Alkaline pectinase mutant with improved heat stability

A pectinase and mutant technology, which is applied in the field of enzyme engineering, can solve the problems of long fermentation period, high energy consumption of low temperature induction, and difficulty in expressing Bacillus subtilis.

Active Publication Date: 2017-05-31
JIANGNAN UNIV
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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 heat stability

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the acquisition of mutant strain

[0028] The method of PCR amplification or chemical synthesis is used to obtain the alkaline pectinase gene whose sequence is shown in SEQ ID NO.2, and then the gene is connected to pET-22b(+), and then transformed into Escherichia coli E.coli BL21( DE3), screened, and the correct transformant was named the recombinant strain E.coli BL21(DE3)(pET-22b(+) / PGL-3S1).

Embodiment 2

[0029] Embodiment 2: the verification of mutant strain

[0030] Seed culture: Inoculate an appropriate amount of recombinant bacteria E.coli BL21(DE3)(pET-22b(+) / PGL-3S1) from a glycerol tube 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.

[0031] 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 bacterial concentration OD 600 =0.6, adding a final concentration of 0.04mM IPTG for induction, and induced at 30°C for 48h.

Embodiment 3

[0032] Embodiment 3: the purification of alkaline pectinase

[0033] 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 is further separated and purified by cation exchange chromatography, and the obtained 95% electrophoretic pure protein is desalted by a desalting column, and the obtained pure enzyme liquid is subjected to property determination.

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Abstract

The invention discloses an alkaline pectinase mutant with improved heat stability and belongs to the field of enzyme engineering. The sequence of the alkaline pectinase mutant provided by the invention is as shown in SEQ ID NO. 1; with regard to an existing PGL, the enzyme activity of the mutant is improved by 3.4 times; and the half-life period at 60 DEG C is improved to 19min from previous 5.2min and is improved by 3.6 times. The alkaline pectinase disclosed by the invention can be used for catalyzing cracking of an alpha-1,4 glycosidic bond of polygalacturonic acid through a trans-elimination effect under an alkaline condition; and the alkaline pectinase mutant is widely applied to the industries including foods, textiles, papermaking and the like.

Description

technical field [0001] The invention relates to an alkaline pectinase mutant with improved thermal stability, 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 mate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C07K19/00C12N1/21C12N15/70C12R1/19
CPCC07K2319/31C12N9/88C12Y402/02002
Inventor 刘松陈坚堵国成赵伟欣黎青华
Owner JIANGNAN UNIV
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