Alkaline pectinase mutant with improved secretion property

A pectinase and mutant technology, applied in the field of enzyme engineering, can solve the problems of long fermentation period, high energy consumption for low temperature induction, complicated process, etc., and achieve the effect of improving secretion efficiency and thermodynamic stability

Active Publication Date: 2017-05-10
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Comprehensive comparison of different hosts that can express alkaline pectinase. Although Pichia pastoris expresses proteins that are easy to purify and have high yields, the fermentatio...

Method used

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  • Alkaline pectinase mutant with improved secretion property
  • Alkaline pectinase mutant with improved secretion property

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the acquisition of mutant strain

[0025] Use PCR amplification or chemical synthesis to obtain the alkaline pectinase gene whose amino acid sequence is shown in SEQ ID NO.2, then connect the gene to pET-22b(+), and then transform it into Escherichia coli E.coli BL21 (DE3), screened to obtain the correct transformant.

Embodiment 2

[0026] Embodiment 2: the verification of mutant strain

[0027] Seed culture: Pick the recombinant strain E.coli BL21(DE3) from the plate and inoculate it in LB medium (100 μg·mL -1 ampicillin, 2% glucose), the filling volume is 20mL / 250mL. 37℃, 200r·min -1 Incubate on a shaker for 10 h.

[0028] Shake flask fermentation: the seed solution cultivated for 10 h was inserted into the fermentation medium TB (100 μg·mL) with an inoculum of 3% (V / V) -1 Ampicillin), the filling volume is 20mL / 250mL, 37℃, 200r·min -1 Cultivate to cell concentration OD 600 =0.6, adding a final concentration of 0.04mM IPTG for induction, and induced at 30°C for 48h.

[0029] With the unmutated alkaline pectinase as a contrast, the recombinant bacteria and the control were cultured and fermented according to the above method, and the recombinant bacteria fermentation liquid that had been fermented was centrifuged at 8000r / min for 20min, the supernatant was taken, and the enzyme activity of the super...

Embodiment 3

[0030] Embodiment 3: the purification of alkaline pectinase

[0031] Centrifuge the recombinant bacterial fermentation broth at 8000r / min for 20min, take the supernatant, add ammonium sulfate for gradient salting-out, collect 30-50% ammonium sulfate precipitated part by low-temperature centrifugation, and dissolve the salted-out precipitated enzyme in glycine-sodium hydroxide buffer Solution (pH7.5), dialyzed with 20mmol / L glycine-sodium hydroxide buffer solution for 24h. The supernatant obtained by centrifugation was further separated, purified and desalted by cation exchange chromatography. Dilute the purified alkaline pectinase, measure the enzyme activity according to the above method, and measure the protein concentration with the BSA protein concentration assay kit. The results show that the specific enzyme activity of the mutant PGL-ADR is 448.36±4.73U / mg.

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Abstract

The invention discloses an alkaline pectinase mutant with an improved secretion property, and belongs to the technical field of enzyme engineering. The sequence of the provided alkaline pectinase mutant is shown as in SEQ ID NO.1. Compared with the existing alkaline pectinase or the alkaline pectinase mutant PGL-S1, the activity of an extracellular enzyme of the mutant PGL-ADR is up to 919.88 plus or minus 82.64 U/mL and is 1.7 times higher than that of the PGL-S1 (the average value of three repeated experiments), the specific enzyme activity of the purified PGL-ADR is 448.36 U/mL, the melting temperature is increased by 4.11 DEG C, and the data prove that the thermodynamic stability is remarkably improved. The alkaline pectinase can catalyze the splitting decomposition of glucosidic bond alpha-1,4 of polygalacturonic acid through trans-elimination under the alkaline conditions, and is widely used in the industries of foods, textiles and papermaking, etc.

Description

technical field [0001] The invention relates to an alkaline pectinase mutant with improved secretion performance, which belongs to the field of enzyme engineering. Background technique [0002] Pectinase is a complex enzyme that breaks down pectin polymers into unsaturated oligogalacturonic acids. The enzyme is widely distributed and found in some parasitic nematodes, plants and microorganisms. Pectinase is widely used and has a history of industrial application for more than 40 years. Pectinases are divided into acid pectinases and alkaline pectinases PGL according to the optimum reaction pH. Among them, acid pectinase is mainly used in clarification of fruit juice and wine, extraction of fruit and vegetable juice, fruit peeling and so on. PGL applications are mainly used in textile, food, paper industry and environmental fields. The application of enzymatic method to the above-mentioned field-related reactions has the advantages of environmental protection, saving raw ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C07K19/00C12N1/21C12N15/70C12R1/19
CPCC07K2319/00C12N9/88C12Y402/02002
Inventor 刘松陈坚堵国成赵伟欣黎青华
Owner JIANGNAN UNIV
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