Ribitol dehydrogenase (RDH) derived from Klebsiella oxytoca, and coding gene and application thereof

A technology of ribitol dehydrogenase and Klebsiella, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of poor bacterial substrate specificity, no involvement in the production of allicitol, etc., and achieve low pollution, The effect of small amount of NAD and cost reduction

Inactive Publication Date: 2013-11-20
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that some microorganisms can realize the interconversion between D-psicose and allitol, such as Enterobacter agglomerans221e (Muniruzzaman S, Tokunaga H, Izumori K. Conversion of D-psicose to allitol by Enterobacter agglomerans strain221e.J Ferment BioengVolume.1995,79(4):323-327), which reported that D-psicose was used as a substrate to transform and produce allitol, but the substrate specificity of the bacteria was poor, such as The conversion of D-fructose and D-psicose mixture may generate other by-products; another example is Enterobacter aerogenes IK7 (Gullapalli P, Takata G, Poonperm W, Rao D, Morimoto K, Akimitsu K, Tajima S, Izumori K.Bioproduction of D-psicose from allitol with Enterobacter aerogenes IK7: a new frontier in rare ketose production.Biosci Biotechnol Biochem.2007,71(12):3048-3054), the literature only reported that allitol was based on allitol The production of D-psicose by chemical transform

Method used

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  • Ribitol dehydrogenase (RDH) derived from Klebsiella oxytoca, and coding gene and application thereof
  • Ribitol dehydrogenase (RDH) derived from Klebsiella oxytoca, and coding gene and application thereof
  • Ribitol dehydrogenase (RDH) derived from Klebsiella oxytoca, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, expression of ribitol dehydrogenase (RDH)

[0049] The expression method of ribitol dehydrogenase (RDH) of the present invention may comprise the following steps:

[0050] 1) Obtaining the ribitol dehydrogenase gene (RDH): using the genomic DNA of Klebsiella oxytoca G4A4 with the deposit number CGMCC No.7662 as a template, in primer 1:

[0051] 5'-CG GGATCC ATGAATACTTCCCCTTAGC-3' (sequence 3 in the sequence listing, the underlined base is the BamH I restriction site) and primer 2: 5'-CCC AAGCTT Under the guidance of GAGATCCACGCTGTTCG-3' (sequence 4 in the sequence listing, the underlined base is the Hind III restriction site), the ribitol dehydrogenase gene (RDH) was amplified by PCR, and the PCR reaction system was: acid-producing Klebsiella Bacteria (Klebsiella oxytoca) G4A4CGMCC No.7662 genomic DNA 1μl, 10×PCR buffer 5μl, 2mM dNTP 5μl, 10mM primer 11μl, 10mM primer 21μl, high-fidelity DNA polymerase 0.5μl, add water to make up to a total volume of 50...

Embodiment 2

[0055] Embodiment 2, using ribitol dehydrogenase (RDH) to convert and produce allicitol

[0056] 1) Collect the bacteria in Example 1 by centrifugation, and use 50mL pH8.0, 50mM Tris-HCl buffer solution (recipe: 50mM Tris aqueous solution, adjust the pH to 8.0 with hydrochloric acid) [phosphate buffer solution (recipe: 50mM hydrogen phosphate Aqueous solution of disodium and sodium dihydrogen phosphate, pH 8.0) or HEPES buffer solution (recipe: 50mM HEPES aqueous solution, pH 8.0 adjusted with sodium hydroxide) can also be] resuspended bacteria, sonicated, centrifuged at 15000rpm for 30min to collect The clear liquid is the crude enzyme liquid. The crude enzyme solution is purified by affinity chromatography and ion exchange chromatography to obtain pure enzyme (purity over 95%).

[0057] 2) Add ribitol dehydrogenase (RDH) crude enzyme solution or purified ribitol dehydrogenase (RDH) to pH 8.0, 50mM Tris-HCl buffer (phosphate buffer or HEPES buffer is also acceptable) , the ...

Embodiment 3

[0061] Embodiment 3, using ribitol dehydrogenase (RDH) to convert and produce allicitol

[0062] The method is the same as in Example 2, except that the concentration of D-psicose in the allicitol conversion reaction system is 3% (mass / volume percent concentration, g / 100ml), and the concentration of sodium formate in the NADH regeneration system is 200mM.

[0063] The reaction process curve of ribitol dehydrogenase purified protein and concentration of 3% substrate D-psicose and 200mM sodium formate is as follows Figure 4 As shown, after 48 hours of reaction, the conversion rate of allicitol reached 90.1%, and the conversion rate continued to prolong the reaction time, and the conversion rate did not change, indicating that the reaction had reached equilibrium.

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Abstract

The invention discloses a ribitol dehydrogenase (RDH) derived from Klebsiella oxytoca G4A4CGMCC No.7662, and realizes expression thereof in Escherichia coli. The experiment proves that the enzyme can realize the biotransformation production of allitol, and D-allulose can be converted into the functional rare sugar alcohol (allitol) with high conversion rate (96% or above at most) and less pollution; and an NADH (reduced nicotinamide adenine dinucleotide) regeneration system is added in the conversion process, and the NAD (nicotinamide adenine dinucleotide) consumption is low, thereby greatly lowering the cost. The allitol can be used in multiple fields such as preparation of pharmaceuticals (such as diabetes treatment medicaments and the like), health products and other raw materials of rare sugar and the like, and has wide application prospects.

Description

technical field [0001] The invention relates to an enzyme, its coding gene and its application, in particular to a ribitol dehydrogenase (RDH) derived from Klebsiella oxytoca and its application in producing allicitol. Background technique [0002] Rare sugar (Rare Sugar) is a kind of monosaccharide and its derivatives that exist in nature but are very low in content (defined by the International Rare Sugar Society ISRS in 2002). Its taste is similar to sucrose, but it has low calories, high stability, sweet It has the advantages of harmonious taste, no hygroscopicity, no cariogenicity, and high tolerance, which plays an important role in improving the diet of special populations. In addition, a large number of research results also show that rare sugars play an important physiological role in many aspects such as anti-cancer, scavenging free radicals, neuroprotection, etc., and can also undergo Maillard reactions with active substances such as proteins and peptides to optim...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/18C12R1/22
Inventor 朱玥明孙媛霞李泓漪韩文佳马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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