CN116676295A - Organic reagent-resistant beta-agarase mutant and application of immobilized enzyme - Google Patents
Organic reagent-resistant beta-agarase mutant and application of immobilized enzyme Download PDFInfo
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- CN116676295A CN116676295A CN202310626698.7A CN202310626698A CN116676295A CN 116676295 A CN116676295 A CN 116676295A CN 202310626698 A CN202310626698 A CN 202310626698A CN 116676295 A CN116676295 A CN 116676295A
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- agarase
- mutant
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- 101710110830 Beta-agarase Proteins 0.000 title claims abstract description 49
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- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 2
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- TWNIBLMWSKIRAT-FPRJBGLDSA-N (1r,2r,3s,4r,5r)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol Chemical compound O1[C@@]2([H])OC[C@]1([H])[C@H](O)[C@H](O)[C@H]2O TWNIBLMWSKIRAT-FPRJBGLDSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
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- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01081—Beta-agarase (3.2.1.81)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Molecular Biology (AREA)
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- Inorganic Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The application discloses an organic reagent-resistant beta-agarase mutant and application of immobilized enzyme, belonging to the fields of genetic engineering technology and enzyme engineering. The application mutates and immobilizes the wild-type beta-agarase from Saccharophagus degradans-40 to obtain the agarase mutant which has the advantages of enhanced thermal stability, repeated utilization and capability of effectively resisting organic reagents, and endows the beta-agarase with larger industrial application value.
Description
Technical Field
The application relates to an organic reagent-resistant beta-agarase mutant and application of immobilized enzyme, belonging to the fields of genetic engineering technology and enzyme engineering.
Background
Agar, also called agar, is a polysaccharide with gel property extracted from marine red algae such as gracilaria and agar. The agar is composed of galactosan family with heterozygous structure, and its main components are agarose and agar. Agarose is a nearly neutral polysaccharide whose main structure is a linear polysaccharide molecule formed by repeated alternating connection of two residues of beta-D-galactose and 3, 6-anhydrous-alpha-L-galactose. Research shows that agarase can degrade agarase polysaccharide into agarase oligosaccharide (NAOS) with polymerization degree of 2-10, and the agarase oligosaccharide has various physiological activities of oxidation resistance, blood fat reduction, immunoregulation, allergy resistance, glycosidase inhibition and the like, and is a functional oligosaccharide with great development potential.
The enzymolysis method is considered as a main method for sustainable and large-scale production of the agaro-oligosaccharide because of the advantages of being capable of specifically hydrolyzing the agarose, having no generation of harmful compounds and the like. Most of agarases from natural sources have an optimal temperature of 30-40 ℃ and agar is in a gel state at 38 ℃, so that the hydrolysis efficiency of the agarases is greatly affected. In chemical synthesis and exchange reactions, biochemical detection projects, enzymes are exposed to a number of organic reagents, such as: methanol, dimethyl sulfoxide, etc., which results in poor effective activity of free enzymes, and other disadvantages of free agarase, such as poor thermostability, narrow pH range, and loss of catalytic activity after one cycle, affect its industrial application value. The enzyme immobilization technology can effectively improve the heat stability of the enzyme, and also realize the effective separation of the enzyme and the product, thereby being more beneficial to the design of an enzyme reactor and realizing the regeneration and the recycling of the enzyme.
Enzyme immobilization refers to the restriction of free enzymes in a specific space or the complete loading of the enzyme on a specific support by some means such that the enzyme is not free to move but is still able to maintain its original spatial structure and complete active center. The technology can obtain stable, reusable and higher-activity enzyme by immobilizing the enzyme on the surface or in holes of the carrier. Immobilized enzymes generally have more excellent properties such as higher enzyme activity and reusability under severe industrial conditions, and thus immobilization of enzymes is a successful strategy to achieve high reaction yields at low cost. These advantages have led to a great deal of attention in the technology of immobilized enzymes and the realization of related industrial applications.
Covalent bonding is the most widely used method in immobilized enzyme research and is classified into random covalent immobilization and directional covalent immobilization. However, random covalent immobilization is usually a random immobilization of amino acid residues (e.g., lysine residues) on the enzyme molecule on a carrier, which may disrupt the natural conformation of the enzyme or cause steric hindrance resulting in a substantial decrease in the activity of the immobilized enzyme, thereby decreasing production efficiency. Compared with random immobilization, directional immobilization can enable enzymes to be immobilized on a proper carrier in a certain direction (away from an active center), so that the enzymes can be better combined with a substrate, and the problems that the natural conformation of the enzymes is damaged or steric hindrance is caused, the substrate enters an active site of the enzymes, the enzyme activity is reduced and the like due to random immobilization are avoided. With the rapid development of structural biology, bioinformatics and computer computing, protein computing design has become a reliable means of modifying protein properties. Therefore, the specific site of the beta-agarase is directionally immobilized by a computer auxiliary means, so that the higher enzyme activity retention rate can be ensured, the thermal stability, the operation stability and the effective activity in an organic reagent of the enzyme can be improved, and the beta-agarase with higher practical industrial application potential can be finally obtained.
Disclosure of Invention
The application uses the computer auxiliary means to mutate the wild type beta-agarase from Saccharophagus degradans-40, mutates the selected mutation site into cysteine, then uses the sulfydryl of the cysteine at the specific site on the surface of the beta-agarase to combine with the carrier specifically, realizes the directional immobilization of the specific site of the beta-agarase, thereby ensuring higher enzyme activity retention rate, improving the thermal stability and operation stability of the enzyme, obtaining the immobilized beta-agarase which has obviously enhanced thermal stability, can be repeatedly used and can effectively resist the influence of organic reagents on the enzyme activity, and endows the beta-agarase with larger industrial application value.
The application provides an agarase mutant, which is obtained by mutating lysine at 308 th position of an agarase parent enzyme with an amino acid sequence shown as SEQ ID NO.2 into cysteine, and is named as E308C;
or the asparagine at 558 of the parent enzyme of agarase with the amino acid sequence shown in SEQ ID NO.2 is mutated into cysteine, and the mutation is named S558C.
Or mutation of asparagine at 588 of parent enzyme of agarase with amino acid sequence shown in SEQ ID NO.2 into cysteine, named K588C.
In one embodiment of the application, the nucleotide sequence of the agarase parent enzyme is shown in SEQ ID NO. 1.
The application also provides a gene for encoding the mutant.
The application also provides a recombinant vector carrying the gene.
In one embodiment of the present application, the recombinant vector is pET-28a as an expression vector.
The application also provides a mutant, a recombinant cell containing the gene or the recombinant vector.
In one embodiment of the application, the recombinant cell uses a prokaryotic cell or a eukaryotic cell as an expression host.
In one embodiment of the application, the expression host is E.coli.
The application also provides a recombinant escherichia coli, which is characterized in that the recombinant escherichia coli expresses the agarase mutant.
The application also provides an immobilized agarase, which is prepared by mixing and incubating the agarase mutant and the sulfhydryl modified magnetic nano-particles.
The application provides a method for selectively fixing a beta-agarase mutant, which comprises the following steps:
(1) Preparing an aminated magnetic nanoparticle carrier;
(2) Preparing sulfhydryl modified magnetic nano particles;
(3) The free sulfhydryl group on the surface sulfhydryl group of the carrier and the CYS group on the surface of the beta-agarase can form intermolecular disulfide bond so as to fix the beta-agarase on the magnetic nano-particles, namely: and (3) incubating the magnetic nano particles prepared in the step (2) with the beta-agarase mutant to prepare the immobilized beta-agarase mutant.
In one embodiment of the application, the method comprises the following specific steps:
(1) Preparation of an aminated magnetic nanoparticle carrier:
preparing magnetic nano particles by a solvothermal method: feCl is added 3 ·6H 2 O is dissolved in glycol, then sodium acetate and chitosan are added, the mixture is stirred, and then the mixture is sealed in an autoclave; heating at 200deg.C for 8 hr, cooling to room temperature, washing with ethanol for several times, and drying at 60deg.C for 6 hr;
(2) Preparation of thiol-modified magnetic nanoparticles:
adding a traut reagent into the aminated magnetic nano particles, and oscillating the mixture in a water bath at 25 ℃ for 12 hours to obtain the sulfhydryl modified magnetic nano particles;
(3) Disulfide bonds can be formed between sulfhydryl groups and CYS residues of mutant enzyme so as to directionally fix beta-agarase on the magnetic nano-particles:
adding the magnetic nano particles prepared in the step (2) into an agarase mutant solution, oscillating the mixture at 25 ℃ for 24 hours, and repeatedly washing to obtain the immobilized beta-agarase.
The application also provides a method for improving the thermal stability of the immobilized enzyme and effectively resisting the influence of the organic reagent on the immobilized enzyme, which comprises the following steps:
(1) Preparing a beta-agarase mutant:
taking plasmid containing original enzyme as a template, and carrying out site-directed mutagenesis by adopting a primer sequence to obtain E308C, S558C and K588C mutants respectively;
(2) Mixing and incubating the agarase mutant obtained in the step (1) with the magnetic nano particles modified by amino groups and sulfhydryl groups to obtain the immobilized agarase.
The application also provides application of the agarase mutant, the gene, the recombinant vector, the recombinant cell or the immobilized agarase in preparation of agarase oligosaccharide or an agarase oligosaccharide-containing product.
In one embodiment of the application, the application is that the agarase mutant or recombinant cells or immobilized agarase is added into a reaction system containing agar to prepare the agarase.
Advantageous effects
(1) According to the application, after the wild agarase from Saccharophagus degradans-40 and the mutant disclosed by the application are characterized and immobilized, the immobilized beta-agarase mutant which has the advantages of enhanced thermal stability, reusability and capability of effectively resisting the influence of an organic reagent on enzyme activity is obtained, and the beta-agarase has a larger industrial application value. Compared with free enzyme, the mutant immobilized enzyme which has higher heat resistance and can effectively resist the influence of organic reagents on enzyme activity is obtained after characterization and immobilization, and the industrial application prospect of agarase is greatly improved.
(2) For immobilized mutant enzyme E308C, S558C, K588C, after water bath for 30min at 40 ℃, the relative enzyme activities of the immobilized enzymes are 95.0%, 88.3% and 95.0%, respectively, and the free enzyme is 79.8%; after 60min of water bath, the relative enzyme activity of the free enzyme is further reduced to 37.8%, and 85.5%, 78.1% and 95.0% of the relative enzyme activities of the immobilized mutant enzyme E308C, S558C, K588C are respectively reserved; after 6h in the water bath, immobilized mutant E308C, S558C, K588C still retained 63.1%, 66.2% and 69.6% of the original enzyme activity, respectively, while free enzyme lost almost all enzyme activity; the stability of the immobilized enzyme of the application is obviously improved.
(3) After incubation for 30min in 50% methanol, n-propanol, n-butanol, isopropanol, dioxane, absolute ethanol, dimethyl sulfoxide and acetone organic reagent, the free enzymes lost all activity. Immobilized mutant beta-agarase, after incubation for 30min in the same organic reagent, all immobilized mutant beta-agarase almost recovered the enzyme activity. In the organic reagent of 50% of normal propanol and dimethyl sulfoxide, all immobilized mutant beta-agarase recovers more than 80% of enzyme activity. Therefore, the immobilized enzyme provided by the application can effectively resist the influence of the organic reagent on the enzyme activity.
Drawings
Fig. 1: SDS-PAGE map of wild enzyme Aga50D and mutant enzyme after purification; wherein in the figure, M: protein molecular weight standard, 1: purified wild type Aga50D,2-4: the purified mutants were E308C, S558C and K588C in this order.
Fig. 2: enzyme activity characterization of wild enzyme Aga50D and mutants thereof; wherein, fig. 2A: relative enzymatic activity of mutant versus wild type, fig. 2B: tm values for wild-type and mutant; fig. 2C: optimum temperature characterization of wild type and its mutants, fig. 2D: optimum pH characterization of wild type and its mutants, fig. 2E: characterization of the thermostability of wild type and its mutants at 40 ℃, fig. 2F: characterization of the thermal stability of the wild type and its mutants at 45 ℃.
Fig. 3: schematic of the preparation process of thiol-modified magnetic nanoparticles.
Fig. 4: fourier infrared spectrogram; wherein fig. 4A: fourier infrared spectrograms of thiol-modified magnetic nanoparticles and amino-modified magnetic nanoparticles immobilized on different mutant enzymes, wherein a, b, c, d is NH respectively 2 -MNPS、E308C-SH-NH 2 -MNPS、S558C-SH-NH 2 -MNPS、K588C-SH-NH 2 -an infrared spectrum of MNPS; fig. 4B: fourier infrared spectrograms of amino-modified magnetic nanoparticles and thiol-modified magnetic nanoparticles immobilized by K588C mutant enzyme, wherein a and d are NH respectively 2 -MNPS、K588C-SH-NH 2 -an infrared spectrum of MNPS.
Fig. 5: X-RD and VSM maps; FIG. 5A is an X-RD diagram of thiol-modified magnetic nanoparticles and amino-modified magnetic nanoparticles immobilized with different mutant enzymes, a, b, c, d is NH, respectively 2 -MNPS、E308C-MAL-NH 2 -MNPS、S558C-MAL-NH 2 -MNPS and K588C-MAL-NH 2 -X-RD profile of MNPS; FIG. 5B is a VSM graph of various mutant enzymes immobilized on thiol-modified magnetic nanoparticles and amino-modified magnetic nanoparticles.
Fig. 6: scanning electron microscope with amino-modified magnetic nanoparticles immobilized on thiol-modified magnetic nanoparticles with mutant K588C enzyme, wherein FIG. A, B, C is a graph of amino-modified magnetic nanoparticles (NH 2 -MNPS) at 100nm, 200nm, 500nm, respectively; FIG. D, E, F is K588C-SH-NH, respectively 2 Electron microscopy at 100nm, 200nm, 500nm respectively of MNPS.
Fig. 7: enzymatic properties of different mutant immobilized agarases; fig. 7A: immobilization ratios of different mutant immobilized enzymes, fig. 7B: enzyme activity retention of different mutant immobilized enzymes, fig. 7C: thermal stability of different mutant immobilized enzymes at 40 ℃, fig. 7D: thermal stability of different mutant immobilized enzymes at 45 ℃, fig. 7E: reusability of different mutant immobilized enzymes.
Detailed Description
pET28A-Aga50D (construction method is described in the Chinese patent publication No. CN 114836405A) and its mutant related in the following examples were synthesized by Soviet's Anshengda biotechnology Co., ltd. The main reagents involved in the following examples: the BSA concentration determination kit was purchased from Nanjinouzan Biotechnology Co., ltd, and the gene synthesis was performed by Souzhou Anshengda Biotechnology Co., ltd, 2-iminothiolane hydrochloride (tout), shanghai leaf Biotechnology Co., ltd.
The following examples relate to the following media:
LB liquid medium: 1g peptone, 0.5g yeast extract, 1g NaCl were weighed out. Dissolving with deionized water, fixing volume to 100mL, and sterilizing at 121deg.C for 20min.
LB solid medium: 1.8% agar powder was added on the basis of LB liquid medium.
The purification method of the enzyme involved in the following examples is as follows:
(1) Balance: the nickel column was equilibrated with 50mM Tris-HCl,500mM NaCl,pH 7.5 buffer;
(2) Loading: the pretreated sample is loaded at a flow rate of 1 mL/min;
(3) Flushing: buffer with 50mM Tris-HCl,500mM NaCl,pH 7.5, 20mM imidazole;
(4) Eluting: eluting with 50mM Tris-HCl,500m M NaCl,300mM imidazole, pH 7.5 buffer, and collecting the target protein to obtain purified enzyme.
The detection method involved in the following examples is as follows:
mutant enzyme activity assay:
the enzyme activity was determined by the 3, 5-dinitrosalicylic acid method (DNS). The agarase is used for catalyzing and hydrolyzing agarose under a certain condition to generate reducing sugar, 3, 5-dinitrosalicylic acid and the reducing sugar are reduced under a hot condition to generate a brownish red amino complex, the color depth is in direct proportion to the reducing sugar amount in a certain range, and the activity of the agarase can be measured under the wavelength of 520 nm.
Definition of enzyme activity unit:
the amount of enzyme required to catalyze the production of 1. Mu. Mol of D-galactose per minute at 30℃and pH 7.5 is defined as one activity unit.
And (3) enzyme activity measurement:
(1) Preheating: 2mL of 1mg/mL agarose solution (pH 7.5) was taken in a cuvette.
(2) The reaction: adding 0.1mL of enzyme solution, shaking and mixing uniformly, reacting for 5min, adding 1.5mL of DNS to terminate the reaction, bathing in boiling water for 5min, and immediately cooling.
(3) Measurement: absorbance was measured at 520nm and enzyme activity was calculated.
Tm value measurement:
differential scanning fluorescence quantification (DSF) was used. The natural protein is in a folded state, the hydrophobic part is hidden inside, the protein structure is gradually disintegrated along with the temperature rise, the hydrophobic part is exposed, at the moment, the dye which is compatible with the hydrophobic part is combined with the protein, and the fluorescence signal intensity of the system is increased; when the temperature reaches a certain point, the developed protein chains aggregate, and the fluorescent dye cannot be combined and then returns to the environment again or fluorescence is quenched at high temperature, so that the fluorescence signal is reduced, and the Tm of the protein can be determined by tracking and detecting the change of the fluorescence signal. The SYPRO Orange dye was diluted 100-fold, 5. Mu.L of dye was mixed with 20. Mu.L of protein and placed in a 96-well thin-walled PCR plate. And heated from 25 ℃ to 99 ℃ in an ABI StepOnePlus real-time fluorescent quantitative PCR instrument system to monitor fluorescence changes.
Enzyme thermostability determination procedure at 40, 45 ℃):
(1) Preheating: taking 2mL of agarose solution (1 mg/mL, pH 7.5) in a colorimetric tube, and placing in a 40 ℃ water bath for heat preservation of 30, 60, 90, 120, 180min and 6h respectively, or 45 ℃ water bath for heat preservation of 30, 60, 90, 150, 270min and 6h respectively;
(2) The reaction: adding 0.1mL of enzyme solution, shaking and mixing uniformly, reacting for 5min, adding 1.5mL of DNS to terminate the reaction, bathing in boiling water for 5min, and immediately cooling.
(3) Measurement: absorbance was measured at 520nm and enzyme activity was calculated.
Calculation of enzyme activity retention:
enzyme activity retention (%) =free enzyme activity/immobilized enzyme activity×100.
And (3) calculating the immobilization rate:
immobilization ratio (%) = (enzyme mass added-enzyme mass in first wash supernatant-enzyme mass in second wash supernatant)/enzyme mass added×100.
The present application will be described in detail below with reference to the drawings and examples.
Example 1: construction of mutants
The method comprises the following specific steps:
construction of recombinant vectors containing mutants:
and (3) designing a site-directed mutagenesis primer, and carrying out site-directed mutagenesis by taking the recombinant plasmid pET28a-Aga50D as a template to obtain mutants E308C, S558C and K588C respectively.
The primer sequences involved are as follows:
the site-directed mutagenesis primer for introducing the E308C mutation is:
E308C-R:5'-TGTCTAAACCCGTAGCAAAGTAAGGGTACCCTTCTGGGTCTACTAGC ATCCATTTACCGTTAATTTTGCATGTGCGAAAGTACCCTGTA-3'；
the site-directed mutagenesis primer for introducing the S558C mutation is:
S558C-F:5'-TTCCCATTCATACCCTCGGTCGCCCATGCGAAGGTGTGCCTACTAGGC AGGCGTTT-3'；
S558C-R:5'-AAACGCCTGCCTAGTAGGCACACCTTCGCATGGGCGACCGAGGGTAT GAATGGGAA-3'；
the site-directed mutagenesis primer introducing the K588C mutation was:
K588C-F:5'-TAGCCGCGTTAAACAATGCCTGGGGGTTATGCCTTAGTTCTTGGGCTG AGTTTGATTT-3'；
K588C-R:5'-AAATCAAACTCAGCCCAAGAACTAAGGCATAACCCCCAGGCATTGTT TAACGCGGCTA-3'；
the PCR reaction system is shown in Table 1:
TABLE 1 mutation PCR reaction System TM
The PCR reaction conditions were: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, annealing at 56℃for 30s, and extension at 72℃for 3min 48s for 30 cycles. After the target fragment gel is recovered, the obtained mutant plasmids are respectively transformed into E.coli BL21 (DE 3), the transformants are coated on kanamycin (50 mug/mL) LB plates, standing culture is carried out at 37 ℃ for overnight, after bacterial colonies grow out, single colonies are picked up to liquid LB culture medium containing kanamycin (50 mug/mL), and cultured at 200rpm for overnight at 37 ℃, and bacterial solutions are sent to Suzhou Ansheng reach biotechnology Co.
Respectively obtaining mutant engineering bacteria containing correct mutants: e.coli BL21 (DE 3)/pET 28a-E308C, E.coli BL21 (DE 3)/pET 28a-S558C, E.coli BL21 (DE 3)/pET 28a-K588C.
As a control: pET28a-Aga50D is converted into E.coli BL21 (DE 3), and the engineering bacteria containing wild agarase are prepared according to the method: e.coli BL21 (DE 3)/pET 28a-Aga50D.
Example 2: purification of the enzyme and determination of the enzymatic Properties
(1) Shake flask fermentation to produce enzyme:
the genetically engineered bacteria obtained in example 1 were streaked on LB plates containing kanamycin (50. Mu.g/mL), cultured at 37℃for 12 hours, and single colonies were picked up and inoculated in LB liquid medium containing kanamycin (50. Mu.g/mL) for shake flask fermentation, and cultured at 37℃and 200rpm for 12 hours to obtain seed liquid.
1mL of the seed solution was pipetted into 100mL of kanamycin (50. Mu.g/mL) LB liquid medium for shaking flask fermentation, and cultured at 37℃to OD 600 When the concentration reaches 0.6-0.8, adding IPTG with the final concentration of 0.5mM, reducing the temperature to 16 ℃, inducing enzyme production, culturing for 12 hours, and centrifugally collecting thalli.
(2) Purification of enzyme:
adding an appropriate amount (2-3 times volume) of lysis buffer (50 mmol/L Tris-HCl,100mmol/LNaCl, pH 7.5) into the collected thalli after centrifugation to resuspend the thalli, carrying out ultrasonic disruption on ice for 15-20 min, carrying out 30% power, carrying out ultrasonic disruption for 2s, stopping 3s, carrying out ultrasonic disruption, centrifuging at 4 ℃ for 10min at 8000rpm, collecting supernatant, and filtering by a 0.22 mu m filter membrane. Ni is carried out 2+ Purifying by column affinity chromatography to obtain mutant enzyme.
Pure enzyme solutions containing wild-type agarase and mutant E308C, S558C, K588C are prepared respectively. The results of SDS-PAGE electrophoresis are shown in FIG. 1.
As can be seen from fig. 1: wild-type WT is a single band with no other hetero-protein bands, the target protein band is located between Marker bands in 97.2kDa and 66.4kDa, consistent with the theoretical molecular weight of Aga50D (84 kDa). In lanes 2-4, mutant E308C, S558C, K588C, respectively, was represented, and both were seen as single bands, and both the molecular weight and the wild type were identical, demonstrating that both the wild type and mutant enzymes were expressed correctly intracellularly.
(3) Relative enzyme activity assay:
the enzyme activities of the wild-type agarase and mutant enzymes E308C, S558C and K588C were measured at 30℃and pH 7.5, and the relative enzyme activities of the wild-type and mutant enzymes were calculated by setting the wild-type enzyme activities to 100%, and the results are shown in FIG. 2.
Relative enzyme activities of mutant enzymes As shown in FIG. 2A, in the 3 mutant designs, specific enzyme activities of WT, E308C, S558C and K588C were 13.+ -. 0.8U/mg, 17.8.+ -. 0.2U/mg, 14.9.+ -. 0.4U/mg and 16.8.+ -. 0.3U/mg, respectively. The relative enzyme activity of all mutants was increased compared to the wild-type enzyme, with the relative enzyme activity of E308C type increased to 137.0%. The relative enzyme activity of the mutant is generally improved, which indicates that the mutant design can improve the catalytic stability of the beta-agarase Aga50D. The thermal melting temperature (Tm) of the mutants is shown in fig. 2B: the wild-type hot melt (Tm) temperature is: the Tm value of S558C alone was higher than that of the wild type (mutant S558C having a heat melting temperature of 43.4 ℃ C.) at 42.9 ℃.
As can be seen from the graph of FIG. 2C, D, the optimal temperatures of the wild type and mutant type were 30℃and the optimal pH was 7.
FIG. 2E shows the heat resistance of the enzyme measured at 40℃and FIG. 2F shows the heat resistance of the enzyme measured at 45 ℃. After incubation at 40℃for 30min and 180min, the wild-type enzyme retained 90.9% and 12.4% of the original enzyme activity. Mutant E308C, S558C, K588C, after 30min incubation, retained 89.4%, 87.0%, 84.9% of the original enzyme activity, respectively; after 180min incubation, 8.5%, 14.2%, 6.8% of the original enzyme activity was retained. Incubation was carried out at 45℃for 30min, with the wild-type enzyme retaining 62.7% of the original enzyme activity. Whereas mutant E308C, S558C, K588C, after incubation at 45℃for 30min, retained 53.2%, 60.5%, 44.4% of the original enzyme activity, respectively; after incubation at 45℃for 150min, both the wild type and the mutant lost all enzyme activity. All the data above demonstrate that: the thermostability of mutant beta-agarase Aga50D was slightly reduced, but the mutant design as a whole had little effect on the enzymatic properties of beta-agarase Aga50D.
Example 3: immobilization of mutant enzymes:
(1) Selection of the reactive groups:
the sulfhydryl of cysteine residue on the surface of the enzyme molecule can spontaneously form disulfide bond with sulfhydryl on the carrier, so that the space structure of the enzyme is stable, and the directional covalent immobilization of the enzyme is carried out through the spontaneous disulfide bond formation between molecules.
(2) Preparation of aminated magnetic nanoparticle carrier (NH 2 -MNPS)：
Preparing magnetic nano particles by a solvothermal method:
6.25mM FeCl 3 ·6H 2 O was dissolved in 30mL of ethylene glycol, followed by addition of 50mM sodium acetate and 0.02g of chitosan. The resulting mixture was vigorously stirred for 30min, then sealed in an autoclave (capacity 50 mL). Heating at 200deg.C for 8 hr, cooling to room temperature, washing with ethanol for multiple times, and drying at 60deg.C for 6 hr to obtain aminated magnetic Nanoparticle (NH) 2 -MNPS)。
(3) Thiol-modified magnetic nanoparticles (SH-NH 2 -MNPS) preparation:
10mg of 2-iminothiolane hydrochloride (tout) is added to 50mg of the aminated magnetic nanoparticle NH prepared in the step (2) 2 In MNPS, the obtained mixture is oscillated for 12 hours in a water bath at 25 ℃ to obtain the sulfhydryl modified magnetic nano particle SH-NH 2 MNPS, the reaction procedure is shown in fig. 3.
(4) Immobilized beta-agarase:
50mg of the sulfhydryl modified magnetic nano-particles prepared in the step (3) are respectively added into a wild-type agarase solution and a mutant agarase solution with the mass of 1mg of enzyme, so that the final content is that the carrier and the enzyme content are 50:1 (w/w), the mixture is oscillated for 24 hours at 25 ℃, and washed twice by PBS buffer solution, thus obtaining immobilized enzyme wild type beta-agarase and immobilized mutant type beta-agarase.
(5) FT-IR test:
and (3) analyzing the structure of the sample by using a KBr tabletting method and using a Fourier transform infrared spectrometer. Wavenumber range of 4000cm -1 ～400cm -1 Resolution of 0.01cm -1 . The results are shown in FIG. 4.
From the FT-IR spectrum, it can be seen that: E308C-SH-NH 2 -MNPS、S558C-SH-NH 2 -MNPS、K588C-SH-NH 2 -MNPS groups are all identical; FIG. 4B is a FT-IR spectrum of mercapto-modified magnetic Fe 3 O 4 Nanoparticle (NH) 2 -MNPS) and E308C-SH-NH 2 -an infrared spectrum of MNPS. It can be seen at 3413cm -1 And 1634cm -1 The peak at the position is obviously enlarged, 3413cm -1 And 1634cm -1 For the stretching vibration of primary amine N-H, proved NH 2 MNPS has successfully added new amino groups; 1072cm -1 Location and 436cm -1 At E308C-SH-NH respectively 2 Absorption peaks of C-S and S-S in MNPS units; while NH is 2 The absence of C-S and S-S absorption peaks in the MNPS demonstrates that the enzyme has been successfully loaded onto the aminated magnetic nanoparticle.
(6) X-RD test
NH after lyophilization 2 MNPS and all magnetic nanopowders with immobilized mutant β -agarase were spread on slides, tested with X-ray diffractometer, test conditions: scanning range: 10-80 DEG, scanning step length: 0.5 deg.. The results are shown in FIG. 5A.
As can be seen from FIG. 5A, E308C-SH-NH 2 -MNPS、S558C-SH-NH 2 -MNPS、K588C-SH-NH 2 MNPS and NH 2 The data of MNPS are highly fitting and all show diffraction peaks at 2θ=18.19 °,30.08 °,35.31 °,43.00 °,53.59 °,57.02 °,62.52 °, indicating E308C-SH-NH 2 -MNPS、S558C-SH-NH 2 -MNPS、K588C-SH-NH 2 The MNPS all have inverse spinel structure, and diffraction peaks of other impurity-free crystal phases are basically free of impurity diffraction peaks in the figure, which proves that immobilization of enzyme is opposite to NH 2 -MNPS has no modification of the crystalline structure.
(7) VSM testing
The magnetic properties of the sample are characterized by adopting a vibrating sample magnetometer at room temperature, and the magnetic field strength is as follows: 2T, the field rate is 25-30 Oe/s. The results are shown in the figure.
As can be seen from fig. 5B: the coercive forces of E308C-SH-NH2-MNPS, S558C-SH-NH2-MNPS, K588C-SH-NH2-MNPS and NH2-MNPS are still 0, and the saturation magnetization is respectively: 70.38emu/g,61.92emu/g,65.29emu/g,67.59emu/g. The above data demonstrates that: the thiol-modified magnetic nanoparticle still has good magnetic properties after immobilization.
(8) Scanning electron microscope test:
weighing an appropriate amount of aminated Fe 3 O 4 Magnetic nano-and inclusion immobilizationAdding magnetic nano particles of beta-agarase into a centrifuge tube, adding absolute ethyl alcohol, dispersing for 5min by ultrasonic, dripping into a silicon wafer, volatilizing the alcohol, adhering the volatilized alcohol to the surface of conductive adhesive of an SEM copper table, and performing scanning test after metal spraying. The results are shown in FIG. 6.
Scanning electron microscope analysis showed that: aminated magnetic Nanoparticles (NH) 2 MNPS) has a uniform particle size with few inter-nanoparticle gaps; agarase (K588C-SH-NH) 2 MNPS) immobilization, the nanoparticles become dispersed, the voids become large, and the particle surface has a thin film. These results indicate that: the enzyme is immobilized on the thiol-modified magnetic nanoparticle.
Example 4: determination of the enzymatic Properties of immobilized wild-type and mutant beta-agarases
1. Immobilization ratio of immobilized enzyme:
(1) 2mL of agarose solution (1 mg/mL, pH 7.5) was taken, and 100. Mu.L of immobilized wild-type (initial enzyme activity: 13.0.+ -. 0.8U/mg) and immobilized mutant beta-agarase (initial enzyme activity: E308C: 17.7.+ -. 0.2U/mg; S558C: 14.9.+ -. 0.4U/mg; K588C: 16.8.+ -. 0.3U/mg) prepared in example 3 were added;
the above reaction system was reacted at 30℃for 5 minutes, and the enzyme activity was measured.
(2) After the completion of the measurement, the enzyme activities of the immobilized wild-type agarase and immobilized mutant enzyme E308C, S558C, K588C were determined, and the enzyme activities of the free-type wild-type agarase and the free-type mutant enzyme prepared in example 2 were set to 100%, respectively, to calculate the immobilization rates of the wild-type and mutant enzymes.
As can be seen from fig. 7A: the immobilization rates of the wild-type agarase and the mutant enzyme WT, E308C, S558C, K588C are respectively as follows: 0.0%,42.3%,61.9%,55.1%. From the figures it can be seen that: wild-type agarase cannot be immobilized on SH-MH due to the absence of CYS groups 2 On the MNPS vector, other mutant agarases were all immobilized on SH-MH as expected 2 -MNPS carrier. The immobilization rate of mutant agarase is 40% -65%.
2. Enzyme activity retention rate of immobilized enzyme:
2mL of agarose solution (1 mg/mL, pH 7.5) was taken, 100. Mu.L of immobilized wild-type (initial enzyme activity: 13.0.+ -. 0.8U/mg) and immobilized mutant type beta-agarase (initial enzyme activity: E308C: 17.7.+ -. 0.2U/mg; S558C: 14.9.+ -. 0.4U/mg; K588C: 16.8.+ -. 0.3U/mg) prepared in example 3 were added and reacted at 30℃for 5 minutes, and the enzyme activities were measured.
The enzyme activities of the immobilized mutant enzymes E308, C, S, 558, C, K and 588C were measured, the free mutant enzyme activities were set to 100%, and the enzyme activity retention rates of the immobilized mutant enzymes were calculated.
The results show that, as can be seen from fig. 7B: the relative enzyme activities of mutant agarases of mutant E308C, S558C, K588C were 68.3%, 81.3% and 96.8%, respectively. The retention rate of the enzyme activity of the immobilized mutant type enzyme is above 65%, and the relative enzyme activity of the K588C mutant type agarase can reach 96.8%. The immobilization strategy has little influence on enzyme activity of enzyme, and the method is successful.
(3) Thermal stability of immobilized enzyme and free enzyme at 40℃and 45 ℃C
Taking 2mL of agarose solution (1 mg/mL, pH 7.5), adding 100 mu L of free wild-type enzyme (initial enzyme activity is 13.0+/-0.8U/mg) and immobilized mutant beta-agarase (initial enzyme activity is respectively E308C: 17.7+/-0.2U/mg; S558C: 14.9+/-0.4U/mg; K588C: 16.8+/-0.3U/mg) prepared in the example 3, placing in a water bath at 40 ℃ for heat preservation respectively for 30, 60, 90, 120, 180min and 6h, reacting for 5min at 30 ℃, and measuring the enzyme activity;
meanwhile, according to the method, the free wild enzyme and the immobilized mutant beta-agarase are respectively placed in water baths at 45 ℃, and after heat preservation for 30, 60, 90, 150, 270min and 6h respectively, the free wild enzyme and the immobilized mutant beta-agarase react for 5min at 30 ℃ to measure the enzyme activity.
It can be seen from FIGS. 7C and 7D that the enzyme activity of the immobilized enzyme gradually decreased with the increase of the water bath time, but the enzyme activity loss rate of the immobilized mutant beta-agarase was significantly smaller than that of the free wild-type enzyme.
From fig. 7C, it can be obtained that: at 40℃the wild-type free enzyme retained 79.8% and 37.8% of the original enzyme activity, respectively, after 30 and 60min incubation, whereas after 6h incubation almost all enzyme activity was lost. Immobilized mutant enzyme E308C, S558C, K588C, after incubation for 30min at 40 ℃, retains 95.0%, 88.3% and 95.0% of the original enzyme activity respectively; after incubation for 60min, 85.5%, 78.1% and 95.0% of the original enzyme activities were retained respectively; after 6h incubation, immobilized mutant enzymes E308C, S558C and K588C retained 63.1%, 66.2%, 69.6% of the original enzyme activity, respectively.
From fig. 7D, it can be derived that: at 45℃the wild-type enzyme retains 60.8% and 22.2% of the original enzyme activity after incubation for 30min and 60min, respectively, whereas at 6h incubation the wild-type enzyme loses all of its enzyme activity. Immobilized mutant enzyme E308C, S558C, K588C, after incubation for 30min at 45 ℃, 88.8%, 77.0% and 86.8% of the original enzyme activity are retained respectively; after incubation for 60min, 82.7%, 66.8% and 78.6% of the original enzyme activities were retained respectively; after 6h incubation, immobilized mutant enzymes E308C, S558C and K588C retained 56.4%, 56.6%, 55.6% of the original enzyme activity, respectively. The above data illustrate: the thermal stability of the immobilized enzyme is obviously improved at different temperatures.
(4) Reusability of immobilized enzyme:
2mL of agarose solution (1 mg/mL, pH 7.5) was taken, 100. Mu.L of immobilized mutant beta-agarase E308C, S C and K588C prepared in example 3 was added, and reacted at 30℃for 5 minutes, and the enzyme activities were measured, and the reusability of the immobilized enzyme was observed for 4 times with respect to the first enzyme activity of each immobilized mutant beta-agarase as 100%.
The results are shown in fig. 7E: after the immobilized enzyme is repeatedly used for 4 times, more than 30% of original enzyme activity is still maintained, and the immobilized enzyme has the same activity as E308C-SH-NH 2 MNPS and K588C-SH-NH 2 Residual enzyme activity after 4 uses of MNPS can reach around 50%, whereas free enzyme cannot be recycled. After repeated use of the immobilized enzyme, the enzyme activity is reduced, namely, because the solid-liquid separation can not be 100% measured under the laboratory condition, and part of the immobilized enzyme is lost due to agar solidification and the like once each time, so that the enzyme activity is reduced to a certain extent, a proper amount of immobilized enzyme can be supplemented, and the maintenance effect of the enzyme activity is more obviously influenced.
(5) Influence of organic reagent on the directed immobilized mutant β -agarase Aga 50D:
taking 100 mu L of immobilized mutant beta-agarase E308C, S C and K588C prepared in the embodiment 3, incubating for 30min at 30 ℃ in an organic reagent, adding 2mL of agarose solution (1 mg/mL, pH 7.5) into a colorimetric tube, shaking and mixing uniformly at 30 ℃, reacting for 5min, adding 1.5mL of DNS to terminate the reaction, boiling for 5min, and cooling at room temperature; absorbance was measured at 520nm and relative enzyme activity was calculated.
The results are shown in Table 2: incubation in 50% methanol, n-propanol, n-butanol, isopropanol, dioxane, absolute ethanol, dimethyl sulfoxide and acetone organic reagents lost all activity of the free enzyme. This demonstrates that the presence of the organic reagent has a considerable effect on the enzymatic activity of the free enzyme. Immobilized mutant beta-agarase almost all recovered the enzyme activity in the presence of the same organic reagent. This demonstrates that immobilization significantly improves the structural stability of the enzyme and can effectively resist the influence of organic reagents on enzyme activity. In the organic reagent of 50% of normal propyl alcohol and dimethyl sulfoxide, all immobilized mutant beta-agarase recovers more than 80% of enzyme activity, and the enzyme activity is obviously improved compared with the enzyme activity of the prior free enzyme. This demonstrates that the spatial conformation of the immobilized mutant beta-agarase is changed, so that the enzyme action is more stable. The specific data are shown in Table 2.
TABLE 2 influence of organic reagents on the directed immobilization of mutant agarases by thiolation
Comparative example 1:
specific embodiments refer to example 3, except that the magnetic nanoparticles are modified with maleimide, and enzyme immobilization and enzymatic property characterization are performed, and the results show that the immobilization rate of the magnetic nanoparticles modified with thiol groups is 40% -65%, while the immobilization rate of the magnetic nanoparticles modified with maleimide is 30% -50%. The use of thiol-modified magnetic nanoparticles proved to bind more strongly to mutant enzymes.
Comparative example 2:
specific embodiments referring to example 3, except that the magnetic nanoparticles were modified with maleimide and immobilization and enzymatic characterization of mutant enzymes R66C, K C and N452C, respectively, showed that at 40 ℃ wild-type enzyme lost almost all of its enzymatic activity after 6h incubation, and that maleimide-modified magnetic nanoparticles immobilized mutant enzymes R66C, K211C, N452C retained 59.25%, 45.10%, 46.88% of the original enzymatic activity, respectively. Compared with the thiol-modified magnetic nanoparticle immobilized mutant enzymes E308C, S558C and K588C, the retained enzyme activity is reduced by 10-15%. The above data illustrate: the thermal stability of immobilized mutant enzymes E308C, S558C and K588C is better through thiol-modified magnetic nanoparticles.
While the application 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 application as defined in the appended claims.
Claims (10)
1. A mutant of agarase, characterized in that the mutant is:
the arginine at the 308 th position of the agarase with the amino acid sequence shown as SEQ ID NO.2 is mutated into cysteine to obtain the agarase; or (b)
The amino acid sequence is shown as SEQ ID NO.2, and the 558 th lysine of the agarase is mutated into cysteine; or (b)
The amino acid sequence is shown as SEQ ID NO.2, and asparagine at 588 of agarase is mutated into cysteine.
2. A gene encoding the mutant of claim 1.
3. A recombinant vector carrying the gene of claim 2.
4. The recombinant vector of claim 3, wherein the recombinant vector is a pET-28a expression vector.
5. A recombinant cell expressing the mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3 or 4.
6. The recombinant cell of claim 5, wherein the recombinant cell is an expression host that is a prokaryotic cell or a eukaryotic cell.
7. The recombinant cell of claim 6 wherein the expression host is e.
8. An immobilized agarase, which is characterized in that the immobilized agarase is prepared by mixing and incubating an agarase mutant according to claim 1 and thiol-modified magnetic nanoparticles.
9. A method for improving the thermal stability of immobilized enzyme is characterized in that the method comprises the following steps of,
(1) Preparing a beta-agarase mutant: the 308 th arginine of agarase with the amino acid sequence shown as SEQ ID NO.2 is mutated into cysteine; or mutating the 558 th lysine of agarase with the amino acid sequence shown in SEQ ID NO.2 into cysteine; or mutating the asparagine at 588 of agarase with the amino acid sequence shown as SEQ ID NO.2 into cysteine;
(2) And (3) mixing and incubating the agarase mutant obtained in the step (1) with the sulfhydryl modified magnetic nano particles to obtain the immobilized agarase.
10. Use of an agarase mutant according to claim 1, or a gene according to claim 2, or a recombinant vector according to claim 3 or 4, or a recombinant cell according to any one of claims 5 to 7, or an immobilized agarase according to claim 8, for the preparation of agarase disaccharide or an agarase disaccharide-containing product.
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