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L-ribose isomerase and application thereof for biologically preparing L-ribose

An isomerase and ribose technology, which is applied to L-ribose isomerase and its application field in biological preparation of L-ribose, can solve the problem of low catalytic efficiency of L-ribulose, and achieves wide application prospects and Economic value, good stability, high catalytic efficiency

Inactive Publication Date: 2016-07-20
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 for biologically preparing L-ribose
  • L-ribose isomerase and application thereof for biologically preparing L-ribose
  • L-ribose isomerase and application thereof for biologically preparing L-ribose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Genome extraction from fermentative Actinoleafermentans NX-1.

[0040] Actinoleafermentans NX-1 was preserved in our laboratory. Genomic DNA of actinoleafermentans NX-1 in the logarithmic growth phase was extracted with GenomicDNA Purification Kit (Takara, Dalian), and analyzed by agarose gel. The obtained bacterial genomes were detected by electrophoresis.

Embodiment 2

[0041] Example 2: Cloning of L-ribose isomerase coding gene (afri) and construction of recombinant bacteria.

[0042] 2.1 PCR amplification of afri gene.

[0043] 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:

[0044] Primer 1: 5'-CG GGATCC( BamHI ) ATGACCCGTACGTATGTGACCCGTC-3';

[0045] Primer 2: 5'-CCC AAGCTT( Hind III )TTAGCGAATGTGCGTCACCAGACGG-3';

[0046] Using the genome obtained in Example 1 as a template, the gene fragment of Actinocytocellum fermentum was amplified.

[0047] 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 O14 μL.

[0048] 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, e...

Embodiment 3

[0066] Example 3: Induced expression of L-ribose isomerase.

[0067] The recombinant E. 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 (containing 25μg / mL kanamycin) in a 500mL shake flask, cultured 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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Abstract

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

Description

technical field [0001] The invention belongs to a 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, only three L-ribose isomerases are reported in the literature, which are derived from Acinetobacter sp...

Claims

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Application Information

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