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Gene and application of L-sorbose/L-sorbosone dehydrogenase

A technology of sorbone dehydrogenase and sorbose, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of high energy consumption and large consumption

Inactive Publication Date: 2012-09-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method has disadvantages such as high energy consumption, large amount of organic solvent consumption, and serious environmental pollution. Compared with the "Lei's method", the two-step fermentation method invented by Yin Guanglin and others has the advantages of simple process, short production cycle, and low cost.

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  • Gene and application of L-sorbose/L-sorbosone dehydrogenase
  • Gene and application of L-sorbose/L-sorbosone dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The construction of embodiment 1 expression vector

[0032]The sdh / sndh gene sequence annotated in the whole genome sequencing results of K.vulgare WSH-001 in our laboratory was amplified and connected to the cloning vector pMD19-T vector to transform E.coli JM109 competent, and the transformants were screened by antibiotics Colony PCR verification was carried out, positive transformant plasmids were extracted for sequencing, the correct sequenced transformants were cultured and plasmids were extracted, and the sdh / sndh gene fragment was recovered by HindIII and NdeI double digestion and connected to the same HindIII and NdeI double digestion On the pET28a(+) vector, construct the expression vector pET28a(+)-sdh / sndh, transform the constructed expression vector into E.coli JM109 competent, screen the transformants by antibiotics and verify the colony PCR of the transformants, and pick positive After the transformant was cultured, the plasmid was extracted, and a 1740bp ...

Embodiment 2

[0033] Example 2 Expression of L-sorbose / L-sorbosone dehydrogenase and assay of its enzyme activity

[0034] Inoculate E.coli-pET28a-sdh / sndh and control E.coli-pET28a in TB medium respectively, one group was cultured until the end, and the other group was OD 600 When it reaches 0.6, add IPTG to a final concentration of 0.5mM to induce the expression of L-sorbose / L-sorbone dehydrogenase. After culturing for 20 hours, take the 1OD bacterial solution and centrifuge at 12000rpm for 5 minutes, and then use 0.1M pH7.0 phosphate The buffer solution was washed twice, and then resuspended in 0.5 mL phosphate buffer solution for 20 min of ultrasonic wall breaking, and the whole cells, broken wall supernatant and broken wall pellet were subjected to SDS-polyacrylamide gel electrophoresis detection, and E. In contrast to coli-pET28a, a protein band with a molecular weight of about 60kD appeared in the recombinant strain E.coli-pET28a-sdh / sndh.

[0035] The basic reaction solution for th...

Embodiment 3

[0036] The determination of the Michaelis constant Km value of embodiment 3L-sorbose / L-sorbosone dehydrogenase

[0037] Prepare L-sorbose solutions with concentrations of 0.0625M, 0.125M, 0.25M, 0.5M, and 1M respectively, and detect the OD of the reaction solution at each concentration of sorbose according to the detection method of the above-mentioned enzyme activity. 600 Change the value in real time, and then calculate the slope k in the linear range under each sorbose concentration, according to V=(k×60) / 1.91×10 4 (unit: mol.L -1 .min -1 ) Find the initial velocity v of the reaction, take the reciprocal 1 / v of the initial velocity i The reciprocal 1 / [S] of the concentration of L-sorbose is plotted to obtain a straight line, and the Michaelis constant Km value of this sorbose dehydrogenase / sorbosone dehydrogenase is obtained as 0.35±0.06mmol / L.

[0038] The experiment proves that the light absorption-time curve of this enzymatic reaction is basically linear in the first ...

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Abstract

The invention discloses a novel gene of L-sorbose / L-sorbosone dehydrogenase. The gene has a nucleotide sequence shown in a first sequence. The gene is expressed in an escherichia coli expression system, through detection of a dichlorodiisopropylether (DCIP) method, an expression product has enzyme activity, also has dual-functional effects of sorbose dehydrogenase and sorbosone dehydrogenase, can be used for converting L-sorbose to 2-keto-L-gulonic acid (2-KLG) and can cooperate with the L-sorbosone dehydrogenase to perform the conversion from L-sorbose to the 2-KLG.

Description

technical field [0001] The invention discloses an L-sorbose / L-sorbone dehydrogenase gene, in particular a brand new L-sorbose / L-sorbone dehydrogenase gene. Background technique [0002] Vitamin C, also known as ascorbic acid, as an essential vitamin and antioxidant, is widely used in pharmaceutical, food, beverage, cosmetic and feed industries. 2-KLG is an important precursor for the synthesis of vitamin C. The earliest process to realize the industrial production of vitamin C was the so-called "Leys method" invented by Reichstein in Germany in 1934, which included five steps of chemical reaction and one step of biotransformation to convert glucose into 2-KLG, and then generate vitamin C through esterification. However, this method has disadvantages such as high energy consumption, large amount of organic solvent consumption, and serious environmental pollution. Compared with the "Lei's method", the two-step fermentation method invented by Yin Guanglin and others has the adv...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N5/10C12N1/15C12N1/19C12N1/21C12N15/63
Inventor 陈坚周景文高丽丽刘杰堵国成
Owner JIANGNAN UNIV
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