CN104630248A - Aryl sulfatase gene, protein encoded by aryl sulfatase gene as well as immobilization method and application of protein - Google Patents

Abstract

The invention discloses an aryl sulfatase gene, a protein encoded by the aryl sulfatase gene as well as an immobilization method and application of the protein. The immobilization method and application comprise the following steps of firstly expressing the aryl sulfatase gene of pseudoalteromonas carrageenovora sp in escherichia coli and purifying to obtain a recombinant aryl sulfatase; by adopting carboxyl-functionalized Fe3O4 magnetic nanoparticles as carriers and glutaraldehyde as a cross-linking reagent and immobilizing the free recombinant aryl sulfatase to obtain the immobilized recombinant aryl sulfatase; treating agar with the immobilized recombinant aryl sulfatase, carrying out shaking reaction for 1 hour at 40 DEG C and measuring the physical properties of agar such as gel strength, the content of sulfate groups and the like. The immobilized recombinant aryl sulfatase disclosed by the invention can be repeatedly used, still maintains more than 60% of the enzyme activity after being repeatedly used for eight times and shows good operating stability and feasibility of practical application.

Description

Arylsulfatase gene, encoded protein and method and application of immobilization thereof

technical field

The invention belongs to the field of enzyme engineering, in particular to a method for assisting the extraction of agar by using carboxyl functionalized magnetic nanoparticles to immobilize enzymes.

Background technique

Agar is a natural organic polysaccharide colloid derived from the cell wall of red algae. It is widely used in food, medicine, chemical industry and other fields. Agar is a mixture of agarose and agaropectin, with agarose as the main ingredient. Agarose is a chain polymer composed of (1-3)-β-D-galactose and (1-4)-3,6-inner ether-α-L-galactose, etc., and the structure of agarose gel is relatively It is complex, and its main structure is the same as that of agarose, except that the hydroxyl group on (1-4)-3, 6-inside ether-α-L-galactose is easily replaced by sulfate groups, methoxy groups, acetonyl groups and other groups. Relevant studies have shown that desulfurization treatment of agar can effectively improve the gel strength of agar or produce agarose with high gel strength, thereby greatly improving the application value of the product.

At present, the main method for removing sulfuric acid groups in agar is chemical method. Since a large amount of strong alkali is used to remove the sulfuric acid groups in agar, it is necessary to add a large amount of acid to neutralize the excess alkali, and to rinse with a large amount of water to remove it. Salt has the disadvantages that the production process is not easy to control, the product yield is low, the environment is polluted, and the process is complicated. Compared with chemical methods, enzymatic modification of agar has the advantages of mild reaction conditions, high substrate conversion rate, no pollution, and simple process. Therefore, arylsulfatase-assisted extraction of agar will gradually replace traditional chemical degradation methods and become the best method for preparing agar. However, the current research on the effect of arylsulfatase on agar is still insufficient and there are problems such as low enzyme activity and cumbersome purification steps, which restrict the use of arylsulfatase to modify agar to produce high value-added products. application and development. In view of this, the inventors researched and designed a method for immobilizing recombinant arylsulfatase and its application method, and this case arose from it.

Contents of the invention

The first object of the present invention is to provide an arylsulfatase gene atsA .

The second object of the present invention is to provide a recombinant arylsulfatase encoded by the arylsulfatase gene atsA .

The third object of the present invention is to provide a preparation method of the recombinant arylsulfatase.

The fourth object of the present invention is to provide a method for immobilizing the free recombinant arylsulfatase with carboxyl-functionalized magnetic nanoparticles.

The fifth object of the present invention is to provide an application of the free recombinant arylsulfatase and the immobilized recombinant arylsulfatase in degrading sulfate groups in agar.

In order to achieve the above object, the technical solution of the present invention to solve its technical problems is:

Pseudoalteromonas carrageenonovora sp. ASY5, the strain comes from the China Center for Industrial Microorganism Culture Collection (CICC), and the preservation number is CICC 23819.

An arylsulfatase gene ats A isolated from Pseudoalteromonas carrageenans CICC 23819, the nucleotide sequence of which is shown in SEQ ID NO.1.

An aryl sulfatase encoded by the aryl sulfatase gene ats A, the amino acid sequence of which is shown in SEQ ID NO.2.

A preparation method for recombinant arylsulfatase, comprising the following steps:

Step 1: Cloning the arylsulfatase gene ats A from Pseudoalteromonas carrageenovora sp. ASY5;

Step 2: inserting the arylsulfatase gene ats A into the pET-28a(+) vector to construct a recombinant plasmid;

Step 3: Transform the recombinant plasmid into Escherichia coli ( E. coli ) BL21 (DE3); select positive clones and cultivate them in Luria-Bertani medium containing 50 μg/mL of Kanna Mycin, the culture condition is temperature 37°C, shaker culture to OD ₆₀₀ =0.8, then add isopropylthio-β-D-galactoside (IPTG) to the final concentration of 50 mmol/L, at 15 Induce for 26 h at °C;

Step 4: Collect the fermented E. coli BL 21 (DE3) cells by centrifugation, resuspend the pellet in the lysis buffer and lyse it with ultrasonic waves; the formula of the lysis buffer can be: 50mmol/L NaH ₂ PO ₄ , 300mmol/L NaCl, 15 mmol/L imidazole, pH 8.0;

Step 5: Put the lysed suspension in step 4 at 4°C and 15000×g for 20 min, collect the supernatant, mix with Ni-NTA Agarose, and purify to obtain the Recombinant arylsulfatase.

A method for immobilizing recombinant arylsulfatase, comprising the following steps:

Step 1: Preparation of carboxyl-functionalized magnetic nanoparticles by chemical co-precipitation method: Fe ³⁺ and Fe ²⁺ solutions were mixed, ammonia water was quickly added under vigorous stirring conditions, and oleic acid was added dropwise after 1 minute, at 70°C Continue to stir rapidly. After the reaction is over, a black sol-like substance is obtained. Use an external magnetic field to separate the resulting precipitate from the reaction system, wash with ethanol and ion water, then add KMnO ₄ solution, and ultrasonically oscillate with an ultrasonic cleaner. After magnetic separation Washing with deionized water to obtain a magnetic fluid, and vacuum freeze-drying the magnetic fluid to prepare magnetic nanoparticles with carboxyl groups on the surface;

Step 2: Immobilization of recombinant arylsulfatase: take 10 mg of magnetic nanocarriers, ultrasonically disperse them, add 5 ml of 1.0% by volume glutaraldehyde solution, shake at 4°C for 3 hours, and wash with 50 mM pH 7.0 Tris- Wash the cross-linked carrier with HCl buffer; add 30 μL recombinant aryl sulfatase solution, the ratio of magnetic nanocarriers to free aryl sulfatase is 1mg:0.7U; immobilize at low temperature, the immobilization time is 3h, and the temperature is 4°C ; Afterwards, wash with Tris-HCl buffer solution and vacuum freeze-dry to obtain immobilized enzyme.

As a preferred embodiment of the embodiment, in the second step, the free recombinant arylsulfatase solution is the supernatant obtained by centrifuging Escherichia coli cells at low temperature and high speed after the cells are broken by ultrasonic waves.

The application of a free recombinant arylsulfatase and an immobilized recombinant arylsulfatase to degrade sulfuric acid groups in agar respectively.

After the above-mentioned technical scheme is adopted in the present invention, the arylsulfatase can be stably expressed in E. coli , and the enzyme activity after purification is high, which overcomes the problems of low activity of the existing arylsulfatase and cumbersome enzyme separation and purification process, etc. The recombinantly expressed arylsulfatase has a good degradation effect on SO4 ^2- in agar, and can replace or partially replace alkali treatment in the process of agar extraction and production, thereby reducing pollution and saving water resources.

Description of drawings

Figure 1: SDS-PAGE diagram of expression and purification of Pseudoalteromonas carrageenovora arylsulfatase gene in Escherichia coli; among them, M is the protein standard marker, and lane 1 is the plasmid pET-28a(+) in E. coli BL21 (DE3) Expression in E. coli BL21 (DE3) in lane 2 (induction); Lane 2 is the expression of recombinant plasmid in E. coli BL21 (DE3); Lane 3 is the expression of recombinant plasmid in E. coli BL21 (DE3) (not induced); Lane 4 is the expression of The target protein purified by Ni-NTA has a molecular weight of 35.1 kDa;

Figure 2: Investigation of the repeatability of the immobilized recombinant arylsulfatase operation.

Detailed ways

The detection method adopted in the following examples:

Determination of recombinant arylsulfatase activity: the reaction system included 80 μL 20 mmol/L p-nitrophenyl potassium sulfate (p-NPS, prepared with 50 mmol/L Tris-HCl (pH 7.0) buffer), 20 μL enzyme After incubation at 50°C for 10 min, 25 μL of 1 mol/L NaOH was added to terminate the reaction, the volume was made up to 1 mL with pre-cooled distilled water, and the absorbance was measured at 410 nm. The activity (U) of arylsulfatase is defined as: under the above conditions, the amount of enzyme required to catalyze the generation of 1 μmoL p-nitrophenol (p-NP) per minute.

Determination of sulfate group content: 0.5 mL enzyme solution and 1 mL 0.2% agar solution were shaken at 50°C for 1 h, then centrifuged to get 0.9 mL supernatant, then added 0.2 mL concentrated HCl and 0.6 mL 13.3% barium chloride -2.67% Tween-80 solution, mix well, let it stand at room temperature for 30 min, and measure the absorbance at a wavelength of 420 nm.

Determination of gel strength: prepare 1.5% agar solution, and place it at room temperature for 15 hours after completely dissolving, place the prepared sample on the center of the sample seat of the gel strength tester, and follow the instructions of the gel strength tester Operation, move the handle to make the lower end of the weight adding device rod contact the surface of the gel, increase the force to break the surface of the gel (the end point enters the gel by about 4mm or more), record the value of the gel strength at this time.

Embodiment 1: Amplification and cloning of arylsulfatase gene

The forward primer sequence of the arylsulfatase gene is: 5'-CGC GGATCC CAAAAAATTAGTATTAT-3' (the underline is the Bam HI recognition sequence), and the reverse primer sequence is: 5'-CCC AAGCTT GCGTTTTAGTTCGTAAC-3' (the underline is the Hin d III recognition sequence). The arylsulfatase gene was amplified using Pseudoalteromonas carrageenonovora genomic DNA as a template.

Amplification reaction program: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 1 min, annealing at 55°C for 45 s, extension at 72°C for 1 min, 30 cycles; 72°C for 10 min. The size of the PCR product was detected by agarose gel electrophoresis, the purified PCR product was ligated with the pMD18-T vector, and the ligated product was transformed into E. coli DH5α competent cells. The cells were smeared on LB culture plates (containing 100 μg/mL ampicillin). After colony PCR identification, the target gene sequence was identified by sequencing. The specific nucleotide sequence and amino acid sequence are shown in GenBank accession number KJ509595.

Embodiment 2: the extraction of arylsulfatase

Transform the recombinant plasmid pET-28a(+) containing the arylsulfatase gene into competent cells E. coli BL21(DE3), culture on LB plate medium containing 50 μg/mL kanamycin at 37°C After 12 h, select a single colony of the positive clone and culture it in 5 mL LB liquid medium (containing 50 μg/mL kanamycin) at 37°C until OD ₆₀₀ =0.8, then add isopropylthio-β-D- Galactoside (IPTG) to a final concentration of 50 mmol/L, induced at 15°C for 26 h, collected by centrifugation, suspended in 200 mL of 50 mM pH 7.0 Tris-HCl buffer, and lysed by ultrasonic waves . Centrifuge at 15,000×g for 20 min at 4°C to collect the supernatant, which is the crude enzyme solution of arylsulfatase. The SDS-PAGE technique was used to analyze the expression and purification of the arylsulfatase gene in E. coli, and the results are shown in Figure 1.

Example 3: Preparation of Carboxyl Functionalized Magnetic Nanoparticles

FeCl ₃ ·6H ₂ O solution and FeCl ₂ ·4H ₂ O solution were mixed, ammonia water was quickly added under vigorous stirring, and oleic acid was added dropwise after 1 minute, and rapid stirring was continued for 1 hour at 70°C. After the reaction, a black sol-like substance was obtained, and the resulting precipitate was separated from the reaction system by applying an external magnetic field. Wash with ethanol twice to remove excess oleic acid, and then wash with deionized water to about pH=7. Then add the KMnO ₄ solution, ultrasonically oscillate for 8 h with an ultrasonic cleaner, and wash with deionized water three times after magnetic separation to obtain a magnetic fluid. Vacuum freeze-drying for 24 h to obtain magnetic nanoparticles with surface-modified carboxyl groups.

Example 4: Immobilization of recombinant arylsulfatase

Accurately weigh 10 mg of magnetic nanocarriers, add 5ml of glutaraldehyde solution with a volume percentage of 1.0% after ultrasonic dispersion, shake at 4°C for 3 hours, wash the cross-linked carrier with 50 mM pH 7.0 Tris-HCl buffer solution; add 30 μL (0.27 mg/mL) recombinant arylsulfatase solution, the ratio of carrier to free arylsulfatase is 1mg:0.7U; immobilized at low temperature, the immobilization time is 3h, the temperature is 4℃; after that, use Tris-HCl The buffer was washed, and the immobilized enzyme was vacuum freeze-dried.

Example 5: Operation repeatability of immobilized recombinant arylsulfatase

Measure immobilized enzyme activity according to the mensuration method of arylsulfatase activity, the immobilized enzyme after magnetic separation washes 3 times with 50mM pH 7.0 Tris-HCl buffer solution, continues to measure immobilized enzyme activity, repeats operation 9 times, the result is as follows Figure 2 shows.

Example 6: Immobilized recombinant arylsulfatase removes agar sulfate group

The agar was treated with free recombinant arylsulfatase and immobilized recombinant arylsulfatase respectively, and the content of sulfuric acid group and gel strength were determined as the key factors to determine the quality of agar products.

Free enzyme treatment group: take 1 mL of 0.2% agar solution, add 50 U (0.5 mL) of free arylsulfatase, shake at 40°C for 1 h, and measure the sulfate group content and gel strength of the product.

Immobilized enzyme treatment group: Take 1 mL of 0.2% agar solution, add 50 U (25 mg) of immobilized arylsulfatase, shake at 40°C for 1 h, and measure the sulfate group content and gel strength of the product.

Blank group: Take 1 mL of 0.2% agar solution, add 0.5 mL of distilled water, shake at 40°C for 1 h, and measure the content of sulfate groups and gel strength of the product. The measurement results are shown in Table 1 (the site temperature is 28°C, and the temperature correction coefficient is 1.27).

Table 1

group Gel strength (g/cm ² ) Sulfate content (μg/mg crude polysaccharide) Sulfate group removal rate (%) blank group 205 13.372 1.2 free enzyme treatment group 428 2.883 78.4 immobilized enzyme treatment group 417 2.990 77.6

Both free aryl sulfatase and immobilized aryl sulfatase can remove sulfate groups in agar and improve the gel strength of the agar obtained. Since the immobilized aryl sulfatase can be used repeatedly, the separation is simple , and the product quality is stable, and has good application value in the agar production process.

All deformations that can be derived or associated directly from the disclosure content of the present invention by those skilled in the art should be considered as the protection scope of the present invention.

sequence listing

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Ala Gly Asp Gly Thr Ala Gly Gln Leu Ala Lys Val Gly Leu Asp Ile

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Gly Gly Leu Pro Ala Ile Leu Ser Leu Arg Trp Gln Thr Ser Ala Arg

       

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Gly Ile Phe Glu Tyr Met Thr Tyr Gly Thr Leu Gly His Tyr Gly Val

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Claims (6)

1. one kind is separated the arylsulfatase gene obtained from food siliquosa Pelvetia Pseudoalteromonas CICC 23819 atsa, is characterized in that: its nucleotide sequence is as shown in SEQ ID NO.1.

2. an arylsulfatase gene as claimed in claim 1 atsthe arylsulfatase of A coding, is characterized in that: its aminoacid sequence is as shown in SEQ ID NO.2.

3. recombinate the preparation method of arylsulfatase, it is characterized in that: comprise the following steps:

Step one: from food siliquosa Pelvetia Pseudoalteromonas ( pseudoalteromonas carrageenovorasp.) arylsulfatase gene is cloned in ASY5 atsa;

Step 2: by described arylsulfatase gene atsa inserts pET-28a (+) vector construction recombinant plasmid;

Step 3: by described recombinant plasmid transformed to intestinal bacteria ( e. coli) in BL21 (DE3); Select positive colony to cultivate in Luria-Bertani substratum, described Luria-Bertani substratum is containing the kantlex of 50 μ g/mL, and culture condition is temperature 37 ° of C, and shaking table is cultured to oD ₆₀₀when=0.8, then to add isopropylthio-β-D-galactoside (IPTG) to final concentration be 50 mmol/L, induces 26 h under 15 ° of C conditions;

Step 4: after collected by centrifugation fermentation e. colibL 21 (DE3) thalline, resuspended being deposited in dissolving damping fluid utilizes supersonic wave wall breaking cracking; The formula of described dissolving damping fluid can be: 50mmol/L NaH ₂pO ₄, 300mmol/L NaCl, 15 mmol/L imidazoles, pH 8.0;

Step 5: by the suspension liquid after cracking in step 4 in 4 ° of C, under 15000 × g condition, after frozen centrifugation 20 min, collects supernatant liquor, mixes, carry out purifying, obtain described restructuring arylsulfatase with Ni-NTA Agarose.

4. a process for fixation for restructuring arylsulfatase as claimed in claim 3, is characterized in that: comprise the following steps:

Step one: utilize chemical coprecipitation to prepare the magnetic nano-particle of carboxyl-functional: Fe ³⁺and Fe ²⁺solution mixes, add ammoniacal liquor fast under intense agitation, after 1 minute, dropwise add oleic acid, under 70 ° of C, continue rapid stirring, after reaction terminates, obtain black sol shape material, separate from reaction system by the precipitation of externally-applied magnetic field by gained, with ethanol, deionized water, then add KMnO ₄solution, ultrasonic washing instrument sonic oscillation, obtains magnetic fluid with deionized water wash after Magneto separate, magnetic fluid described in vacuum lyophilization, and obtained finishing has the magnetic nano-particle of carboxyl;

Step 2: immobilization restructuring arylsulfatase: get 10 mg magnetic nano-carriers, add the glutaraldehyde solution that 5ml volume percent is 1.0% after ultrasonic disperse, 4 ° of C vibrate after 3h, are cross-linked carrier with 50 mM pH 7.0 Tris-HCl wash buffer; Add 30 μ L restructuring arylsulfatase liquid, magnetic nano-carrier is 1mg:0.7U with the ratio of free arylsulfatase; Low temperature immobilization, immobilization time 3h, temperature is 4 DEG C; Use Tris-HCl wash buffer afterwards, vacuum lyophilization obtains immobilized enzyme.

5. the process for fixation of a kind of arylsulfatase of recombinating as claimed in claim 4, it is characterized in that: in described step 2, described free restructuring arylsulfatase liquid is the centrifugal gained supernatant liquor of Bacillus coli cells low-temperature and high-speed after supersonic wave wall breaking.

6. the application of restructuring arylsulfatase and the sulfate group of immobilization restructuring arylsulfatase respectively in degraded agar that dissociates as claimed in claim 5.