be greatly promoted in the presence of low-concentration irons such as Mn<2+> and Co<2+>. Under the optimal catalytic conditions, by means of the L-arabinose isomerase, the conversion rate of the L-arabinose can reach 95 percent or above, and the L-arabinose isomerase has very important significance and economic value to biological production of the L-ribulose.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to L-arabinose isomerase from Paenibacillus polymyxa NX-1 and application thereof. Background technique [0002] L-ribulose is a rare sugar that does not exist in nature. The main value of L-ribulose is that it can be used as a precursor to synthesize L-ribose, and L-ribulose can be converted into L-ribose under the action of mannose-6-phosphate isomerase. And L-ribose is an extremely rare monosaccharide, which is an important pharmaceutical intermediate and can synthesize the anti-hepatitis B drug Telbivudine (Telbivudine), which has important medical and economic value. Various L-ribose synthesized by L-ribose -Ribose derivatives are widely used in the field of anti-tumor and anti-virus. [0003] L-Arabinose Isomerase (EC5.3.1.4, L-Arabinose Isomerase, L-AI) not only catalyzes D-galactose to generate D-tagatose but also catalyzes the isomerization of L-arabinose to L-ribo...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P19/24C12P19/02
CPCC12N9/90C12P19/02C12P19/24
Inventor 徐虹刘超徐铮王笑李莎冯小海
Owner NANJING UNIV OF TECH
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