CN111304188A - Gel type biocatalyst, preparation method thereof and application thereof in production of D-lysine - Google Patents
Gel type biocatalyst, preparation method thereof and application thereof in production of D-lysine Download PDFInfo
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- CN111304188A CN111304188A CN202010120312.1A CN202010120312A CN111304188A CN 111304188 A CN111304188 A CN 111304188A CN 202010120312 A CN202010120312 A CN 202010120312A CN 111304188 A CN111304188 A CN 111304188A
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Abstract
The invention discloses a gel-type biocatalyst, a preparation method thereof and application thereof in producing D-lysine, wherein the preparation method comprises the following steps: (1) mixing the aqueous polyurethane gel powder with an enzyme-producing strain; (2) and (2) stirring the material obtained in the step (1), adding a buffer solution into the material, and obtaining the gel-type biocatalyst after the water-based polyurethane gel powder absorbs water, swells and forms. Compared with the prior art, the invention has the following advantages: (1) after the hydrogel material with the buffering effect is mixed with the microorganisms, the tolerance of the microorganisms to temperature and pH is effectively improved; (2) the method improves the yield of D-lysine produced by a biological racemization method.
Description
Technical Field
The invention belongs to the technical field of chiral amino acid and biocatalysis, and particularly relates to a gel type biocatalyst, a preparation method thereof and application thereof in producing D-lysine.
Background
D-lysine has better antibacterial property and important application in medicine, can be used as a precursor to participate in the synthesis of luteinizing hormone releasing hormone analogues, and can inhibit the glycosylation of some proteins which are not enzymes, thereby preventing some complications of diabetes. Some drugs (oral and intravenous) in which D-lysine participates in synthesis can greatly reduce the uptake of radioactive peptides into the kidney, and thus D-lysine is more suitable for the treatment of cancer than L-lysine. In addition, the polymer of D-lysine can stimulate the proliferation of human brain astrocytes and chondrocytes, and is a good carrier of medicines. There are two main methods for racemization of amino acids: one is chemical racemization, and the racemization method has harsh conditions and causes certain pollution to the environment; the other is biological enzyme racemization, which means that a specific amino acid racemase is utilized to realize the configuration conversion. However, the weakness and the volatility of the microorganisms or enzyme proteins are one of the main problems restricting the racemization development of the biological method. Therefore, the main difficulty of producing the D-lysine by using a biological racemization method is to improve the tolerance of a biocatalyst.
The hydrogel can be swelled by water but not be dissolved by water, has polymer with three-dimensional structure network, has good biocompatibility, and has good protection effect on wrapped microorganism or protein. Compared with a hard material made of a solid material, the hydrogel provides a soft buffer area with high water content, and is more beneficial to improving the temperature and pH tolerance of microorganisms. The hydrogel is utilized to improve the service performance of the biocatalyst, and further improve the yield of D-lysine produced by a biological racemization method.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a gel-type biocatalyst aiming at poor biological racemization effect caused by high pH and temperature sensitivity and poor tolerance of enzyme protein in the prior art.
The technical problem to be solved by the invention is to provide a preparation method of the gel-type biocatalyst.
The technical problem to be solved finally by the invention is to provide the application of the gel-type biocatalyst in the production of D-lysine, wherein the gel-type biocatalyst has a certain improvement on the yield of the D-lysine produced by a biological racemization method and has an obvious improvement effect on the tolerance of temperature and pH.
In order to solve the technical problems, the invention discloses a preparation method of a gel type biocatalyst, which comprises the following steps:
(1) grinding the dry waterborne polyurethane gel particles into powder, and quickly and uniformly mixing the waterborne polyurethane gel powder with the enzyme-producing strain;
(2) and (2) stirring the material obtained in the step (1), adding a buffer solution into the material, and obtaining the gel-type biocatalyst after the water-based polyurethane gel powder absorbs water, swells and forms.
In the step (1), the aqueous polyurethane gel is adopted, so that the biocompatibility is good, and the price is low.
In step (1), the enzyme-producing strain is preferably a lysine racemase-producing bacterium, more preferably a lysine racemase-producing Escherichia coli, and most preferably a strain obtained by the following construction method
(a) Construction of the strains: gene LYR from P.mirabilis BCRC10725 was synthesized by Kingchi into the vector pET-28a (+) between the NcoI/XhoI cleavage sites. This recombinant plasmid was then transformed into e.coli BL21(DE3) to construct the LYR strain. Wherein, the nucleotide sequence of LYR is shown in SEQ ID NO. 1.
(b) Culturing
Plate culture: LYP strain was taken out, streaking was performed in a clean bench, and the plate was incubated at 37 ℃ for 12 hours in a incubator.
Seed liquid culture: 5mL of seed medium (LB medium) and 10. mu.L of 25g/L kanamycin were added to a 50mL centrifuge tube, and about 10. mu.L of glycerol cryopreserved cells were selected and inoculated, followed by culture at 37 ℃ at 200r/min for 8 hours.
And (3) shake flask fermentation culture: in a 500mL shake flask containing 100mL LB medium, 200. mu.L of 25g/L kanamycin was added, the volume of the inoculum size of the seed solution was 1%, and the mixture was cultured at 37 ℃ at 200r/min to OD600Adding isopropyl- β -D-thiogalactoside (IPTG) to give final concentration of 1mmol/L, inducing at 30 deg.C for 12 hr, centrifuging, and collecting the strainWhole cell LYR (lysozyme) of lysine racemase.
In the step (1), the aqueous polyurethane gel is aliphatic isocyanate type aqueous polyurethane or aromatic isocyanate type aqueous polyurethane; the mesh number of the water-based polyurethane gel powder is 500-1000 meshes; the grinding is in the interval, the operation difficulty is low, and the mixing effect is good; the ground dry hydrogel fine powder and the microbial sludge have a better mixing interface, so that the dry hydrogel fine powder and the microbial sludge have a certain protection effect on microorganisms, and meanwhile, the dry hydrogel fine powder cannot excessively embed the microorganisms, which is favorable for mass transfer of substances.
In the step (1), the mass ratio of the aqueous polyurethane gel powder to the enzyme-producing strain is 0.1-1: 1-10.
In the step (1), the mixing is carried out for 5-10 min at 4 ℃ and at a rotating speed of 100-500 rpm.
In the step (2), the buffer solution is any one or a combination of more of a PBS buffer solution, a Tris-HCl buffer solution and an LB culture medium; wherein, the LB medium (L)-1) Comprises the following steps: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 0.05g of kanamycin and water as a solvent; wherein the buffer can provide corresponding pH while protecting the enzyme.
In the step (2), the adding speed of the buffer solution is 10-100 mL/min; the amount of the buffer solution is 100-300 mL/0.1-1 g of the aqueous polyurethane gel powder.
In the step (2), the stirring is carried out at 200-500 rpm until the water-based polyurethane gel powder absorbs water to expand and be molded.
The gel-type biocatalyst prepared by the above method is also within the protection scope of the present invention.
The application of the gel-type biocatalyst in the production of D-lysine is also within the scope of the present invention.
Wherein, the application comprises the following steps: mixing potassium phosphate buffer solution, gel type biocatalyst and L-lysine, and reacting.
Wherein the concentration of the potassium phosphate buffer solution is 0.2M, and the pH value is 7.0; potassium phosphate buffer and L-lysine dosageThe ratio is 10 mL: 1.8 g; controlling the amount of gel-type biocatalyst to make the concentration of enzyme-producing bacteria OD6000.5 to 2 (preferably OD)6000.5); the reaction is carried out for 10-30 min at 37 ℃ and 200-500 rpm in an oscillating way.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. after the hydrogel material with the buffering effect is mixed with the microorganisms, the tolerance of the microorganisms to temperature and pH is effectively improved.
2. The method has the advantages of good applicability, simple operation, easily obtained materials and low cost, and effectively reduces the production cost of the biocatalyst.
3. The method improves the yield of D-lysine produced by a biological racemization method.
Drawings
FIG. 1 is a graph showing comparative data of relative enzyme activity U at different temperatures;
FIG. 2 is a graph showing comparative data of enzyme activity U at different pH values;
FIG. 3 shows gel-type biocatalyst reuse performance.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
EXAMPLE 1 Strain construction and cultivation Process
1.1 construction of the strains
Gene LYR from P.mirabilis BCRC10725 was synthesized by Kingchi into the vector pET-28a (+) between the NcoI/XhoI cleavage sites. This recombinant plasmid was then transformed into e.coli BL21(DE3) to construct the LYR strain. Wherein, the nucleotide sequence of the LYR protein is shown as SEQ ID NO. 1.
Wherein the preparation process of E.coli BL21(DE3) competence refers to the operation of Escherichia coli competence preparation kit of Taori physician technology company;
1.2 culture Medium
Plate Medium (L)-1): 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 20g of agar, 0.05g of kanamycin and water as a solvent.
LB Medium (L)-1): 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 0.05g of kanamycin and water as a solvent.
The sterilization method comprises the following steps: filtering antibiotic for sterilization, and sterilizing the rest materials with high pressure steam at 121 deg.C for 15 min.
1.3 cultivation and Collection of microorganisms
1.3.1 plate culture
LYP strain stored in a refrigerator at-80 deg.C was taken out, streaking was performed on the plate in a clean bench, and the plate was incubated in a 37 deg.C incubator for 12 hours.
1.3.2 seed culture
5mL of seed medium (LB medium) and 10. mu.L of 25g/L kanamycin were added to a 50mL centrifuge tube, and about 10. mu.L of glycerol cryopreserved cells were selected and inoculated, followed by culture at 37 ℃ at 200r/min for 8 hours.
1.3.3 Shake flask fermentation culture
In a 500mL shake flask containing 100mL LB medium, 200. mu.L of 25g/L kanamycin was added, the volume of the inoculum size of the seed solution was 1%, and the mixture was cultured at 37 ℃ at 200r/min to OD600When the concentration is 0.6, adding isopropyl- β -D-thiogalactoside (IPTG) to the mixture to make the final concentration of IPTG reach 1mmol/L, inducing at 30 ℃ for 12h, and centrifuging to collect the strain, wherein the obtained strain is whole cell LYR (lysozyme) containing lysine racemase.
1.3.4 cell Density (OD)600) Measurement of (2)
Diluting the fermentation broth with deionized water to a certain degree to obtain absorbance value (A)600) Between about 0.2 and about 0.8, as measured by an ultraviolet-visible spectrophotometer. Cell density (OD)600)＝A600X dilution factor.
Example 2: preparation of gel-type biocatalyst
Grinding dry PDI type waterborne polyurethane gel particles into powder, and sieving the powder by a 1000-mesh molecular sieve to obtain uniform fine powder. Weighing 1g of fine powder and 10g of bacterial sludge of the enzyme-producing strain (the whole cell LYR containing lysine racemase prepared in example 1), stirring and mixing at the temperature of 4 ℃ and the rotating speed of 100rpm for 5-10 minutes, after the two are uniformly mixed, adding a mixed buffer (mixed solution of an LB culture medium and a PBS buffer solution with the volume ratio of 1: 5) while stirring at the rotating speed of 100rpm, wherein the adding speed of the mixed buffer is 100mL/min, and the adding volume of the mixed buffer is 300mL until the dry waterborne polyurethane fine powder absorbs water and swells to form gel with the bacterial sludge, thus preparing the gel type biocatalyst.
[1]The influence of the NCO/OH ratio and the 1,6-hexanediol/dimethylol propionic acid molar ratio on the properties of waterbornepolyurethane dispersions based on1,5-pentamethylene diisocyanate
Example 3 analytical method
3.1 detection method of enzyme Activity
10mL of 0.2M potassium phosphate buffer (pH 7.0), OD 7, was added to a 50mL centrifuge tube600Whole cell LYR containing lysine racemase of 5, L-lysine of 1.8g, shaking at 37 deg.C and 200r/min for 10min, sampling, and detecting D-lysine yield by liquid phase assay.
In the experiment, the Dry Cell Weight (DCW) required for catalyzing 1 mu mol L-lysine to generate D-lysine per minute is defined as an enzyme activity unit; the content of DCW in 1L fermentation broth is the cell density (OD)600) 0.4 times of the total weight of the powder.
3.2 analytical methods
D-lysine and L-lysine detection method: high performance liquid chromatography (Agilent 1200 high performance liquid chromatography); the chromatographic column is Chirex 3126(D) -penicilamine 150X 4.6mm, and the pre-column is 30X 4.6 mm; mobile phase: CuSO4·5H2Mixed solution of O, water and isopropanol, the volume ratio of the water to the isopropanol is 95:5, and CuSO4·5H2The concentration of O is 1 mM; the flow rate is 0.8 mL/min; the temperature is 25 ℃; the wavelength is 254 nm.
Example 4 catalytic Activity and tolerance comparison
1. The reaction is carried out at the temperature of 20-45 ℃ (temperature interval is 5 ℃), the pH is 7, and other reaction conditions refer to the biopsy method of 3.1 enzyme in example 3, so as to obtain the data of whole cells;
furthermore, the lysine racemase-containing whole-cell LYR in 3.1 was replaced with the gel-type biocatalyst prepared in example 2, and the amount was controlled so that the concentration of the enzyme-producing bacterium was OD600＝0.5。
The results of the above experiments are shown in FIG. 1. The data in figure 1 show that the optimal reaction temperature of the gel-type biocatalyst is 35 ℃, and 1500U of relative enzyme activity can be still maintained at 45 ℃, which is 1.55 times of that of a whole cell at the same temperature, so that the heat resistance of the gel-type biocatalyst is obviously improved, and the maximum catalytic activity of the gel-type biocatalyst at the optimal temperature is 2679U of relative enzyme activity, which is 1.6 times of that of the whole cell, so that the yield of D-lysine is obviously improved.
2. Two types of buffer solutions are selected to adjust the pH (acid buffer solution or alkaline buffer solution), the reaction is respectively carried out in the range of pH 4-8 (the interval of the pH is 1), the reaction temperature is 35 ℃, and the other conditions of the reaction refer to the biopsy method of 3.1 enzyme in the example 3;
furthermore, the lysine racemase-containing whole-cell LYR in 3.1 was replaced with the gel-type biocatalyst prepared in example 2, and the amount was controlled so that the concentration of the enzyme-producing bacterium was OD600＝0.5。
The results of the above experiments are shown in FIG. 2. The data in figure 2 show that the pH tolerance of the gel-type biocatalyst is significantly improved compared with that of whole cells, and at pH 5, the gel-type biocatalyst can maintain the relative enzyme activity of about 2000U, which is 2.61 times of that of whole cells at the same pH; the gel-type biocatalyst was able to maintain a relative enzyme activity of approximately 1917U at pH 8, which is 3.83 times that of whole cells at the same pH. The preparation technology of the gelation is shown, so that the microorganism has better tolerance to pH and better protection effect on the microorganism.
3. According to the above reaction system, i.e., the reaction temperature of 35 ℃ and pH 7, and other conditions of the reaction, see the biopsy method of enzyme 3.1 in example 3, the gel type biocatalyst was centrifuged, added to a new reaction system, and this was repeated 5 times, to obtain FIG. 3. After 5 using cycles, the gel-type biocatalyst can keep 90% of relative enzyme activity, the gel-type biocatalyst still keeps better catalytic activity, show that the preparation technology of this kind of gelatinization prolongs the service life of the microorganism-type biocatalyst, has saved the actual production cost of preparing D-lysine of biological racemization method.
In conclusion, the method for producing D-lysine by using the gel-type biocatalyst improves the tolerance of microorganisms to temperature and pH based on the protection effect of hydrogel on the microorganisms, further widens the application range of the gel-type biocatalyst, reduces the use limitation of the gel-type biocatalyst, reduces the production cost of preparing D-lysine by using a biological racemization method, and also improves the catalytic efficiency of preparing D-lysine by using the biological racemization method under the environment with the optimal temperature and the optimal pH.
The present invention provides a gel-type biocatalyst, a method for preparing the same, and a concept and a method for using the same in producing D-lysine, and a method and a way for implementing the technical scheme are numerous, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Sequence listing
<110> Nanjing Kano Biotech Co., Ltd
<120> a gel type biocatalyst, its preparation method and application in producing D-lysine
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1233
<212>DNA
<213> Artificial sequence (Artificial)
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catgggcatg tctctgggta tccgttacct ggctctgctg ccgctgttcg ttatcaccgc 60
ttgccagcag ccggttaact acaacccgcc ggctacccag gttgctcagg ttcagccggc 120
tatcgttaac aactcttgga tcgaaatctc tcgttctgct ctggacttca acgttaaaaa 180
agttcagtct ctgctgggta aacagtcttc tctgtgcgct gttctgaaag gtgacgctta 240
cggtcacgac ctgtctctgg ttgctccgat catgatcgaa aacaacgtta aatgcatcgg 300
tgttaccaac aaccaggaac tgaaagaagt tcgtgacctg ggtttcaaag gtcgtctgat 360
gcgtgttcgt aacgctaccg aacaggaaat ggctcaggct accaactaca acgttgaaga 420
actgatcggt gacctggaca tggctaaacg tctggacgct atcgctaaac agcagaacaa 480
agttatcccg atccacctgg ctctgaactc tggtggtatg tctcgtaacg gtctggaagt 540
tgacaacaaa tctggtctgg aaaaagctaa acagatctct cagctggcta acctgaaagt 600
tgttggtatc atgtctcact acccggaaga agacgctaac aaagttcgtg aagacctggc 660
tcgtttcaaa cagcagtctc agcaggttct ggaagttatg ggtctggaac gtaacaacgt 720
taccctgcac atggctaaca ccttcgctac catcaccgtt ccggaatctt ggctggacat 780
ggttcgtgtt ggtggtatct tctacggtga caccatcgct tctaccgact acaaacgtgt 840
tatgaccttc aaatctaaca tcgcttctat caactactac ccgaaaggta acaccgttgg 900
ttacgaccgt acctacaccc tgaaacgtga ctctgttctg gctaacatcc cggttggtta 960
cgctgacggt taccgtcgtg ttttctctaa cgctggtcac gctctgatcg ctggtcagcg 1020
tgttccggtt ctgggtaaaa cctctatgaa caccgttatc gttgacatca cctctctgaa 1080
caacatcaaa ccgggtgacg aagttgtttt cttcggtaaa cagggtaact ctgaaatcac 1140
cgctgaagaa atcgaagaca tctctggtgc tctgttcacc gaaatgtcta tcctgtgggg 1200
tgctaccaac cagcgtgttc tggttgacct cga 1233
Claims (10)
1. A preparation method of a gel-type biocatalyst is characterized by comprising the following steps:
(1) mixing the aqueous polyurethane gel powder with an enzyme-producing strain;
(2) and (2) stirring the material obtained in the step (1), adding a buffer solution into the material, and obtaining the gel-type biocatalyst after the water-based polyurethane gel powder absorbs water, swells and forms.
2. The method for preparing a gel-type biocatalyst as claimed in claim 1, wherein in step (1), said aqueous polyurethane gel is an aliphatic isocyanate-type aqueous polyurethane or an aromatic isocyanate-type aqueous polyurethane; the mesh number of the water-based polyurethane gel powder is 500-1000 meshes.
3. The method for preparing the gel-type biocatalyst according to claim 1, wherein in step (1), the mass ratio of the aqueous polyurethane gel powder to the enzyme-producing strain is 0.1-1: 1-10.
4. The method for preparing a gel-type biocatalyst as claimed in claim 1, wherein in the step (1), the mixing is performed by stirring and mixing at a rotation speed of 100 to 500rpm at 4 ℃ for 5 to 10 min.
5. The method for preparing a gel-type biocatalyst as claimed in claim 1, wherein in step (2), the buffer is any one or a combination of PBS buffer, Tris-Hcl buffer, LB medium; the adding speed of the buffer solution is 10-100 mL/min; the amount of the buffer solution is 100-300 mL/0.1-1 g of the aqueous polyurethane gel powder.
6. The method for preparing the gel-type biocatalyst as claimed in claim 1, wherein in the step (2), the stirring is performed at 200-500 rpm until the aqueous polyurethane gel powder absorbs water to swell and form.
7. A gel-type biocatalyst prepared by the process of any one of claims 1 to 6.
8. Use of the gel-type biocatalyst as claimed in claim 7 for the production of D-lysine.
9. Use according to claim 8, characterized in that it comprises the following steps: mixing potassium phosphate buffer solution, gel type biocatalyst and L-lysine, and reacting.
10. The use according to claim 9, wherein the potassium phosphate buffer has a concentration of 0.2M and a pH of 7.0; the dosage ratio of the potassium phosphate buffer solution to the L-lysine is 10 mL: 1.8 g; controlling the amount of gel-type biocatalyst to make the concentration of enzyme-producing bacteria OD6000.5-2; the reaction is carried out for 10-30 min at 37 ℃ and 200-500 rpm in an oscillating way.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113214503A (en) * | 2021-04-13 | 2021-08-06 | 北京科技大学 | Preparation and application of amino acid-based polyurethane supermolecule high-viscosity gel patch |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113214503A (en) * | 2021-04-13 | 2021-08-06 | 北京科技大学 | Preparation and application of amino acid-based polyurethane supermolecule high-viscosity gel patch |
| CN113214503B (en) * | 2021-04-13 | 2022-06-10 | 北京科技大学 | Preparation and application of amino acid-based polyurethane supermolecule high-viscosity gel patch |
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Application publication date: 20200619 |
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| WW01 | Invention patent application withdrawn after publication |