Thermally stable mutant aromatic sulfatase and its gene and use

A base sulfate and aromatic technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of affecting the removal efficiency of agar sulfate and low thermal stability of enzymes

Active Publication Date: 2017-02-15
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The aryl sulfatase of Pseudoalteromonas carrageenovora has the hydrolysis activity of agar sulfate and can remove the sulfate groups on the agar, but the

Method used

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  • Thermally stable mutant aromatic sulfatase and its gene and use
  • Thermally stable mutant aromatic sulfatase and its gene and use
  • Thermally stable mutant aromatic sulfatase and its gene and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Thermostability improves the screening of mutant arylsulfatase

[0024] Using the recombinant plasmid (WT) containing the wild-type Pseudoalteromonas carrageenovora arylsulfatase gene as a template, perform error-prone PCR to amplify arylsulfatase:

[0025] Upstream primer (SEQ ID NO.3): 5'-CGC GGATCC TTTACGTTTAACGGCAGC-3′;

[0026] Downstream primer (SEQ ID NO.4): 5'-CCC AAGCTT GCGTTTTAGTTCGTAAC-3';

[0027] 50μL amplification system contains: 5μL 10× buffer, 0.2μmol / L primer, 0.5μmol / L dTTP, 0.5μmol / L dGTP, 0.1μmol / L dATP, 0.1μmol / L dCTP, 1U rTaq polymerase, 7mmol / L MgCl 2 and 2ng of recombinant plasmid template containing the wild-type enzyme gene. The PCR reaction conditions were: 95°C for 5 min; 30 cycles of 94°C for 1 min, 55°C for 45 sec, 72°C for 1 min; 72°C for 10 min. Recombinant expression plasmids were constructed by enzyme digestion cloning, that is, PCR products were double-digested with BamHI and HindIII, and the digested fragments w...

Embodiment 2

[0030] Example 2: Expression and purification of recombinant mutant arylsulfatase using recombinant expression strains

[0031] After culturing the recombinant expression strain overnight, transfer it to 250mL LB liquid medium (containing 50mg / mL kanamycin) at a volume ratio of 1:100, and culture it to OD at 37°C and 180r / min 600 When it reaches 0.6–0.8, add IPTG at a final concentration of 0.05mmol / L, and culture at 25°C and 180r / min for 10h. The cells were collected by centrifugation and resuspended in Buffer A (50mmol / L NaH 2 PO 4 , 300mmol / L sodium chloride, 15mmol / L imidazole, pH 8.0), ultrasonication was performed on ice. Centrifuge at 4°C, collect the supernatant, then perform Ni-NTA affinity chromatography, wash buffer (50mmol / L NaH 2 PO 4 , 300mmol / L sodium chloride, 30mmol / L imidazole, pH 8.0) after washing, use elution buffer (50mmol / L NaH 2 PO 4 , 300mmol / L sodium chloride, 250mmol / L imidazole, pH 8.0) elution, collect eluate. Detected by SDS-PAGE, the molec...

Embodiment 3

[0032] Embodiment 3: the detection of the activity of arylsulfatase

[0033] Add 20 μL enzyme solution (400ng enzyme) to 80 μL 20mmol / L p-NPS substrate solution. After incubating at 55°C for 10 min, 25 μL of NaOH (5 mol / L) was added to terminate the reaction, the volume was made up to 1 mL with distilled water, and the absorbance at 410 nm was measured. The activity of arylsulfatase is defined as the amount of enzyme required to catalyze the production of 1 μmoL p-nitrophenol (p-NP) per minute under the above conditions.

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Abstract

The invention discloses thermally stable mutant aromatic sulfatase and its gene and use. Through an error-prone PCR technology, random mutagenesis is introduced, a P. carrageenovora aromatic sulfatase mutant library is constructed and through screening, the mutant aromatic sulfatase with improved thermal stability is obtained. The result shows that compared with WT, H260L thermal stability is obviously improved. Based on potassium p-nitrophenyl sulfate as a substrate, the H260L has the optimal reaction temperature of 55 DEG C and pH of 8.0. The H260L is stable in the pH range of 6.0-9.0. EDTA has a strong inhibitory effect on the activity of the mutant enzyme and it is proved that a metal ion produces an important effect in the catalytic process of the mutant sulfatase. H260L has good tolerance to a detergent and has a gracilaria lemaneiformis crude polysaccharide sulfuric acid group desulfurization rate of 82%. The invention also discloses genetic engineering bacteria containing the mutant aromatic sulfatase gene. The genetic engineering bacteria realize the heterologous expression of the sulfatase and provide a good foundation for the industrial production and application of the sulfatase.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and enzyme engineering, in particular to a heat-stable mutant arylsulfatase, gene and application thereof. Background technique [0002] Agar, also known as agar gel, is a natural polysaccharide extracted from marine red algae. It has excellent gelling and thickening properties and is widely used in food, light industry, medicine and bioengineering. The natural agar molecules in red algae contain a large number of sulfate groups, which is the main reason for affecting the gel strength, electroosmosis and protein adsorption capacity of agar. Removing sulfate groups is a necessary and key link in agar production. At present, the alkaline method is commonly used in industrial production to remove sulfate groups in agar, which not only causes serious environmental problems, but also causes a large amount of agar to be degraded and lost. Compared with the alkali treatment process, the use ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/16C12N15/70
CPCC12N9/16C12Y301/06001
Inventor 朱艳冰乔超超倪辉肖安风杨远帆李利君杜希萍
Owner JIMEI UNIV
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