CN118147123A - D-psicose-3-epimerase and D-psicose-3-epimerase directional immobilized enzyme - Google Patents
D-psicose-3-epimerase and D-psicose-3-epimerase directional immobilized enzyme Download PDFInfo
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- 108030002106 D-psicose 3-epimerases Proteins 0.000 title claims abstract description 89
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- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 claims abstract description 21
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- AEMOLEFTQBMNLQ-UHFFFAOYSA-N 3,4,5,6-tetrahydroxyoxane-2-carboxylic acid Chemical compound OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- OIHQCYYSCXMHOH-UHFFFAOYSA-N C(C1=CC=CC=C1)=O.C=1(O)C(O)=CC=CC1 Chemical compound C(C1=CC=CC=C1)=O.C=1(O)C(O)=CC=CC1 OIHQCYYSCXMHOH-UHFFFAOYSA-N 0.000 description 1
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- 206010022489 Insulin Resistance Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
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- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/091—Phenol resins; Amino resins
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Abstract
The invention relates to the technical field of biology, in particular to D-psicose-3-epimerase and a D-psicose-3-epimerase directional immobilized enzyme. The immobilized enzyme of the D-psicose-3-epimerase provided by the invention has the advantages that the surface of the enzyme molecule contains cysteine residues by utilizing site-directed mutation, and then the site-directed mutated D-psicose-3-epimerase is directionally immobilized by utilizing amino resin modified by catechol groups. In the invention, the catechol group can be specifically and covalently combined with the sulfhydryl group, so that the enzyme can be directly immobilized in the crude enzyme solution without purifying the enzyme. The directional immobilized enzyme of the D-psicose-3-epimerase has more excellent performance and better enzyme activity stability, and the continuous reaction device reduces experimental operation flow, obviously improves the yield of the D-psicose, and has good industrial application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to D-psicose-3-epimerase and a D-psicose-3-epimerase directional immobilized enzyme.
Background
Sweetness is a taste pursuit inherent to humans. However, chronic diseases such as overweight, diabetes, "three high" and the like, which have long been caused by excessive ingestion of sugars, are also increasingly prominent as well as a range of secondary health problems that result therefrom. The delicacy and health are always the double demands of the masses towards a good life, so the development and application of low-calorie sweet substitutes are effective ways to ensure delicacy and health at the same time.
D-psicose (D-allose) is a low-calorie sweetener closest to sucrose in physicochemical properties, texture characteristics and processability, has sweetness of 70% of sucrose, but has calorie of only 0.4kcal/g, and has physicochemical properties of reducing absorption of dietary D-fructose and D-glucose, enhancing insulin resistance, resisting obesity activity, reducing blood lipid and the like. D-psicose has become a research hotspot in the field of rare sugar biosynthesis worldwide due to its higher sweetness and lower energy as well as unique physiological functions and potential health benefits.
D-psicose (D-allose) belongs to ketohexose, is a C-3 epimer of D-fructose, and is mainly divided into a chemical synthesis method and a biocatalysis method at present, but the chemical synthesis method has few common defects which are difficult to overcome due to separation difficulty, more byproducts, production of chemical waste and the like, and the environment-friendly bioenzyme method is used in industry, can catalyze D-fructose to produce D-psicose by using D-psicose-3-epimerase (DPease), has no byproduct, and is a mode for efficiently producing D-psicose.
However, in the process of synthesizing D-psicose by the traditional biological enzyme method, the free crude enzyme has the defects of poor chemical stability, difficult collection after the enzyme reaction is finished, and the like, so that the automatic production is not facilitated, and the production cost is overlarge. Therefore, the immobilized enzyme technology is used for immobilizing the D-psicose-3-epimerase, and compared with the free enzyme, the immobilized enzyme not only can keep the characteristics of high efficiency and specificity of enzyme catalysis, but also can greatly improve the thermal stability and chemical stability of the enzyme. The prior art reported DPease immobilized enzyme technology is mainly characterized in that an embedding method with sodium alginate as a carrier and an adsorption method with single resin as a carrier are used for fixation, for example, a Zhangming station and the like are used for immobilizing DPease enzyme through sodium alginate embedding and CaCl 2 crosslinking, and the enzyme activity recovery rate is maintained to be more than 65% of the initial enzyme activity after the DPease immobilized enzyme is repeatedly used for 10 times (publication No. CN 116355888A); yi and the like, the macroporous resin is used as a carrier to immobilize DPease, and the activity of 10 batches of enzyme does not obviously decline (publication No. CN 110438113A); zhuming and the like, through adding a section of label rich in lysine into the C-terminal fusion of psicose 3 epimerase, the covalent bonding between the enzyme and the epoxy group of a carrier is improved, thereby enhancing the immobilization effect and improving the immobilization efficiency and the reuse rate (publication No. CN 112831489A). Although these methods solve the problems of poor chemical stability of the free enzyme and difficult collection after the reaction is completed, they generally have the problems of greatly decreasing the activity of the enzyme, reducing the immobilization amount of the enzyme, and the like, and require the separation and purification of the enzyme first, thus increasing the immobilization cost. Therefore, the development of the immobilized carrier can specifically combine the enzyme and the immobilized carrier to realize the directional immobilization of the enzyme, thereby being beneficial to the entry of a substrate into an active site of the enzyme, avoiding the reduction of the enzyme activity and increasing the stability of the enzyme, and simultaneously, repeatedly utilizing the enzyme, thereby having great significance for the industrial development of the D-psicose.
Disclosure of Invention
In order to solve the above problems, an object of the present invention is to provide a D-psicose-3-epimerase and a D-psicose-3-epimerase-directed immobilized enzyme.
The invention makes the surface of the enzyme molecule mutate a plurality of cysteine residues by carrying out site-directed mutation on the gene of D-psicose-3-epimerase (DsDPEase), and uses the specific combination of sulfhydryl and catechol group on the surface of resin to prepare the directional immobilized enzyme and provide a mode for producing the D-psicose in a high-efficiency continuous way,
The aim of the invention can be achieved by the following technical scheme:
It is a first object of the present invention to provide a D-psicose-3-epimerase selected from the group consisting of proteins having the amino acid sequences:
(1) Replacing serine at position 44 of the amino acid sequence shown in SEQ ID No.1 with cysteine;
(2) Substitution of serine at position 193 of the amino acid sequence shown in SEQ ID No.1 with cysteine;
(3) Serine at position 44 of the amino acid sequence shown in SEQ ID No.1 is replaced with cysteine, and serine at position 264 is replaced with cysteine.
In one embodiment of the invention, the one D-psicose-3-epimerase is selected from the group consisting of proteins having the amino acid sequence:
Serine at position 44 of the amino acid sequence shown in SEQ ID No.1 is replaced with cysteine, and serine at position 264 is replaced with cysteine.
It is a second object of the present invention to provide an isolated nucleic acid which is a nucleic acid molecule encoding the above D-psicose-3-epimerase.
It is a third object of the present invention to provide a recombinant expression vector comprising the above-described nucleic acid sequence.
It is a fourth object of the present invention to provide a recombinant expression vector transformant comprising the above recombinant expression vector.
A fifth object of the present invention is to provide an application of the above-mentioned D-psicose-3-epimerase in preparing a D-psicose-3-epimerase-directed immobilized enzyme.
In one embodiment of the present invention, the use of D-psicose-3-epimerase in the preparation of a D-psicose-3-epimerase-directed immobilized enzyme specifically comprises the steps of:
Reacting amino resin with 3, 4-dihydroxybenzaldehyde to obtain catechol modified resin; and then evenly mixing catechol modified resin with D-psicose-3-epimerase, and obtaining the D-psicose-3-epimerase directional immobilized enzyme after crosslinking reaction.
In one embodiment of the present invention, the amino resin is selected from one of LX1000HA, LX1000EPN, LX1000HFA, HFA 001.
In one embodiment of the present invention, the mass ratio of the amino resin to 3, 4-dihydroxybenzaldehyde is 8 to 12:1, a step of; in the reaction process of the amino resin and the 3, 4-dihydroxybenzaldehyde, the temperature is room temperature and the time is 5-8 h.
In one embodiment of the invention, the ratio of catechol modified resin to D-psicose-3-epimerase crude enzyme solution is 1g: 15-30 mg.
In one embodiment of the invention, the temperature is between 0 and 4℃and the time is between 5 and 7 hours during the crosslinking reaction.
The sixth object of the present invention is to provide a D-psicose-3-epimerase-immobilized enzyme directed thereto, which is prepared by the above-mentioned D-psicose-3-epimerase.
The seventh object of the invention is to provide an application of the directional immobilized enzyme of D-psicose-3-epimerase in preparing D-psicose.
In one embodiment of the present invention, the use of a D-psicose-3-epimerase directed immobilization enzyme in the preparation of D-psicose specifically comprises the steps of:
In a continuous reaction device, fructose solution is taken as a substrate, and is uniformly mixed with D-psicose-3-epimerase directional immobilized enzyme, and D-psicose is obtained through catalytic reaction.
In one embodiment of the invention, fructose solution is mixed with D-psicose-3-epimerase directional immobilized enzyme at a flow rate of 1-1.5 mL/min;
In the catalytic reaction process, the temperature is 65-75 ℃.
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, the site-directed mutation is carried out on the D-psicose-3-epimerase, the surface of an enzyme molecule is mutated into a plurality of cysteine residues, and the specific combination of sulfhydryl groups in the cysteine residues and catechol groups on the surface of resin is utilized to realize the directional immobilization of the D-psicose-3-epimerase, thereby being beneficial to the enzyme activity center of a substrate and a product entering and exiting the D-psicose-3-epimerase and furthest retaining the catalytic activity of the D-psicose-3-epimerase. The method is simple and convenient to operate, the D-psicose-3-epimerase is not required to be purified, the target enzyme can be specifically immobilized in the crude enzyme liquid, and the recovery rate of the enzyme activity is high. Compared with free enzyme, the directional immobilized enzyme of D-psicose-3-epimerase has the advantages of remarkably improved stability, good continuous conversion capability and capability of converting high-concentration fructose solution in a packed column device to produce psicose; the D-psicose-3-epimerase directional immobilized enzyme prepared by using the B.subtilis 168/PMATE-DP-C44-C264 mutant strain still keeps the conversion rate of 30% after continuous reaction for 30 hours, and the residual enzyme activity of the directional immobilized enzyme remains 95%.
Drawings
FIG. 1 is a plasmid map of recombinant plasmid PMATE-DP.
FIG. 2 is an active site diagram of the protein structure of D-psicose-3-epimerase.
FIG. 3 is a protein expression pattern for recombinant strain B.subilis 168/PMATE-DP and mutant strain B.subilis 168/PMATE05-DP-C44-C193 and B.subilis 168/PMATE 05-DP-C44-C264.
FIG. 4 is a graph showing the catalytic reaction conversion rate of D-psicose-3-epimerase prepared from recombinant strain B.subilis 168/PMATE-DP and D-psicose-3-epimerase prepared from each mutant strain.
FIG. 5 is a diagram showing a device for the directional immobilized enzyme reaction of D-psicose-3-epimerase; reference numerals in the drawings: 1. a water bath kettle; 2. a packed bed reactor; 3. a packed column; 4. a peristaltic pump; 5. a substrate accommodating groove; 6. and a heat preservation jacket.
FIG. 6 is a graph showing D-psicose conversion rate of a continuous catalytic reaction of D-psicose-3-epimerase-directed immobilized enzyme for 30 hours.
Detailed Description
The invention provides a D-psicose-3-epimerase, which is selected from proteins with the following amino acid sequences:
(1) Replacing serine at position 44 of the amino acid sequence shown in SEQ ID No.1 with cysteine;
(2) Substitution of serine at position 193 of the amino acid sequence shown in SEQ ID No.1 with cysteine;
(3) Serine at position 44 of the amino acid sequence shown in SEQ ID No.1 is replaced with cysteine, and serine at position 264 is replaced with cysteine.
In one embodiment of the invention, the one D-psicose-3-epimerase is selected from the group consisting of proteins having the amino acid sequence:
Serine at position 44 of the amino acid sequence shown in SEQ ID No.1 is replaced with cysteine, and serine at position 264 is replaced with cysteine.
The present invention provides an isolated nucleic acid which is a nucleic acid molecule encoding the above-described D-psicose-3-epimerase.
The present invention provides a recombinant expression vector comprising the above-described nucleic acid sequence.
The present invention provides a recombinant expression vector transformant comprising the above recombinant expression vector.
The invention provides an application of the D-psicose-3-epimerase in preparing the D-psicose-3-epimerase directional immobilized enzyme.
In one embodiment of the present invention, the use of D-psicose-3-epimerase in the preparation of a D-psicose-3-epimerase-directed immobilized enzyme specifically comprises the steps of:
Reacting amino resin with 3, 4-dihydroxybenzaldehyde to obtain catechol modified resin; and then evenly mixing catechol modified resin with D-psicose-3-epimerase, and obtaining the D-psicose-3-epimerase directional immobilized enzyme after crosslinking reaction.
In one embodiment of the present invention, the amino resin is selected from one of LX1000HA, LX1000EPN, LX1000HFA, HFA 001.
In one embodiment of the present invention, the mass ratio of the amino resin to 3, 4-dihydroxybenzaldehyde is 8 to 12:1, a step of; in the reaction process of the amino resin and the 3, 4-dihydroxybenzaldehyde, the temperature is room temperature and the time is 5-8 h.
In one embodiment of the invention, the ratio of catechol modified resin to D-psicose-3-epimerase crude enzyme solution is 1g: 15-30 mg.
In one embodiment of the invention, the temperature is between 0 and 4℃and the time is between 5 and 7 hours during the crosslinking reaction.
The invention provides a D-psicose-3-epimerase directional immobilized enzyme, which is prepared by the D-psicose-3-epimerase.
The invention provides an application of the D-psicose-3-epimerase directional immobilized enzyme in preparation of D-psicose.
In one embodiment of the present invention, the use of a D-psicose-3-epimerase directed immobilization enzyme in the preparation of D-psicose specifically comprises the steps of:
In a continuous reaction device, fructose solution is taken as a substrate, and is uniformly mixed with D-psicose-3-epimerase directional immobilized enzyme, and D-psicose is obtained through catalytic reaction.
In one embodiment of the invention, fructose solution is mixed with D-psicose-3-epimerase directional immobilized enzyme at a flow rate of 1-1.5 mL/min;
In the catalytic reaction process, the temperature is 65-75 ℃.
The invention will now be described in detail with reference to the drawings and specific examples.
In the examples below, unless otherwise specified, all reagents used were commercially available, and all detection means and methods used were conventional in the art.
Example 1
This example provides a method for constructing recombinant strain B.subtilis 168/PMATE-DP.
(S1) taking a DPease gene (synthesized by Optimum in Optimum of Praeparatum) with a nucleotide sequence shown as SEQ ID NO.1 as a template, obtaining a target gene by a conventional PCR cloning technology, performing agarose gel electrophoresis, and recovering the agarose gel to obtain a PCR product DPease gene.
Wherein the upstream primer sequence is F1 (the nucleotide sequence of which is shown as SEQ ID NO. 2); SEQ ID No.2:5'-GGCGACACTAGGGGGAATAATTCATATGAAACATGGTATCTACTAC GCTTACTGGG-3';
The sequence of the used downstream primer is R1 (the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 3); SEQ ID NO.3:5'-TGTCGGAACGAGACTTCTCTACTCGAGCTTCCAGTCCAGCATGT-3';
(S2) plasmid PMATE (available from Chun Yao biology Co., ltd., product number CYB 005) was subjected to agarose gel electrophoresis using high fidelity enzyme P515 PCR as a linearization carrier, and the agarose gel was recovered to obtain a pure PMATE05 linearization plasmid.
Wherein the upstream primer sequence is F2 (the nucleotide sequence of which is shown as SEQ ID NO. 4); SEQ ID NO.4:5'-AATTATTCCCCCTAGTGTCGCCAAATCGAAGTAGTCAC-3';
The sequence of the upstream primer is R2 (the nucleotide sequence of which is shown as SEQ ID NO. 5); SEQ ID NO.5:5'-TAGAGAAGTCTCGTTCCGACAGTTGGCATATGAGTTATG-3';
(S3) ligation of the DPease gene obtained in step (S1) with the PMATE linearization vector obtained in step (S2) gives a recombinant plasmid PMATE-DP (FIG. 1).
The connection reaction system is as follows: ligation was performed using ABclonal company 2X MultiF Seamless Assembly Mix. Mu.L, DPease gene fragment c/(0.02 base pair). Mu.L, PMATE05 linearized vector c/(0.02 base pair) 2). Mu.L, ddH 2 O to 20. Mu.L, and ligation was performed at 50℃for 30min to obtain ligation products; the ligation product is transformed into escherichia coli DH5 alpha, positive strains PMATE-DP are screened by PCR and DNA sequencing is carried out, after verification of the construction correctness of the recombinant plasmid, the positive strains PMATE-DP are inoculated into 5mL LB liquid medium containing 50mg/L KanR of final concentration for overnight culture to obtain bacterial liquid, recombinant plasmids PMATE-DP are extracted according to the operation instruction of a plasmid extraction kit (Omiga), the obtained recombinant plasmids PMATE-DP are transformed into B.subtilis 168 competent cells, a constant temperature incubator at 37 ℃ is used for overnight culture, single bacterial colony is picked for PCR verification, and the recombinant strains B.subtilis 168/PMATE05-DP are obtained.
Wherein SEQ ID NO.1 is specifically ：CATATGAAACAAGGCATCTACTACAGCTAC TGGGAACACGAATGGTCCGCTAAATTCGGTCCATACATCGAGAAAGTGGCCAAACTGGGCTTCGACATCATCGAAGTAGCAGCACACCACATCAACGAATACTCTGATGCAGAACTGGCAACTATCCGTATGAGCGCGAAAGACAACGGTATCATTCTGACCGCTCGTATCGGTCCGTCCAAGACGAAGAACCTGAGCAGCGAAGACGCTGCTGTTCGTGCAGCAGGTAAAGCCTTCTTCGAACGCACCTTGTCCAATATAGCTAAACTGGACATCGACACCATCGGTGGCGCACTGCATAGCTACTGGCCAATCGACTACTCTCAGCCAGTTGACATGGCAGGTGATTACGATCGTGGTGTTGAACGTATCAACGGCATCCCAGACTTCGCGAACGACCTGGGCATCAACCTGAGCATCGAGGTTCTGAACCGTTTCGAGAACCACGTACTGAACACCGCTGCTGAAGGTGTTGCATTTGTCAAAGACGTGGGTAAGAACAACGTGAAAGTCATGCTGGACACTTTCGACATGAACATCGAAGAAGATAGCTTCGGTGACGCTATCCGTACTGCTGGTCCACTGCTGGGTCACTTCCACACTGGTGAATCCAACCGTCGAGTGCCAGGTAAAGGTCGTCTGCCGTGGCACGATATCGGTCTGCCACTGCGTGACATCAACTACACTGGTGCGGTTATCATGGAACCGTTTGTCAAGACCGGTGGTACTATCGATAGCGACATCAAAGTGTCGCGTGATCTGTCTCGTGGTGCTGACATCGCTAAGATCGATGAAGATGCTCGTAACGCACTGGCGTTCTCTCGCTTCGACTTGGGCGCTCTCGAG
Example 2
This example provides site-directed mutagenesis and validation of D-psicose-3-epimerase.
(S1) through software simulation such as pymol, autodock, three amino acid residues of TYR6, GLU149 and GLU243 in the protein structure of D-psicose-3-epimerase form a catalytic pocket of an active center and are responsible for recognition and catalysis of fructose (figure 2). In this example, serine at positions 44, 77, 193 and 264 was mutated to cysteine residues by molecular docking simulation selection.
(S2) respectively carrying out PCR on recombinant plasmids PMATE-DP by using corresponding primers, wherein a PCR reaction system is as follows: recombinant plasmid PMATE-DP 3 μL, front primer 1 μL, rear primer 1 μL, P515 enzyme 10 μL, ddH 2 O to 20 μL. After the PCR is finished, the recombinant plasmids PMATE-DP-C44, PMATE-DP-C77, PMATE-DP-C193 and PMATE-DP-C264 after site-directed mutagenesis are obtained by digestion with DpnI enzyme for 1h, agarose gel electrophoresis and agarose gel recovery; the recombinant plasmids obtained above were transformed into E.coli DH 5. Alpha. Respectively, and positive strains PMATE-DP-C44, PMATE-DP-C77, PMATE-DP-C193 and PMATE-DP-C264 were selected by PCR. Then, the positive strain was subjected to extraction of recombinant plasmid according to the protocol of plasmid extraction kit (Omiga), followed by transformation into host cell B.subilis 168, cultured overnight at 37℃in a constant temperature incubator, and single colonies were picked for PCR verification to obtain mutant strains B.subilis 168/PMATE05-DP-C44, B.subilis 168/PMATE05-DP-C77, B.subilis 168/PMATE-DP-C193 and B.subilis 168/PMATE05-DP-C264.
Wherein, the front primer of the recombinant plasmid PMATE-DP-C44 is F3 (the nucleotide sequence of which is shown as SEQ ID NO. 6), and the rear primer is R3 (the nucleotide sequence of which is shown as SEQ ID NO. 7);
The front primer of the recombinant plasmid PMATE-DP-C77 is F4 (the nucleotide sequence of which is shown as SEQ ID NO. 8), and the rear primer is R4 (the nucleotide sequence of which is shown as SEQ ID NO. 9);
The front primer of the recombinant plasmid PMATE-DP-C193 is F5 (the nucleotide sequence of which is shown as SEQ ID NO. 10), and the rear primer is R5 (the nucleotide sequence of which is shown as SEQ ID NO. 11);
The front primer of the recombinant plasmid PMATE-DP-C264 is F6 (the nucleotide sequence of which is shown as SEQ ID NO. 12), and the rear primer is R6 (the nucleotide sequence of which is shown as SEQ ID NO. 13);
SEQ ID NO.6：5’-CCAGAATACTGCACCGACCAGATC-3’；
SEQ ID NO.7：5’-GGTCGGTGCAGTATTCTGGAAGCGG-3’；
SEQ ID NO.8：5’-ACAACATGGGTTGTTCCGACGCT-3’；
SEQ ID NO.9：5’-GCGTCGGAACAACCCATGTTGTGG-3’；
SEQ ID NO.10：5’-CATCGAAGAAGAGTGCATCGGTGA-3’；
SEQ ID NO.11：5’-GCATCACCGATGCACTCTTCTTC-3’；
SEQ ID NO.12：5’-CTGGCGTGACATCTGCCGTGGT-3’；
SEQ ID NO.13：5’-TTCAGATGCACCACGGCAGATGTCAC-3’；
(S3) inoculating mutant strains B.subilis 168/PMATE-DP-C44, B.subilis 168/PMATE-DP-C77, B.subilis 168/PMATE-DP-C193 and B.subilis 168/PMATE-DP-C264, and recombinant strain B.subilis 168/PMATE-DP respectively to LB medium containing final concentration of 50mg/L KanR, culturing overnight to obtain bacterial cells, weighing equal amount of 0.5g wet bacterial cells, re-suspending with 20ml Tris-HCl, performing ultrasonic disruption for 15min to obtain crude enzyme solution, and placing on ice for later use.
(S4) 100ml of 700g/L fructose (containing 1mM MnCl 2, pH 6.0) was used as a substrate, the reaction temperature was set at 70 ℃, and the crude enzyme solution obtained in the step (S3) was used in sequence for 1 hour of reaction. The results showed that the catalytic efficiency of the mutant strains B.subtis 168/PMATE-DP-C44, B.subtis 168/PMATE05-DP-C193 and B.subtis 168/PMATE-DP-C264 was not decreased compared to the catalytic reaction of the recombinant strain B.subtis 168/PMATE-DP strain, and thus the mutant strain could be further mutated (FIG. 4).
(S5) in accordance with the steps (S1) and (S2), site-directed mutagenesis is performed on the mutant B.subilis 168/PMATE-DP-C44 at position 193 and 264 to obtain mutant B.subilis 168/PMATE05-DP-C44-C193 and B.subilis 168/PMATE05-DP-C44-C264; the protein expression diagram is shown in figure 3; the catalytic efficiency of the B.subtilis 168/PMATE05-DP-C44-C264 strain was improved compared to the recombinant strain B.subtilis 168/PMATE-DP (FIG. 4).
Wherein, the front primer in PMATE-DP-C44-C193 recombinant plasmid is F7 (the nucleotide sequence of which is shown as SEQ ID NO. 14), and the rear primer is R7 (the nucleotide sequence of which is shown as SEQ ID NO. 15);
The front primer in PMATE-DP-C44-C264 recombinant plasmid is F8 (the nucleotide sequence of which is shown as SEQ ID NO. 16), and the rear primer is R8 (the nucleotide sequence of which is shown as SEQ ID NO. 17);
SEQ ID NO.14：5’-CATCGAAGAAGAGTGCATCGGTGA-3’；
SEQ ID NO.15：5’-GCATCACCGATGCACTCTTCTTC-3’；
SEQ ID NO.16：5’-CTGGCGTGACATCTGCCGTGGT-3’；
SEQ ID NO.17：5’-TTCAGATGCACCACGGCAGATGTCAC-3’。
Example 3
This example provides a method for preparing a D-psicose-3-epimerase-directed immobilized enzyme.
(S1) culturing B.subilis 168/PMATE-DP-C44-C264 overnight, centrifuging to collect wet thalli, weighing 5g B.subtilis 168/PMATE-DP-C44-C264 strain wet thalli, adding sodium phosphate buffer with pH of 7.5 into the wet thalli, uniformly mixing, performing ultrasonic crushing for 15min, and obtaining the product and placing the product on ice for standby.
(S2) placing the amino resin in phosphate buffer solution, oscillating for 1h at 200rpm at the constant temperature of 25 ℃, repeatedly treating for 3 times, and then cleaning the amino resin by using ultrapure water;
(S3) adding 2.0g of the amino resin washed in the step (S2) into 20ml of ethanol solution containing 0.2g of 3, 4-dihydroxybenzaldehyde, stirring for 8 hours at room temperature, filtering, washing the product with ethanol for 1 time and washing with pure water for 3 times to obtain catechol modified resin;
And (S4) adding the resin prepared in the step (S3) into the DPease crude enzyme liquid obtained in the step (S1), stirring for 6 hours at 4 ℃, filtering, and washing with pure water for 2 times to obtain the D-psicose-3-epimerase directional immobilized enzyme taking amino resin as a carrier and catechol benzaldehyde as a directional cross-linking agent.
Comparative example 1
This comparative example is identical to example 3 except that "B.sub.168/PMATE 05-DP-C44-C264" is replaced with "B.sub.168/PMATE 05-DP-C44".
Comparative example 2
This comparative example is identical to example 3 except that "B.sub.168/PMATE 05-DP-C44-C264" is replaced with "B.sub.168/PMATE 05-DP-C193".
Comparative example 3
This comparative example is identical to example 3 except that "B.sub.168/PMATE 05-DP-C44-C264" is replaced with "B.sub.168/PMATE 05-DP-C264".
Comparative example 4
This comparative example is identical to example 3 except that "B.sub.168/PMATE 05-DP-C44-C264" is replaced with "B.sub.168/PMATE 05-DP".
Example 4
This example provides the optimum reaction temperature for the directed immobilization of D-psicose-3-epimerase
Under the reaction conditions of 40 ℃, 50 ℃,60 ℃ and 70 ℃, 700g/L fructose is taken as a substrate (containing 1mM MnCl 2 and having the pH of 6.0) and the D-psicose-3-epimerase directional immobilized enzyme prepared in the example 3 is added in an equivalent amount for catalytic reaction, and after 2 hours of reaction, the sample is taken to determine the conversion rate. As a result, it was found that the conversion of D-psicose was only 20% at 40℃and about 25% at 50℃and 60℃and that the highest conversion of D-psicose was 30% at 70 ℃.
Example 5
This example provides a method for continuously preparing psicose by using a packed column reaction apparatus by using a D-psicose-3-epimerase directional immobilized enzyme.
The packed column reaction device is shown in fig. 5, and comprises a water bath pot 1, a packed bed reactor 2, a peristaltic pump 4 and a substrate accommodating groove 5; the inside of the packed bed reactor 2 is provided with a packed column 3, the outside of the packed column 3 is provided with a heat preservation jacket 6 connected with the water bath 1 through a pipeline, and the water bath 1 is used for preserving heat of the packed column 3; the bottom end of the packed column 3 is connected with a substrate holding tank 5 (for providing substrate for the packed column) through a pipeline and a peristaltic pump 4 (for controlling the flow rate of the substrate), and the top end of the packed column 3 is a product (psicose) outlet.
The D-psicose-3-epimerase immobilized enzyme prepared in example 3 was packed in a packed column 3 with a heat-insulating jacket 6 at a temperature of 70℃and 700g/L fructose solution (containing 1mM MnCl 2, pH 6.0) was passed through the packed column 3 at a flow rate of 1.5 ml/min; the conversion rate of psicose in effluent of the packed column reaction device is basically balanced, and the conversion rate is 28-30 percent (figure 6); 530g of psicose can be obtained after continuous catalysis of the immobilized enzyme directed by the D-psicose-3-epimerase by using a packed column reaction apparatus for 30 hours.
Comparative example 5
This comparative example was identical to example 5 except that the "D-psicose-3-epimerase directionally immobilized enzyme prepared in example 3" was replaced with the "D-psicose-3-epimerase directionally immobilized enzyme prepared in comparative example 1".
Comparative example 6
This comparative example was identical to example 5 except that the "D-psicose-3-epimerase-immobilized enzyme prepared in example 3" was replaced with the "D-psicose-3-epimerase-immobilized enzyme prepared in comparative example 2".
Comparative example 7
This comparative example was identical to example 5 except that "the D-psicose-3-epimerase directionally immobilized enzyme prepared in example 3" was replaced with "the D-psicose-3-epimerase directionally immobilized enzyme prepared in comparative example 3".
Comparative example 8
This comparative example was identical to example 5 except that "the D-psicose-3-epimerase-immobilized enzyme prepared in example 3" was replaced with "the D-psicose-3-epimerase-immobilized enzyme prepared in comparative example 4".
In comparison with example 5, in comparative example 8, under the same conditions, the continuous catalytic time reached only 5h and the conversion of the collection liquid reached only 15.8%; in comparative examples 5 to 7, the continuous catalytic time was only 15 hours under the same conditions, and the conversion rate of the collected liquid was only about 25%. This is because the double mutation site is more stable in catalytic efficiency than the single mutation site, and has a good effect of grasping with resin in the application of the immobilized enzyme, and the immobilized enzyme has better stability and longer catalytic time.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the explanation of the present invention, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. A D-psicose-3-epimerase, characterized in that the one D-psicose-3-epimerase is selected from the group consisting of proteins having the amino acid sequence:
(1) Replacing serine at position 44 of the amino acid sequence shown in SEQ ID No.1 with cysteine;
(2) Substitution of serine at position 193 of the amino acid sequence shown in SEQ ID No.1 with cysteine;
(3) Substitution of serine at position 264 of the amino acid sequence shown in SEQ ID No.1 with cysteine;
(4) Serine at position 44 of the amino acid sequence shown in SEQ ID No.1 is replaced with cysteine, and serine at position 264 is replaced with cysteine.
2. A D-psicose-3-epimerase according to claim 1, characterized in that said D-psicose-3-epimerase is selected from proteins having the amino acid sequence:
Serine at position 44 of the amino acid sequence shown in SEQ ID No.1 is replaced with cysteine, and serine at position 264 is replaced with cysteine.
3. An isolated nucleic acid, wherein said nucleic acid is a nucleic acid molecule encoding a D-psicose-3-epimerase according to any one of claims 1 to 2.
4. A recombinant expression vector comprising the nucleic acid of claim 3.
5. A recombinant expression vector transformant comprising the recombinant expression vector according to claim 4.
6. Use of a D-psicose-3-epimerase according to any one of claims 1 to 2 for the preparation of a D-psicose-3-epimerase-directed immobilized enzyme.
7. The use according to claim 6, characterized in that the preparation of D-psicose-3-epimerase directed immobilization enzyme by D-psicose-3-epimerase comprises in particular the following steps:
Reacting amino resin with 3, 4-dihydroxybenzaldehyde to obtain catechol modified resin; and then evenly mixing catechol modified resin with D-psicose-3-epimerase, and obtaining the D-psicose-3-epimerase directional immobilized enzyme after crosslinking reaction.
8. A D-psicose-3-epimerase-directed immobilized enzyme characterized by being prepared by the D-psicose-3-epimerase according to any one of claims 1 to 2.
9. Use of a D-psicose-3-epimerase-directed immobilized enzyme according to claim 8 for the preparation of D-psicose.
10. The use according to claim 9, characterized in that the preparation of D-psicose by D-psicose-3-epimerase directed immobilization enzyme comprises in particular the following steps:
In a continuous reaction device, fructose solution is taken as a substrate, and is uniformly mixed with D-psicose-3-epimerase directional immobilized enzyme, and D-psicose is obtained through catalytic reaction.
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