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L-ribose isomerase and application thereof in biological preparation of L-ribose

A technology of isomerase and ribose, which is applied to L-ribose isomerase and its application field in biological preparation of L-ribose, and can solve the problem of low catalytic efficiency of L-ribulose.

Active Publication Date: 2020-01-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the catalytic efficiency of the found L-RI to L-ribulose is not high, so mining L-RI with high catalytic ability to L-ribulose is of great significance for the production of L-ribulose

Method used

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  • L-ribose isomerase and application thereof in biological preparation of L-ribose
  • L-ribose isomerase and application thereof in biological preparation of L-ribose
  • L-ribose isomerase and application thereof in biological preparation of L-ribose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Genome extraction from Actinotalea fermentans NX-1.

[0069] Actinotalea fermentans NX-1 was preserved in our laboratory, and the genomic DNA of Actinotalea fermentans NX-1 in logarithmic growth phase was extracted with GenomicDNA Purification Kit (Takara, Dalian) , and the obtained bacterial genomes were detected by agarose gel electrophoresis.

Embodiment 2

[0070] Example 2: Cloning of L-ribose isomerase coding gene (afri) and construction of recombinant bacteria.

[0071] 2.1 PCR amplification of afri gene.

[0072] According to the existing functionally unknown sequence on GeneBank (Genbank Accession No. WP_034244055), use VectorNTI software to design primers Primer1 and Primer2, the primer sequence is:

[0073] Primer 1: 5'-CG GGATCC (BamH I)ATGACCCGTACGTATGTGACCCGTC-3';

[0074] Primer 2: 5'-CCC AAGCTT (Hind III) TTAGCGAATGTGCGTCACCAGACGG-3';

[0075] Using the genome obtained in Example 1 as a template, the gene fragment of Actinocytocellum fermentum was amplified.

[0076] The PCR amplification system is: 2 μL of genomic DNA, 1 μL of each of primers Primer1 and Primer2, 2 μL of dNTP, 2.5 μL of 10×Tag buffer, 0.5 μL of Ex-Tag polymerase, ddH 2 O 14 μL.

[0077] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds; then annealing at 54°C for 2 minutes, ext...

Embodiment 3

[0101] Example 3: Induced expression of L-ribose isomerase.

[0102] Recombinant Escherichia coli BL21-AFRI was inoculated in 5 mL of LB liquid medium supplemented with 25 μg / mL kanamycin, and cultured on a shaker at 37°C overnight; then transferred to 100 mL of LB medium ( In a 500mL shake flask containing 25μg / mL kanamycin), culture on a shaker at 37°C for 2-3h, until OD 600 At about 0.6, add IPTG for induction (IPTG final concentration 1mmol / L), 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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PUM

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Abstract

The invention discloses L-ribose isomerase, an amino acid sequence of the L-ribose isomerase is as shown in SEQ ID NO.1, and the nucleotide sequence of the L-ribose isomerase is as shown in SEQ ID NO.2. The L-ribose isomerase has good thermal stability and pH stability, and the catalytic efficiency on the L-ribulose can reach 85% or above. The invention further discloses recombinant bacteria containing the L-ribose isomerase and a construction method of the recombinant bacteria. The catalytic efficiency of the recombinant bacteria on L-ribulose can reach 81% or above. The L-ribose isomerase described by the invention has good application prospect and economic value for industrial production of L-ribose.

Description

technical field [0001] The invention belongs to the technical field, and in particular relates to L-ribose isomerase from fermenting Actinoleafermentans NX-1 and application thereof. Background technique [0002] L-ribose is an extremely rare monosaccharide. It is an important pharmaceutical intermediate with important medical value. Various L-ribose derivatives synthesized from L-ribose are widely used in the fields of anti-virus and anti-tumor. [0003] At present, the preparation of L-ribose mainly adopts chemical method, which needs to go through at least 7 steps of complex reactions, and faces problems such as many by-products and low yield. L-ribose isomerase (L-RI) can use L-ribulose as raw material to prepare L-ribose, which has the characteristics of mild reaction and high yield, and L-ribulose can be catalyzed from L-arabinose by biological method get. At present, there are only three L-ribose isomerases reported in the literature, which are derived from Acinetob...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P19/24C12P19/02C12R1/19
CPCC12N9/90C12N15/70C12P19/24C12P19/02C12Y503/0102
Inventor 徐虹刘超徐铮王笑李莎冯小海
Owner NANJING UNIV OF TECH
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