CN109295023B - Glutamate oxidase mutant, nucleic acid molecule, application and method for preparing ketoglutaric acid - Google Patents
Glutamate oxidase mutant, nucleic acid molecule, application and method for preparing ketoglutaric acid Download PDFInfo
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Abstract
The invention discloses a glutamate oxidase mutant, a nucleic acid molecule, application and a method for preparing ketoglutaric acid. The amino acid sequence of the glutamate oxidase mutant is shown in SEQ ID NO. 1. The mutant of glutamate oxidase can catalyze glutamic acid to form ketoglutaric acid, and has higher catalytic capability compared with wild type glutamate oxidase.
Description
Technical Field
The invention relates to the technical field of ketoglutaric acid preparation, and in particular relates to a glutamate oxidase mutant, a nucleic acid molecule, application of the glutamate oxidase mutant and a method for preparing ketoglutaric acid.
Background
α -Ketoglutaric acid (α -Ketoglutaric acid, α -KG) is an important biological compound, which is a keto acid product of deamination of glutamic acid, and is an intermediate product of tricarboxylic acid cycle, and participates in synthesis and energy metabolism of amino acids, proteins and vitamins in organisms, α -Ketoglutaric acid has wide application in food, medicine, fine chemical and feed industries, particularly, L-arginine α -ketoglutarate with a molar ratio of 1: 1 and L-arginine α -ketoglutarate with a molar ratio of 2: 1 are amino acid salt health care products with increasing sales in international markets at present, and the product is mainly used as a functional nutrition enhancer and a liver protection drug, and is mainly used for strengthening physique, promoting rapid growth and recovery of muscles, promoting absorption of nutrition and energy by liver cells, maintaining normal state and the like.
The conventional α -ketoglutaric acid production process basically takes a chemical synthesis method and a biological fermentation method as main materials, wherein the chemical synthesis method mainly takes diethyl succinate, diethyl oxalate and the like as raw materials, and synthesizes α -ketoglutaric acid through condensation and hydrolysis, for example, Zhangguoji (China patent application publication No. CN102584568A) takes methyl dichloroacetate and methyl acrylate as raw materials, under the condition of sodium methoxide, intermediate 2, 2-dichloroglutaric acid dimethyl ester is synthesized, and then reacts with alkali solution to obtain α -ketoglutaric acid aqueous solution crude product, α -ketoglutaric acid product is obtained after refining, the chemical synthesis method produces α -ketoglutaric acid, although the yield is high and the raw materials are easy to obtain, the use of a large amount of organic solvent in the reaction process and a large amount of byproducts can be produced in the multi-step complex synthesis reaction process so as to increase the separation cost, so that the total cost of the chemical synthesis method is higher, the environmental unfriendly, the chemical synthesis method is α -ketoglutaric acid prepared by the biological fermentation method, compared with the chemical synthesis method, the biological fermentation method for preparing the ketoglutaric acid, has the production of the bacterial strain which has the characteristics of mild production conditions, simple process, the environmental protection, the production of the industrial production of the glutaric acid, the industrial production of glutaric acid, the industrial products of the industrial products.
However, biological fermentation has the disadvantages of Long production period, low yield, complex extraction and refining process and high total cost due to the mixing of the product and various components in the fermentation broth, and the biological enzyme method can well make up for the defects of the fermentation method, greatly improve the product concentration, simplify the extraction process and reduce the cost, LongLiu et al (Long Liu, G SHOSSain, et al. journal of Biotechnology,2013,164:97-104) use L-glutamic acid as a substrate and remove amino group by using L-amino acid deaminase to generate α -ketoglutaric acid, but the yield is low and is only less than 12.6G/L, and the reaction has product inhibition effect, so the industrialization is difficult at present.
In recent years, the production of fine chemical products by using a whole-cell catalyst is a very efficient method, and compared with a fermentation method, the method has the advantages of short production period, high efficiency and relatively single product. Compared with an enzyme method, the method has simple process and does not need to purify enzyme and immobilized enzyme. Chenjian et al (Chinese patent application No. 201410132063.2) at the university of Jiangnan in 2014, which adopts a method for producing a-ketoglutaric acid by whole-cell transformation, but because the catalytic property of the L-glutamic oxidase adopted by the method is not good enough, the conversion rate is low, within 24 hours, the conversion rate is only 59.6%, and the yield is 7.7 g/L.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a mutant of glutamate oxidase, which can catalyze glutamic acid to form ketoglutaric acid and has higher catalytic capability compared with wild type glutamate oxidase.
It is another object of the present invention to provide an isolated nucleic acid molecule encoding a glutamate oxidase mutant as described above.
It is another object of the present invention to provide a vector.
Another object of the present invention is to provide a recombinant bacterium.
Another object of the present invention is to provide a method for producing a mutant glutamate oxidase.
It is another object of the present invention to provide a process for the preparation of ketoglutaric acid.
The invention is realized by the following steps:
in one aspect, the invention provides a glutamate oxidase mutant, the amino acid sequence of which is shown in SEQ ID NO. 1.
The inventor of the invention finds that the glutamate oxidase mutant not only retains the activity of catalyzing glutamic acid to form ketoglutaric acid, but also has higher catalytic efficiency compared with wild type glutamate oxidase, and achieves 100% of catalytic efficiency.
In another aspect, the invention provides an isolated nucleic acid molecule encoding a glutamate oxidase mutant as described above.
Further, in some embodiments of the invention, the base sequence of the nucleic acid molecule is as shown in SEQ ID NO. 2.
In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above.
Further, in some embodiments of the invention, the vector is an pDK6 plasmid vector.
Further, in some embodiments of the invention, the nucleic acid molecule is linked between the EcoRl and Pstl cleavage sites on the pDK6 plasmid vector.
In another aspect, the present invention provides a recombinant bacterium comprising the vector described above.
In another aspect, the present invention provides a method for preparing the above glutamate oxidase mutant, comprising: and (3) culturing the recombinant strain.
Separating and purifying the culture product to obtain the glutamate oxidase mutant.
The obtained glutamate oxidase mutant can be used for catalyzing glutamic acid to form ketoglutaric acid. Of course, in some embodiments of the invention, the culture may also be used directly to catalyze the formation of ketoglutarate from glutamate.
On the other hand, the invention provides the application of the glutamate oxidase mutant in catalyzing glutamic acid to form ketoglutaric acid.
In another aspect, the present invention provides a method of preparing ketoglutaric acid, comprising: the glutamic acid oxidase mutant or the recombinant bacterium is used for catalyzing glutamic acid to form ketoglutaric acid.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a map of pDK6 plasmid containing gene encoding glutamate oxidase mutant.
FIG. 2 is a gel electrophoresis image of the fermentation broth in the example after centrifugation to remove supernatant and precipitate.
FIG. 3 shows the results of liquid chromatography detection of ketoglutaric acid in the experimental examples.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
Genetically engineered bacterium for constructing and expressing glutamate oxidase mutant
(1) Based on the wild sequence of the streptomycete glutamate oxidase, artificial design is carried out, the designed gene sequence is shown as SEQ ID NO.2, and the amino acid sequence of the encoded glutamate oxidase mutant is shown as SEQ ID NO. 1. The gene sequence is synthesized by a whole-gene synthesis method.
(2) The synthesized gene sequence is cloned into pDK6 plasmid through EcoRl and Pstl 2 restriction enzyme sites, the plasmid constructed above is transferred into Escherichia coli DH5 α competent cells, and after positive transformants are selected and sequenced and identified, a recombinant expression vector is obtained, which is named as pDK6-12 plasmid (see figure 1).
(3) The recombinant expression vector pDK6-12 plasmid is transferred into Escherichia coli BL21(DE3) strain to obtain the genetic engineering bacteria capable of inducing and expressing glutamate oxidase mutant.
Example 2
Preparation of glutamate oxidase mutant
(1) The genetic engineering bacteria for expressing the streptomycete glutamate oxidase mutant prepared in the embodiment 1 are streaked on a kanamycin-resistant LB solid culture medium, cultured overnight in a 37 ℃ biochemical incubator, and a larger colony is selected and inoculated into a kanamycin-resistant LB liquid culture medium, cultured for 6-8 h at the temperature of 220rpm of a shaker 37 ℃ or cultured overnight at the temperature of 200rpm of a shaker 30 ℃ to obtain a primary seed culture solution.
(2) Inoculating the primary seed culture solution into a TB liquid culture medium containing kanamycin resistance according to the inoculation amount of 1%, and culturing for 4-6 h at 37 ℃ and 220rpm of a shaking table until the visual bacterial solution is cloudy, thereby obtaining a secondary seed culture solution.
(3) According to the inoculation amount of 1%, the secondary seeds are inoculated into a fermentation tank for culture. Initial control: and (3) ventilating and maintaining pressure at 37 ℃. When the dissolved oxygen is reduced, the ventilation volume, the rotating speed and the tank pressure are gradually increased to increase the dissolved oxygen, and the tank pressure is not higher than 0.08 MPa.
(4) During fermentation, nutrients are supplemented properly to control dissolved oxygen at 20-40%, and when OD600 reaches about 15-20%, the temperature is respectively reduced to 28 deg.C, 30 deg.C and 25 deg.C, and IPTG with concentration of 0.15mM, 0.2mM and 0.3mM is added.
(5) The fermentation liquor is centrifuged or filtered to collect thallus, the collected thallus contains glutamate oxidase mutant, and the thallus can be directly subjected to enzyme catalytic reaction or refrigerated in a refrigerator for use.
(6) The fermentation broth was centrifuged at 8000rpm at 4 ℃ for 5 minutes, and the supernatant and precipitate obtained after centrifugation were subjected to gel electrophoresis, and the results are shown in FIG. 2.
Examples of the experiments
Experiment for catalyzing glutamic acid to form ketoglutaric acid by using the above-mentioned bacteria
Enzyme reaction: the reaction system is 8 liters
1. 200 g of frozen thallus (about 2.5% of the transformation system), 80mL of 40 ten thousand units of catalase, tap water to 5L (pH about 6.4), heat preservation at 35 ℃, 400rpm, 1vvm and 0.05MPa of tank pressure are weighed.
2. 800g of monosodium glutamate monohydrate (approx. 10% of the conversion system) are weighed out, dissolved in 3 l of tap water and fed into the conversion system by means of a peristaltic pump at a feed rate of 1.2 l/h. During the period, 1% hydrochloric acid is used to control the pH value to be about 6.5. Samples were taken every hour for ketoglutaric acid and conversion was terminated in about 3 hours.
Ketoglutaric acid detection
1. Standard configuration: accurately weighing 0.008 g of ketoglutaric acid standard substance (more than 98%), adding deionized water to a constant volume of 10 ml, shaking up, and storing in a refrigerator at 4 deg.C.
2. Liquid phase detection:
2.1 mobile phase: 0.52 g of concentrated sulfuric acid is weighed, dissolved to 1 liter by deionized water, and subjected to ultrasonic degassing after water film suction filtration.
2.2 detection conditions: amino sugar column, wavelength 215nm, column temperature 50 deg.C, flow rate 0.6mL/min, sample size 20uL, retention time 15 min.
As a result:
the volume of the obtained enzyme reaction solution is 8.1L, and the concentration of ketoglutaric acid detected by liquid phase is 77.11g/L, namely 624.6g of ketoglutaric acid is totally contained in the solution. The liquid phase diagram of ketoglutarate detection is shown in FIG. 3.
The molecular weight of the monosodium glutamate is 187, the molecular weight of the ketoglutaric acid is 146, 800g of monosodium glutamate is added in the reaction, and 624.6g of ketoglutaric acid can be obtained by 100% of the theoretical reaction. I.e., the conversion of the enzymatic reaction is 100%.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Nanjing sequoia Biotech Co., Ltd
<120> glutamate oxidase mutant, nucleic acid molecule, application and method for preparing ketoglutaric acid
<160>2
<170>PatentIn version 3.5
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gtgttaacaa acctttcaaa gaatggcttg ctggttgggc tgacgttgtt cgtgacttcg 780
acggttactc tatgggtcgt ttccttcgtg aatacgctga attctctgac gaagctgttg 840
aagctaaagg tactaaagaa aacaggactt ctcgtcttca ccttgctttc ttccactctt 900
tccttggtcg ttctgacaaa gaccctcgtg ctacttactg ggaaaaagaa ggtggttctc 960
gtatgcttcc tgaaactctt gctaaagacc ttcgtgacca aaaagttatg ggtcaacgta 1020
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ggaaacgtga acttgacgct aaagctcctg gtctttacga ctactaccaa caatggggtg 1380
aagacgacgc tgaagctgct cttgctcttc ctcaatctgt tcgtaacctt cctactggtc 1440
ttcttggtgc tcacccttct gttgacgaat ctcgtaaagg tgaagaacaa gttgaatact 1500
accgtaactc tgaacttcgt ggtggtgttc gtcctgctac taacgcttac ggtggtggtt 1560
ctactactga caaccctaac cgtttcatgt actacccttc tcaccctgtt cctggtactc 1620
aaggtggtgt tgttcttgct gcttactctt ggtctgacga cgctgctcgt tgggactctt 1680
tcgacgacgc tgaacgttac ggttacgctc ttgaaaacct tcaatctgtt cacggtcgtc 1740
gtaaagaagt tttctacact ggtgctggtc aaactcaatc ttggcttcgt gacccttacg 1800
cttgcggtga agctgctgtt tacactcctc accaaaggac tgctttccac cttgacgttg 1860
ttcgtcctga aggtcctgtt tacttcgctg gtgaacacgt ttctcttaaa cacgcttgga 1920
aagaaggtgc tgttgaaact gctgttcgtg ctgctaaagc tgttaacgaa gctcctgttg 1980
gtgacactgg tgttactgct gctgctggtc gtcgtggtgc tgctgctgct actgaaccta 2040
tgcgtgaaga agctcttact tcat 2064
Claims (10)
1. A glutamate oxidase mutant, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. An isolated nucleic acid molecule encoding the glutamate oxidase mutant of claim 1.
3. The isolated nucleic acid molecule of claim 2, wherein the base sequence of said nucleic acid molecule is set forth in SEQ ID No. 2.
4. A vector comprising the nucleic acid molecule of claim 2 or 3.
5. The vector of claim 4, wherein the vector is an pDK6 plasmid vector.
6. The vector of claim 5, wherein said nucleic acid molecule is ligated between the EcoR and Pstl cleavage sites on the pDK6 plasmid vector.
7. A recombinant bacterium comprising the vector according to any one of claims 4 to 6.
8. A method for preparing a mutant glutamate oxidase according to claim 1, comprising: culturing the recombinant bacterium according to claim 7.
9. Use of a mutant glutamate oxidase according to claim 1, for catalysing the formation of α -oxoglutarate from glutamate.
10. A method for producing ketoglutaric acid, characterized in that it comprises using the glutamate oxidase mutant according to claim 1 or the recombinant bacterium according to claim 7 to catalyze the formation of α -ketoglutaric acid from glutamic acid.
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Denomination of invention: Glutamic acid oxidase mutants, nucleic acid molecules and their application and preparation of ketoglutarate Effective date of registration: 20210310 Granted publication date: 20200623 Pledgee: Nanjing Zidong sub branch of Bank of Nanjing Co., Ltd Pledgor: NANJING REDWOOD FINE CHEMICAL Co.,Ltd. Registration number: Y2021980001571 |
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