CN116334152A - 2,5-DKG reductase capable of efficiently converting 2,5-DKG into 2-KLG - Google Patents
2,5-DKG reductase capable of efficiently converting 2,5-DKG into 2-KLG Download PDFInfo
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Abstract
The invention discloses a 2,5-DKG reductase capable of efficiently converting 2,5-DKG into 2-KLG, belonging to the technical fields of genetic engineering and biological engineering. The invention discovers a new 2,5-DKG reductase with stronger catalytic capability and some enzymatic properties thereof by expressing 2,5-DKG reductase and aldehyde ketone reductase with different sources in escherichia coli BL21 (DE 3) and comparing the capability of catalyzing 2,5-DKG. The 2,5-DKG reductase can be used for constructing a one-step reaction of glucose to 2-KLG, so that the 2,5-DKG reductase plays an important role in the fields of cosmetics, textiles and foods.
Description
Technical Field
The invention relates to 2,5-DKG reductase capable of efficiently converting 2,5-DKG into 2-KLG, belonging to the technical fields of genetic engineering and biological engineering.
Background
Vitamin C is also called L-ascorbic acid, is a water-soluble vitamin, has strong reducibility, and is one of vitamins necessary for human body. In recent years, application cases of vitamin C have been developed, which have very important roles in foods, medicines, cosmetics, feeds and medical health. With the continuous development of other functions of vitamin C, the later-stage demand of vitamin C is expected to be further increased, so that the further increase of the productivity of vitamin C is important for the economic promotion of related industries as a mass demand of national people.
The main synthesis methods of vitamin C include Lei's method and two-step fermentation method. The Lei method is the earliest mode applied to vitamin C production, and has the advantages of mature process and higher product yield, but has the defects of complex production procedure, high labor intensity and serious environmental pollution caused by using a large amount of organic solvents in the production process. The two-step fermentation method is proposed by a scientist Yin Guanglin in China in the 70 th century, and a microorganism fermentation method is used for replacing a chemical synthesis step in the Lei method. Firstly, converting glucose into sorbitol by high-pressure hydrogenation, then converting sorbitol into sorbitol by Gluconobacter oxydans fermentation, then converting sorbitol into 2-keto-L-gulonic acid by mixed fermentation, and finally performing lactonization reaction to generate vitamin C. In addition, there are new two-step fermentation process, one-step fermentation process and other processes. Glucose can be converted into 2, 5-diketo-D-gluconic acid by a plurality of microorganisms of genus Erwinia, rahnella, serratia, tatomum, pantoea and the like, and then the 2, 5-diketo-D-gluconic acid is converted into 2-keto-L-gulonic acid by a coryneform 2, 5-diketo-D-gluconic acid reductase, which is a novel two-step fermentation method. In 1982, sonoyama, which is a Japanese salt wild pharmaceutical, fermented an Erwinia strain capable of producing 2, 5-diketo-D-gluconic acid in series with corynebacteria, thereby realizing the production of 2-keto-L-gulonic acid by a novel two-step fermentation method of glucose without high-pressure hydrogenation. In 1985, anderson laboratory in the United states first introduced 2, 5-diketo-D-gluconic acid reductase from corynebacteria into Erwinia which produced 2, 5-diketo-D-gluconic acid, and constructed an engineering strain which realized the direct production of 2-keto-L-gulonic acid from glucose by one-step fermentation. Similar engineering bacteria were constructed in Hardy, switzerland in 1988, but the conversion rate of glucose to 2-keto-L-gulonic acid was lower.
To solve the problems, the application aims to dig a novel source, and the 2,5-DKG reductase with higher catalytic activity has potential guiding value and guiding significance in promoting the development of synthetic biology and the progress of vitamin industry.
Disclosure of Invention
The invention provides a novel 2,5-DKG reductase which is derived from Gluconobacter oxydans, wherein the amino acid sequence of the 2,5-DKG reductase is shown as SEQ ID NO. 1.
In one embodiment of the invention, the 2,5-DKG reductase is cloned from the genome of Gluconobacter oxydans.
In one embodiment of the invention, the nucleotide sequence encoding the 2,5-DKG reductase is shown in SEQ ID NO. 2.
The invention also provides an enzyme activity detection mode of the 2,5-DKG reductase.
In one embodiment of the invention, the substrate for the 2,5-DKG reductase is 2,5-DKG.
In one embodiment of the invention, the cofactor of the 2,5-DKG reductase is NADPH.
In one embodiment of the invention, the activity of 2,5-DKG reductase is measured by decreasing the absorbance of NADPH at 340 nm.
In one embodiment of the invention, the production of the final product 2-KLG is detected by High Performance Liquid Chromatography (HPLC).
The invention provides a method for preparing 2-keto-L-gulonic acid, which comprises the steps of adding 2,5-DKG reductase with an amino acid sequence shown as SEQ ID NO.1 or recombinant bacteria expressing the 2,5-DKG reductase with the amino acid sequence shown as SEQ ID NO.1 into a reaction system containing 2, 5-diketo-D-gluconic acid to prepare the 2-keto-L-gulonic acid.
In one embodiment of the invention, the nucleotide sequence encoding the 2,5-DKG reductase is shown in SEQ ID NO. 2.
In one embodiment of the present invention, the reaction conditions in the reaction system are: 25-30, normal pressure and pH 5.0-7.0.
In one embodiment of the present invention, the reaction conditions in the reaction system are: normal temperature, normal pressure and pH 7.0.
In one embodiment of the present invention, the amount of 2, 5-diketo-D-gluconic acid added to the reaction system is as follows: 2mM; the addition amount of the 2,5-DKG reductase is as follows: 50. Mu.L of NADPH was added to the reaction system in an amount of 2mM.
In one embodiment of the present invention, the recombinant bacterium expressing the 2,5-DKG reductase with the amino acid sequence shown in SEQ ID No.1 is bacterial or fungal as the expression host.
The invention also provides application of the 2,5-DKG reductase with the amino acid sequence shown as SEQ ID NO.1 or the 2,5-DKG reductase with the expressed amino acid sequence shown as SEQ ID NO.1 or the gene for encoding the 2,5-DKG reductase with the nucleotide sequence shown as SEQ ID NO.2 or the recombinant vector carrying the gene for encoding the 2,5-DKG reductase with the nucleotide sequence shown as SEQ ID NO.2 in preparation of 2-keto-L-gulonic acid or products containing the 2-keto-L-gulonic acid.
In one embodiment of the present invention, the recombinant vector is a pET28a or pCDFDUET expression vector.
In one embodiment of the present invention, the recombinant bacterium uses E.coli BL21 (DE 3) as an expression host.
The invention also provides a method for improving the yield of 2-keto-L-gulonic acid, which comprises the steps of adding 2,5-DKG reductase with an amino acid sequence shown as SEQ ID NO.1 or recombinant bacteria expressing the 2,5-DKG reductase with the amino acid sequence shown as SEQ ID NO.1 into a reaction system containing 2, 5-diketo-D-gluconic acid for reaction to prepare the 2-keto-L-gulonic acid.
In one embodiment of the invention, the nucleotide sequence encoding the 2,5-DKG reductase is shown in SEQ ID NO. 2. The invention provides a novel 2,5-DKG reductase which is derived from Gluconobacter oxydans, wherein the amino acid sequence of the 2,5-DKG reductase is shown as SEQ ID NO. 1.
In one embodiment of the invention, the 2,5-DKG reductase is cloned from the genome of Gluconobacter oxydans.
In one embodiment of the invention, the nucleotide sequence encoding the 2,5-DKG reductase is shown in SEQ ID NO. 2.
Advantageous effects
(1) The invention provides a novel 2,5-DKG reductase with higher catalytic capacity than the reported 2,5-DKG reductase from Corynebacterium glutamicum. The related enzymatic properties of the 2,5-DKG reductase are detected based on the enzyme activity detection mode of the enzyme reported in the data, and the enzyme has better stability under the conventional pH and temperature.
(2) The 2,5-DKG reductase discovered by the invention has higher efficient catalytic activity, and provides more enzyme origins for constructing a single-step fermentation path from glucose to 2-KLG.
Drawings
FIG. 1 is a diagram of metabolic pathways for glucose one-step synthesis of 2-keto-L-gulonic acid.
FIG. 2 is a SDS-PAGE diagram of 2,5-DKG reductase and aldehyde-ketone reductase from different sources.
FIG. 3 is a graph comparing specific enzyme activities of 2,5-DKG reductase and aldehyde ketone reductase of different sources using 2,5-DKG as substrate.
FIG. 4 shows the change in enzyme activity of Gluconobacter oxydans-derived 2,5-DKG reductase at different pH.
FIG. 5 shows the change in enzyme activity of Gluconobacter oxydans-derived 2,5-DKG reductase at various temperatures.
FIG. 6 is an enzyme activity effect of different metal ions on Gluconobacter oxydans-derived 2,5-DKG reductase.
Detailed Description
The following examples relate to the following media:
sorbitol medium: 50g/L of D-sorbitol, 10g/L of yeast powder, 1g/L of monopotassium phosphate and MgSO 4 ·7H 2 O 0.25g/L。
LB medium: 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride.
The reaction systems involved in the examples below:
250. Mu.L of Tris-HCl buffer (pH 7.0) contained 2mM 2,5-DKG, 2mM NADPH and 50. Mu.L of 2,5-DKG reductase or aldehyde-ketone reductase.
And (3) solution A: 25mM Tris,150mM NaCl,25mM imidazole, ph=8.0.
And (2) liquid B: 25mM Tris,150mM NaCl,500mM imidazole, ph=8.0.
And C, liquid: 25mM Tris,150mM NaCl,pH =8.0.
Cloning of aldehyde-ketone reductase and 2,5-DKG reductase genes of different origins involved in the examples below
Gluconobacter oxydans ATCC9937,9937 was inoculated into sorbitol medium for culture. The cells were collected and the genome was extracted using an Ezup-column bacterial genomic DNA extraction kit (purchased from the company of biotechnology (Shanghai)), and 2,5-DKG reductase and aldehyde ketone reductase were amplified using PCR, and the amplification primers contained the homology arm sequences required for ligation of plasmids. PCR was performed using 2X Phanta Max Master Mix (available from Nanjinozan Corp.). The PCR product was recovered using a SanPrep column type DNA gel recovery kit (available from Biotechnology (Shanghai) Co., ltd.).
Corynebacterium glutamicum ATCC 13032 was inoculated into LB medium for culture. The cells were collected and the genome was extracted using an Ezup-column bacterial genomic DNA extraction kit (purchased from the company of biotechnology (Shanghai)), and 2,5-DKG reductase was amplified using PCR, and the amplification primers contained the homology arm sequences required for ligation of plasmids. PCR was performed using 2X Phanta Max Master Mix (available from Nanjinozan Corp.). The PCR product was recovered using a SanPrep column type DNA gel recovery kit (available from Biotechnology (Shanghai) Co., ltd.).
Tatumella citrea CICC10802 was inoculated into LB medium for cultivation. The cells were collected and the genome was extracted using an Ezup-column bacterial genomic DNA extraction kit (purchased from the company of biotechnology (Shanghai)), and 2,5-DKG reductase and aldehyde ketone reductase were amplified using PCR, and the amplification primers contained the homology arm sequences required for ligation of plasmids. PCR was performed using 2X Phanta Max Master Mix (available from Nanjinozan Corp.). The PCR product was recovered using a SanPrep column type DNA gel recovery kit (available from Biotechnology (Shanghai) Co., ltd.).
Construction of 2,5-DKG reductase and aldehyde-ketone reductase plasmids and expression of genes involved in the examples
1) The pcr is used to amplify the vector, linearize the vector and make it carry a homology arm sequence that can be complementary to the amplified 2,5-DKG reductase or aldehyde ketone reductase gene.
2) The linearized vector was seamlessly ligated with 2,5-DKG reductase or aldehyde ketone reductase of different sources using the Information-Cloning kit (purchased from Nanjinopran Corp.) to construct a complete plasmid.
3) The constructed plasmid is transferred into escherichia coli BL21 (DE 3) to be competent, and is coated on a flat plate containing corresponding antibiotics, and positive clones are selected for sequencing.
Expression and purification of 2,5-DKG reductase and aldehyde-ketone reductase referred to in the examples below
1) The strain with correct sequence is inoculated to a seed culture medium added with corresponding resistance to prepare seed liquid. After the seed solution is prepared, inoculating the seed solution into an expression culture medium, and carrying out expression of 2,5-DKG reductase or aldehyde ketone reductase by induction.
2) And (3) collecting thalli after centrifuging the sample, and re-suspending the thalli by using the solution A.
3) The sample was crushed using a homogenizer. The sample was centrifuged at 12000 Xg for 60min, the impurities were removed, the supernatant was collected and passed through a 0.22 μm filter, and the sample was placed on ice for use.
4) The sample was passed through a nickel column equilibrated with solution a using an AKTA pure instrument (universal electric company, usa) and the nickel column was again equilibrated with solution a. The sample was eluted with solution B, and the target was collected and concentrated using protein concentration tube (sammer feishi technologies (china) limited). Finally, the concentrated sample was passed through a gel column equilibrated with solution C (superdex 200pg 26/600) and the target peak was collected.
The method for measuring the enzymatic activity of 2,5-DKG reductase in the following examples
The reaction was performed in Tris-HCl buffer (pH 7.0) containing 2mM 2,5-DKG, 2mM NADPH and 50. Mu.L of 2,5-DKG reductase or aldehyde-ketone reductase-pure enzyme solution (concentration about 0.7 mg/mL). The volume of the reaction solution was adjusted to 250. Mu.L with Tris-HCl (pH 7.0), and the absorbance change of the sample at 340nm was measured at room temperature using a 96-well plate for 30 min.
One unit of 2,5-DKG reductase activity is defined as the amount of enzyme required to reduce 1. Mu. Mol of NADPH per minute. In order to avoid interference of 2,5-DKG browning on detection, the substrate concentration is controlled below 3 mM;
the enzyme activity/protein concentration is the specific enzyme activity.
Example 1: gene cloning of 2,5-DKG reductase and aldehyde ketone reductase from different sources, construction of plasmid and expression
(1) 2,5-DKG reductase of different sources
Gluconobacter oxydans ATCC9937 was activated and inoculated into sorbitol medium, cultured at 30℃and 220rpm for 48h, centrifuged at 4000rpm, and the cells were collected to extract Gluconobacter oxydans ATCC9937 genome (No. CP102494-CP102499 on NCBI) and amplified with 2,5-DKG reductase from Gluconobacter oxydans by using primer pair dkgA-G-F/dkgA-G-R1. The primers of Table 1, yhdN-G-F/yhdN-G-R, yeaE-G-F\yeaE-G-R, psakr-G-F\psakr-G-R, pakr-G-F\pakr-G-R, were used to amplify different aldehyde-ketone reductases from Gluconobacter oxydans, respectively. And recovering the PCR product. The result was 2,5-DKG reductase DKGRA-G, aldehyde-ketone reductase YhdN-G, aldehyde-ketone reductase YeaE-G, aldehyde-ketone reductase Psakr-G and aldehyde-ketone reductase Pakr-G derived from Gluconobacter oxydans ATCC 9937.
Corynebacterium glutamicum ATCC 13032 was activated and inoculated into LB medium, cultured at 30℃and 220rpm for 24 hours, centrifuged at 4000rpm, and the cells were collected to extract Corynebacterium glutamicum ATCC 13032 genome (No. CP025533.1 on NCBI) and amplified by using the primer pair dkgA-C-F/dkgA-C-R1 as a 2,5-DKG reductase derived from Corynebacterium glutamicum. And recovering the PCR product. Obtaining the 2,5-DKG reductase DKGRA-C from Corynebacterium glutamicum ATCC 13032.
Tatumella citrea CICC10802 is activated and inoculated into LB medium, cultured at 30℃and 220rpm for 24 hours, centrifuged at 4000rpm, and the cells are collected to extract Tatumella citrea CICC10802 genome (No. CP015579.1 on NCBI) and amplified by using primer pairs dkgA-T-F/dkgA-T-R1 and dkgB-T-F/dkgB-T-R, respectively, for 2,5-DKG reductase derived from Tataricum. The primer pairs akr1-T-F\ akr2-T-R, akr2-T-F\ akr2-T-R, akr3-T-F\ akr3-T-R are used for amplifying different aldehyde-ketone reductases from the Tatarium citricum source respectively. And recovering the PCR product. Obtaining 2,5-DKG reductase DKGRA-T, 2,5-DKG reductase DKGRB-T, aldehyde-ketone reductase AKR1-T, aldehyde-ketone reductase AKR2-T and aldehyde-ketone reductase AKR3-T from Tatumella citrea CICC 10802.
The primer sequences involved are shown in Table 1.
Primers used in Table 1
(2) Construction of expression vectors
The pET28a (+) vector was amplified using the primer pair F/R and the PCR product was recovered.
The PCR reaction system is as follows: 25. Mu.L 2X Phanta Max Master Mix, 1. Mu.L forward primer (10. Mu. Mol. L) -1 ) 1. Mu.L of reverse primer (10. Mu. Mol.L) -1 ) 1 μl of template DNA was added with distilled water to 50 μl. The L-sorbosone dehydrogenase PCR amplification procedure was set as follows: firstly, pre-denaturation at 95 ℃ for 3min; then enter 30 cycles: denaturation at 95℃for 30s, annealing at 56℃for 30s, and extension at 72℃for 1min; finally, the temperature is 72 ℃ for 5 minutes, and the temperature is kept at 4 ℃. The pET28a (+) plasmid linearization PCR amplification procedure was set as follows: firstly, pre-denaturation at 95 ℃ for 3min; then 25 cycles are entered: denaturation at 95℃for 30s, annealing at 56℃for 30s, and extension at 72℃for 3min; finally, the temperature is 72 ℃ and the extension is 10miAnd n, preserving heat at 4 ℃.
The cloned pcr products of different 2,5-DKG reductase and aldehyde ketone reductase were mixed with 20ng pET28a (+) linearization vector, and ligated using the information-Cloning kit to construct plasmid pET28a (+) -DKG/AKR. Transferring 10 mu L pET28a (+) -DKGR/AKR ligation product into BL21 (DE 3) competence, ice-bathing 30min, heat-shocking at 42 ℃ for 90s, ice-bathing 5min, adding 1ml LB culture medium, culturing 45min at 37 ℃ and 220rpm, centrifuging at 3000rpm for 3min, removing supernatant, suspending the strain in 100 mu L LB culture medium, coating on LB plate containing 50mg/L kanamycin sulfate, and culturing overnight. Positive clones were selected for sequencing to verify if the plasmid was correct.
Recombinant strains are prepared respectively: e.coli BL21 (DE 3)/pET 28a (+) -DKGRA-G, E.coli BL21 (DE 3)/pET 28a (+) -DKGRA-C, E.coli BL21 (DE 3)/PET 28a (+) -DKGRA-T, E.coli BL21 (DE 3)/pET 28a (+) -DKGRB-T, E.coli BL21 (DE 3)/pET 28a (+) -yhdN-G, E.coli BL21 (DE 3)/pET 28a (+) -yeaE-G, E.coli BL21 (DE 3)/pET 28a (+) -pakr G, E.coli BL21 (DE 3)/pET 28a (+) -akr-T, E.coli BL21 (DE 3)/pET 28a (+) -akr-T, E.coli BL21 (DE 3)/pET 28a (+) -35T 84-T.
(3) Expression of 2,5-DKG reductase and aldehyde-ketone reductase of different origins
The recombinant strains with correct sequence were transferred to 10ml LB containing 50mg/L kanamycin sulfate for culture at 37℃and 220rpm for 12 hours, respectively, to prepare seed solutions.
Transferring the cultured seed solution into 50ml TB medium containing 50mg/L kanamycin sulfate at 2% (v/v) until the thallus concentration reaches OD 600 Cooling to 20 ℃ at the time of=0.8, and adding IPTG to the final concentration of 0.5mM for induction to express the enzyme of interest; respectively preparing fermentation liquor of 2,5-DKG reductase and aldehyde ketone reductase with different sources.
Example 2: preparation of pure enzymes of 2,5-DKG reductase and aldehyde ketoreductase of different origins
(1) Preparation of crude enzyme solution
The purification of the 2,5-DKG reductase and the aldehyde-ketone reductase used in the examples was carried out at 4 ℃.
Centrifuging the fermentation broth obtained in the example 2, discarding the supernatant to collect the bacterial cells, repeatedly cleaning the bacterial cells for three times by using PBS solution, then crushing the bacterial cells by using a high-pressure homogenizer, wherein the pressure range is 800-1000bar, and repeatedly crushing the bacterial cells for 3 times; the disrupted solution was centrifuged at 12000g for 30min to remove uncrushed cells, and the supernatant was passed through a 0.45 μm filter to obtain a crude enzyme solution (FIG. 2).
(2) Preparation of pure enzyme solution
The crude enzyme solution is filtered by using a Ni-NAT affinity chromatographic column to obtain pure enzyme solution. First, the Ni-NAT chromatographic column is equilibrated with solution A. The crude enzyme solution was loaded at a flow rate of 3 ml/min. And (3) after the crude enzyme liquid is completely loaded, balancing the chromatographic column by using the liquid A again. Then using B liquid to make gradient elution (0-100% B liquid; 30 min). And collecting and verifying the elution peak to obtain the pure enzyme solution.
Example 3: comparison of catalytic Capacity of 2,5-DKG reductase from different sources
The method comprises the following specific steps:
(1) The specific enzyme activities of the 2,5-DKG reductase and the aldehyde ketone reductase pure enzyme solution from different sources were detected, and the results are shown in Table 2 and FIG. 3.
Table 2: specific enzyme activity of 2,5-DKG reductase and aldehyde ketone reductase pure enzyme solution from different sources
| Enzymes | Specific enzyme activity (U/mg) |
| |
0 |
| AKRr1- |
0 |
| AKR2- |
0 |
| AKR3- |
0 |
| YhdN- |
0 |
| YeaE- |
0 |
| psAKR- |
0 |
| pAKR- |
0 |
| DKGRA-T | 234.66667 |
| DKGRB-T | 255.33333 |
| DKGRA-C | 575.33333 |
| DKGRA-G | 1676.66667 |
The results show that the specific activity of the 2,5-DKG reductase (DKGRA-G) from G.oxydans ATCC9937, among the 2,5-DKG reductase and the aldehyde-ketone reductase from three different microorganisms, is far higher than that of the other 2,5-DKG reductases, which is 1677U/mg. This is 2.5 times that reported in the literature for the C.glutamicum-derived 2,5-DKG reductase DKGRA-C (533U/mg). The specific enzyme activities of the 2,5-DKG reductase from citread were the lowest, 235U/mg (DKGRA-T) and 255U/mg (DKGRB-T), respectively. Other aldehyde ketoreductases do not exhibit the ability to catalyze 2,5-DKG. This suggests that the novel 2,5-DKG reductase discovered by the present invention has a stronger catalytic activity on 2,5-DKG.
Example 4: pH stability of 2,5-DKG reductase from oxydans ATCC9937
The method comprises the following specific steps:
the effect of pH on 2,5-DKG reductase activity was determined using the following 20mM buffer at pH 3.5-10.0: sodium acetate buffer (pH 3.5-6.0), tris-HCl buffer (pH 6.0-8.0) and glycine buffer (8.0-10.0), and the detection mode refers to detection of specific enzyme activity. Three measurements were made using a microplate reader.
The results show that, as can be seen from FIG. 4, the optimum pH of the 2,5-DKG reductase from G.oxydans ATCC9937 is between pH5.5 and 8.5, the enzyme activity is hardly affected by different buffers, the catalytic ability is stronger than that of the acid when the pH is alkaline, but the catalytic ability starts to be significantly reduced when the pH is higher than 8.5. When the reaction pH is less than 5.5, the catalytic activity thereof is also rapidly lowered. This suggests that the novel 2,5-DKG reductase discovered by the present invention is relatively stable at conventional pH.
Example 5: influence of temperature on catalytic Activity of G.oxydans ATCC 9937-derived 2,5-DKG reductase
The method comprises the following specific steps:
the 2,5-DKG reductase solution was divided into 5 portions and heated at 20℃to 40℃for 80 minutes, and samples were taken at 20 minute intervals to detect the enzyme activity. The remaining amount of enzyme activity after heating at different temperatures for different times was calculated with the initial enzyme activity as 100%. The enzyme activity detection mode refers to detection of specific enzyme activity.
The results show (FIG. 5) that the 2,5-DKGhuan protoenzyme discovered by the invention can still keep more than 90% of the enzyme activity after 80min, when the enzyme activity is hardly affected by the enzyme activity when the enzyme activity is kept for 80min in a 20mM Tris-HCl buffer (pH 7.0) at the temperature lower than 40 ℃. The DKGA-G can still maintain the catalytic activity of more than 80 percent after being stored for 72 hours at the temperature of 4 ℃.
When the temperature is increased to 50 ℃, the enzyme activity is reduced to 80% of the initial enzyme activity after 20min, and the enzyme activity is not obviously reduced after the operation is continued for 80 min.
When the temperature is higher than 60 ℃, the enzyme activity is reduced rapidly along with the increase of the preservation time, the enzyme activity is about 70% after 20min, and the enzyme activity is about 30% after 40min, until the enzyme activity is completely lost after 80 min. The optimal catalytic temperature is 20-30 ℃, and the catalytic activity of the 2,5-DKG reductase from G.oxydans can be maintained to be more than 95% in the detection time within the temperature range.
Example 6: the effect of common metal ions on the catalytic ability of 2,5-DKG reductase from G.oxydans ATCC9937 is specifically as follows:
the catalytic activity of the enzyme was measured in the presence of various salts. The method for detecting the enzyme activity is described above. Sodium chloride, calcium chloride, magnesium chloride and magnesium chloride are respectively taken to prepare mother liquor with the concentration of 1 mol/L. When diluted in use, the concentration gradient of each metal ion was set to 0, 10mM, 25mM, 50mM, 100mM, 200mM, 250mM. The effect of common metal ions on the catalytic activity of 2,5-DKG reductase was judged by comparing the change in catalytic activity of 2,5-DKG reductase in the presence of different metal ions. The results are shown in Table 3.
Table 3: influence of different Metal ions on enzyme Activity
The results showed (FIG. 6) that the catalytic ability was gradually increased with increasing sodium ion concentration when sodium ions were added to the system, and was greatly improved when the added amount was 250mM.
Whereas the addition of calcium ions clearly inhibited the catalytic activity of the 2,5-DKG reductase from G.oxydans ATCC 9937. At calcium concentrations greater than 100mM, the 2,5-DKG reductase from G.oxydans ATCC9937 was almost completely lost in catalytic capacity.
The potassium ion has obvious promotion effect on the catalytic capacity of the 2,5-DKG reductase from G.oxydans ATCC9937, and the substrate consumption speed is increased along with the increase of the addition concentration.
Whereas magnesium ion concentrations below 200mM promote the catalytic activity of the 2,5-DKG reductase from G.oxydans ATCC9937, the promotion is most pronounced at concentrations above 200mM, and it begins to inhibit the catalytic activity of the 2,5-DKG reductase from G.oxydans ATCC9937 at concentrations above 200 mM.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for preparing 2-keto-L-gulonic acid is characterized in that 2,5-DKG reductase with an amino acid sequence shown as SEQ ID NO.1 or recombinant bacteria expressing 2,5-DKG reductase with an amino acid sequence shown as SEQ ID NO.1 are added into a reaction system containing 2, 5-diketo-D-gluconic acid to prepare the 2-keto-L-gulonic acid.
2. The method of claim 1, wherein the nucleotide sequence encoding the 2,5-DKG reductase is set forth in SEQ ID No. 2.
3. The method according to claim 1 or 2, wherein the reaction conditions in the reaction system are: 25-30, normal pressure and pH 5.0-7.0.
4. The method according to claim 3, wherein the 2, 5-diketo-D-gluconic acid is added to the reaction system in an amount of: 2mM; the addition amount of the 2,5-DKG reductase is as follows: 50. Mu.L of NADPH was added to the reaction system in an amount of 2mM.
5. The method of claim 4, wherein the recombinant bacterium expressing the 2,5-DKG reductase having the amino acid sequence shown in SEQ ID NO.1 is a bacterium or a fungus as an expression host.
6. The application of 2,5-DKG reductase with the amino acid sequence shown as SEQ ID NO.1 or 2,5-DKG reductase with the expression amino acid sequence shown as SEQ ID NO.1 or a gene for encoding the 2,5-DKG reductase with the nucleotide sequence shown as SEQ ID NO.2 or a recombinant vector carrying the gene for encoding the 2,5-DKG reductase with the nucleotide sequence shown as SEQ ID NO.2 in the preparation of 2-keto-L-gulonic acid or products containing the 2-keto-L-gulonic acid.
7. The use according to claim 6 or 7, wherein the recombinant vector is a pET28a or pcdfdue expression vector.
8. The use according to claim 7, wherein the recombinant bacterium is an E.coli BL21 (DE 3) as an expression host.
9. A method for improving the yield of 2-keto-L-gulonic acid is characterized in that 2,5-DKG reductase with an amino acid sequence shown as SEQ ID NO.1 or recombinant bacteria expressing 2,5-DKG reductase with an amino acid sequence shown as SEQ ID NO.1 are added into a reaction system containing 2, 5-diketo-D-gluconic acid for reaction, and the 2-keto-L-gulonic acid is prepared.
10. The method of claim 9, wherein the nucleotide sequence encoding the 2,5-DKG reductase is set forth in SEQ ID No. 2.
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