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Aryl sulfatase gene, protein encoded by aryl sulfatase gene as well as immobilization method and application of protein

A technology of arylsulfatase and arylsulfate, applied in the field of enzyme engineering, can solve the problems of insufficient enzyme activity, restriction, and low level, and achieve the effects of high enzyme activity, pollution reduction, and water conservation

Inactive Publication Date: 2015-05-20
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Aryl sulfatase gene, protein encoded by aryl sulfatase gene as well as immobilization method and application of protein
  • Aryl sulfatase gene, protein encoded by aryl sulfatase gene as well as immobilization method and application of protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Amplification and cloning of arylsulfatase gene

[0032] The forward primer sequence of the arylsulfatase gene is: 5'-CGC GGATCC CAAAAAATTAGTATTAT-3’ (underlined as Bam HI recognition sequence), the reverse primer sequence is: 5'-CCC AAGCTT GCGTTTTAGTTCGTAAC-3' (underlined as Hin d III recognition sequence). by Pseudoalteromonas carrageenovora Genomic DNA was used as a template to amplify the arylsulfatase gene.

[0033] 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 E. coliDH5α competent cells. The cells were smeared on LB culture plates (containing 100 μg / mL ampicillin). After colony PCR identification, the target gene sequence was ...

Embodiment 2

[0034] Embodiment 2: the extraction of arylsulfatase

[0035] Transform the recombinant plasmid pET-28a(+) containing the arylsulfatase gene into competent cells E. coli BL21(DE3), cultured on LB plate medium containing 50 μg / mL kanamycin at 37°C for 12 h, selected positive clones and placed them in 5 mL LB liquid medium (containing 50 μg / mL kanamycin ), cultured at 37°C to OD 600 =0.8, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 50 mmol / L, induce at 15°C for 26 h, collect the cells by centrifugation, and suspend in 200 mL Bacterial cells were lysed by sonication in 50 mM pH 7.0 Tris-HCl buffer. Centrifuge at 15,000×g for 20 min at 4°C to collect the supernatant, which is the crude enzyme solution of arylsulfatase. SDS-PAGE technology was used to analyze the expression and purification of arylsulfatase gene in Escherichia coli, and the results were as follows figure 1 shown.

Embodiment 3

[0036] Example 3: Preparation of Carboxyl Functionalized Magnetic Nanoparticles

[0037] FeCl 3 ·6H 2 O solution and FeCl 2 4H 2 O solution was mixed, ammonia water was quickly added under vigorous stirring conditions, 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 KMnO 4 The solution was ultrasonically oscillated by an ultrasonic cleaner for 8 h, and after magnetic separation, it was washed 3 times with deionized water to obtain a magnetic fluid. Vacuum freeze-drying for 24 h to obtain magnetic nanoparticles with surface-modified carboxyl groups.

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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

technical field [0001] 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 [0002] 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, methoxy, acetonyl and other groups. Relevant studies have shown that desulfurization treatment of agar can effectively improve the gel strength of agar or produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/16C12N15/70C12N11/14C12P19/04
Inventor 肖安风蔡慧农倪辉殷勤杜希萍姜泽东朱艳冰黄高凌杨秋明杨远帆伍菱
Owner JIMEI UNIV
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