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Sorbose reductase OpCR gene, mutant, encoding protein and application in preparation of glassine

A reductase and sorbose technology, applied in oxidoreductase, application, genetic engineering and other directions, can solve the problems of difficulty in industrial scale-up and complicated operation, and achieve the effect of reducing production costs and overcoming difficulties in process scale-up.

Pending Publication Date: 2022-05-17
乐山利源科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the products of these two steps of reaction are all purified by column chromatography, the operation is complicated and industrial scale-up is difficult.

Method used

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  • Sorbose reductase OpCR gene, mutant, encoding protein and application in preparation of glassine
  • Sorbose reductase OpCR gene, mutant, encoding protein and application in preparation of glassine
  • Sorbose reductase OpCR gene, mutant, encoding protein and application in preparation of glassine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The influence of embodiment 1 different catalysts on the preparation reaction of β-acetone xyloside

[0029] Xylose (10g) was completely dissolved in deionized water (40mL), added to a 100mL three-necked flask, added different catalysts (1.25eq), and reacted at 90°C for 18 hours. The chemical reaction formula is as follows, and the reaction results are shown in Table 1 shown.

[0030]

[0031] The influence of table 1 catalyst on the preparation of β-acetone xyloside

[0032] alkali Reaction temperature °C Response time h Yield% NaHCO 3

90 18h 87％ LiOH 90 18h 56％ NaOH 90 18h 88％

[0033] According to the results in Table 1, it can be seen that under the above-mentioned catalysts, the method of the present invention can obtain higher yields. Taken together, the catalysts are NaOH and NaHCO 3 The response is better.

[0034] The influence of embodiment 1 reaction temperature and time on the preparation reaction of β-a...

Embodiment 3

[0040] The preparation of embodiment 3 boson

[0041] 1. Preparation of β-acetone xyloside

[0042] Xylose (100g) was completely dissolved in deionized water (400mL), added to a 1000mL three-necked flask, NaOH (53.2g, 2eq), and reacted at 50°C for 1 hour. The pH value of the solution is adjusted to neutral, and the solvent is removed to obtain the first intermediate product β-acetone xyloside.

[0043] 2. Cloning of β-acetone xyloside reductase gene and construction of recombinant bacteria

[0044] The enzymes required by the present invention are all synthesized by the company and then constructed on the expression plasmid and then produced by E. coli fermentation; it specifically includes the following steps:

[0045] Derived from the sorbose reductase OpCR gene of Ogataea parapolymorpha, the preservation number is JCM 22074 (purchased from the Japan Microorganism Collection Center), the gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID ...

Embodiment 4

[0072] Example 4OpCR E70D / Q92L / M135F Enzymatic preparation of Bosene

[0073] 30g crude product of β-acetone xyloside was dissolved in 1.0L phosphate buffer (K 2 HPO 4 -KH 2 PO 4 ) buffer (50mM, pH 8.0) solution, then add 0.34mM, 30g glucose, 2.5g reduced β-nicotinamide adenine dinucleotide disodium tetrasodium phosphate (NADPH), and 6000U glucose dehydrogenase GDH. Add crude enzyme solution (OpCR E70D / Q92L / M135F ), stirred slowly at 30°C for 24 hours, after the reaction, centrifuged, desalted the supernatant by nanofiltration, evaporated water under reduced pressure, and purified to obtain 29.8g product with a purity of 98.9% and a yield of 98.3%.

[0074] The reaction formula is as follows:

[0075]

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Abstract

The invention discloses a sorbose reductase OpCR gene, a mutant, an encoded protein and application of the sorbose reductase OpCR gene in preparation of a glassine. The nucleotide sequence of the OpCR gene is as shown in SEQ ID NO.1, the amino acid sequence of the protein coded by the OpCR gene is as shown in SEQ ID NO.2, and the nucleotide sequence of the mutant is as shown in SEQ ID NO.3-5. The beta-acetone xyloside can be stereospecifically reduced to obtain the glassine, pollution and dangerous chemical combination reagents are avoided, green production and manufacturing of the glassine can be achieved, the defects that in an existing route, the technology is difficult to amplify, many hazardous waste exists and the like are overcome, and meanwhile the production cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a sorbose reductase OpCR gene, a mutant and its encoded protein and its application in the preparation of Bosein. Background technique [0002] Bosein (Pro-Xylane TM ), the chemical name is hydroxypropyltetrahydropyranotriol (CAS: 439685-79-7), which is a xylose derivative with anti-aging activity and is often used in cosmetics. Bosein (Pro-Xylane TM ) was successfully listed in September 2006. Studies have shown that Boseine has a wide range of biological activities. Boseine can directly affect the extracellular matrix in the three-layer structure of the skin, activate or promote the synthesis of mucopolysaccharides (GAGs) in the skin, and improve the adhesion between the dermis and the epidermis. Promote the regeneration of damaged tissues, help maintain the elasticity of the dermis, effectively keep the skin firm and delicate, and delay the aging of the skin. Addit...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12P17/06
CPCC12N9/0006C12P17/06C12Y101/01289
Inventor 陈建明陈封政付斌鑫李兴德
Owner 乐山利源科技有限公司
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