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Enzyme with function of catalyzing formaldehyde for synthesis of 1,3-dihydroxyacetone and preparation method of enzyme

A technology of condensation of dihydroxyacetone and formaldehyde, applied in the biological field, can solve the problems of incapable of industrialized production and many side reactions, etc.

Active Publication Date: 2015-12-09
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

The formaldehyde condensation method studied in the early stage is mainly limited to the use of inorganic bases as catalysts and formaldehyde aqueous solution as raw material to synthesize 1,3-dihydroxyacetone. Glycan reaction, the products are mainly C2-C7 straight-chain sugars and branched-chain sugars, with as many as 47 components, as well as reversible reactions of carbonyl rearrangement in sugar molecules and between sugar molecules, etc., resulting in many side reactions, which cannot Separation and purification by ordinary methods, the selectivity of DHA is less than 10%
Even though the formaldehyde condensation method has been developed to the present, with paraformaldehyde as raw material and organic base as catalyst, the yield and selectivity of 1,3-dihydroxyacetone have been greatly improved, but it cannot be used in industrial production.

Method used

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  • Enzyme with function of catalyzing formaldehyde for synthesis of 1,3-dihydroxyacetone and preparation method of enzyme
  • Enzyme with function of catalyzing formaldehyde for synthesis of 1,3-dihydroxyacetone and preparation method of enzyme
  • Enzyme with function of catalyzing formaldehyde for synthesis of 1,3-dihydroxyacetone and preparation method of enzyme

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Experimental program
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Effect test

Embodiment 1B

[0035] Example 1 BFD gene acquisition, vector construction

[0036] The source of BFD species is Pseudomonasputida, and its gene sequence is shown in SEQIDNO.2. Under the premise of not changing the amino acid sequence of BF, the codons of the above wild-type gene were replaced with the codons of Escherichia coli preference (high frequency use). After codon optimization, the gene sequence has Escherichia coli preferred codons, and its gene sequence is shown in SEQ ID NO.3.

[0037]The gene sequence was directly synthesized in the pET-28a vector (Novagen, Kan + ,See figure 1 ), located between the restriction sites NdeI and XhoI, the recombinant plasmid was named pET-28a-bfd.

Embodiment 2

[0038] The expression of embodiment 2 gene

[0039] In order to detect the activity of BFD enzyme in vitro, the enzyme was expressed and purified exogenously in Escherichia coli.

[0040] (1) The Escherichia coli expression recombinant plasmid pET-28a-bfd was transferred into E.coliBL21(DE3) to obtain the recombinant bacteria. Positive clones were screened using kanamycin-resistant plates (Kan + , 100mg / mL), cultivate overnight at 37°C;

[0041] (2) pick a single clone into 5mL LB liquid medium (Kan + , 100mg / mL), cultured at 37℃, 220r / min until OD 600 It is 0.6-0.8. The bacterium liquid in 5mLLB culture medium is transferred in the 800mL2YT culture medium (Kan + , 100mg / mL), cultured at 37℃, 220rpm to OD 600 When the temperature is 0.6-0.8, cool down to 16°C, add IPTG to a final concentration of 0.5mM, and induce expression for 16h;

[0042] (3) The above-mentioned cultured bacteria liquid is collected in the bacteria collection bottle, and centrifuged at 5500r / min for...

Embodiment 3

[0044] Example 3 protein purification

[0045] (1) Bacteria destruction: use a high-pressure and low-temperature crusher to destroy bacteria twice at a pressure of 1200 bar and 4°C. Centrifuge at 4°C and 10000r / min for 45min, take the precipitate and supernatant after centrifugation, and prepare samples;

[0046] (2) Purification: the supernatant was suction-filtered through a 0.45 μm microporous membrane, and then purified by nickel affinity chromatography. The specific steps were as follows:

[0047] a: column balance: before hanging the supernatant, first use ddH 2 O washes 2 column volumes, then equilibrates 1 column volume of Ni affinity chromatography column with protein buffer;

[0048] b: Sample loading: slowly pass the supernatant through the Ni affinity chromatography column at a flow rate of 0.5mL / min, and repeat again;

[0049] c: Elution of impurity proteins: Rinse 1 column volume with protein buffer, then use 50mL protein buffer containing 50mM imidazole to el...

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Abstract

The invention provides an enzyme with a function of catalyzing formaldehyde for synthesis of 1,3-dihydroxyacetone. The enzyme is selected from following polypeptides (a) to (c): (a) polypeptides with amino acid sequences shown as SEQ ID NO.1; (b) polypeptides derived from (a) by replacing, deleting or adding one or more amino acids to the amino acid sequences in (a), wherein the polypeptides has a function of catalyzing formaldehyde condensation for synthesis of 1,3-dihydroxyacetone; (c) polypeptides with amino acid sequences in 95% similarity with those in (a), wherein the polypeptides has the function of catalyzing formaldehyde condensation for synthesis of 1,3-dihydroxyacetone. By the enzyme with the function of catalyzing formaldehyde for synthesis of 1,3-dihydroxyacetone, a novel method for synthesis of 1,3-dihydroxyacetone is established.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enzyme capable of catalyzing the condensation of formaldehyde to synthesize 1,3-dihydroxyacetone and its preparation and application. Background technique [0002] 1,3-Dihydroxyacetone (1,3-dihydroxyacetone) is the simplest three-carbon ketose that exists in nature and has a wide range of uses. 1,3-Dihydroxyacetone itself has a sunscreen function, which can effectively prevent excessive evaporation of water in the skin into the air, and has the functions of moisturizing, sunscreen and preventing ultraviolet radiation in the sun, so 1,3-Dihydroxyacetone can be used Raw materials for the manufacture of cosmetics. In addition, 1,3-dihydroxyacetone is one of the intermediate products of carbohydrate metabolism in the body. It plays a very important role in the process of carbohydrate metabolism in the body. It can reduce body fat in pigs, promote body fat consumption, and reduce protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N15/81C12P7/26
CPCC12N9/88C12P7/26C12Y401/01007
Inventor 江会锋刘玉万逯晓云燕志慧马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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