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Alkaline pectinase mutant with improved specific enzyme activity and heat stability

A thermostable and mutant technology, applied in the field of enzyme engineering, can solve the problems of low expression enzyme activity, difficult expression of Bacillus subtilis, and high energy consumption of low temperature induction

Active Publication Date: 2016-02-10
JIANGNAN UNIV
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  • 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 fermentation cycle is long, the process is complicated, and the energy consumption of low temperature induction is high; Bacillus subtilis is difficult to express or has low enzyme activity. shortcoming

Method used

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  • Alkaline pectinase mutant with improved specific enzyme activity and heat stability
  • Alkaline pectinase mutant with improved specific enzyme activity and heat stability
  • Alkaline pectinase mutant with improved specific enzyme activity and heat stability

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: the acquisition of mutant strain

[0024] Use PCR amplification or chemical synthesis to obtain the alkaline pectinase gene whose amino acid sequence is shown in SEQIDNO.1, then connect the gene to pET-22b(+), and then transform it into Escherichia coli E.coliBL21(DE3) During screening, the correct transformant was named the recombinant strain E.coliBL21(DE3)(pET-22b(+) / PGL-(GS) 3 -S1).

Embodiment 2

[0025] Embodiment 2: the verification of mutant strain

[0026] Seed culture: the recombinant strain E.coliBL21(DE3)(pET-22b(+) / PGL-(GS) 3 -S1) Take an appropriate amount from a glycerol tube and inoculate it into 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.

[0027] 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 until the cell concentration OD600 = 0.6, add a final concentration of 0.04mMIPTG for induction, and induce at 30°C for 48h.

Embodiment 3

[0028] Embodiment 3: the purification of alkaline pectinase

[0029] 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 and purified by cation exchange chromatography.

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Abstract

The invention discloses an alkaline pectinase mutant with improved specific enzyme activity and heat stability, and belongs to the field of enzyme engineering. Compared with an existing mutant PGL-S1, the specific enzyme activity of the mutant PGL-(GS)3-S1 is improved by 6 times and the half-life period at the temperature of 60 DEG C is prolonged by 1.3 times. Alkaline pectinase can be catalyzed under the alkaline condition, the alpha-1,4 glucosidic bond of polygalacturonic acid is split through the reverse eliminating effect, and the alkaline pectinase mutant can be widely applied to industries such as the food industry, the textile industry and the papermaking industry.

Description

technical field [0001] The invention relates to an alkaline pectinase mutant with improved specific enzyme activity and thermal stability, belonging 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 pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N1/21C12R1/19
CPCC07K2319/00C12N9/88C12Y402/02002
Inventor 刘松陈坚堵国成赵伟欣
Owner JIANGNAN UNIV
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