CN102492027A - Method for extracting Bt insecticidal protein crystal - Google Patents

Abstract

The invention provides a method for extracting Bt insecticidal protein crystal, which is low in cost, safe and simple to operate. The method includes the step of pre-processing fermented basic solution and extracting protein crystal. The method for extracting the Bt insecticidal protein crystal is large in extraction amount, simple to operate, low in cost, safe and suitable for industrial production.

Description

A kind of extraction Bt insecticidal proteins crystalline method

Technical field

The present invention relates to a kind of extraction Bt insecticidal proteins crystalline method, belong to biological pesticide technical field.

Background technology

Bacillus thuringiensis ( Bacillus thuringiensis,Bt) be important biological pesticide.Nineteen fifty-seven thuricade-1 Initial Public Offering is sold, and the commodity preparation has reached kind more than 100 at present, is that output is maximum in the world, uses the widest microbial pesticide.The ratio that Bt preparation output accounts for all living microorganism sterilants reaches more than 90%.

Sporeine preparation is a major ingredient with the insecticidal proteins crystal, and it is generally acknowledged that its insecticidal mechanism is: the insecticidal proteins crystal is dissolved as preceding toxin after being got food by sensitive insect in insect midgut; Form the toxicity peptide with insecticidal activity through the midgut proteinase hydrolysis again, the toxicity peptide passes peritrophic membrane, with combining of midgut epithelial cell BBM; And further insert in the film, form hole or ionic channel, cause the ion seepage; Destroy intestines wall epithelial cell; Make the enteron aisle Dissolve things inside infiltrate haemocoele, cause septicemia, thereby cause the insect general paralysis and dead.

At genetically engineered, toxicologic study, the insecticidal mechanism of Bt and aspect the mankind's scientific researches such as security, all Bt insecticidal proteins crystal has been proposed great demand.Some separation and purification insecticidal proteins crystalline methods are arranged at present: like the spore suspension method, density gradient centrifugation, extraction process, isoelectric point precipitation, alkaline lysis etc.Though these methods finally also can be extracted the insecticidal proteins crystal, the spore suspension method need repeat many times, wastes time and energy; The protein content that density gradient method extracts is few, and time-consuming of institute is long, and cost is high, complicated operation; Extraction process need use highly toxic organic solvent CCl ₄, being unfavorable for environment protection, production process is also caused danger easily, and the phenomenon that the insecticidal proteins crystal is degraded by the basic protein lytic enzyme that self produces appears in alkaline lysis easily.

Summary of the invention

In order to overcome above-mentioned defective, the invention provides a kind of cost extraction Bt insecticidal proteins crystalline method low, safe, simple to operate.

Technical scheme of the present invention is following:

A kind of extraction Bt insecticidal proteins crystalline method, concrete steps are following:

(1) pre-treatment of fermenation raw liquid:

After the bacillus thuringiensis fermentation finishes, with centrifugal 1 min of fermented liquid 1500-3000 r/min; Filtrating is removed deposition through 1000 r/min, 4 ℃ of centrifugal 1.5-2min; Supernatant is through 10000 r/min, 4 ℃ of centrifugal 15min; The deposition of collecting is dissolved in the saline water that contains 0.1% (v/v) TritonX-100; Continue the centrifugal 15min down 10000r/min, 4 ℃ then, deposition is with the saline water washing, get rid of excessive moisture after collecting precipitation subsequent use;

(2) extract protein crystal

The deposition that step (1) is collected is mixed with PEG, potassium primary phosphate, potassium hydrogenphosphate, and the formation mixed solution that is dissolved in water is respectively ultimate density: deposition 24g/L, PEG 120 g/L, potassium hydrogenphosphate 90 g/L; Potassium primary phosphate 30g/L regulates pH of mixed to 8.5, while stirring at 1440r/min; Centrifugal 2min under 15 ℃ removes supernatant, adds water to complement to and above-mentioned mixed solution equal-volume; And then the potassium primary phosphate that adds the potassium hydrogenphosphate and 3% (w/v) account for mixed solution TV 9% (w/v) carries out reextraction, at 4500r/min, and 4 ℃ of centrifugal 15-20 min down; The collecting precipitation part adds the saline water washing, collects the Bt crystal proteins; It is dissolved in the saline water, frozen down in-20 ℃.

The said PEG of step (2) is PEG4000.

Beneficial effect of the present invention:

Bt insecticidal proteins crystalline extracted amount of the present invention is big, simple to operate, and low, the safety of cost is fit to suitability for industrialized production.

Embodiment

Embodiment 1

(1) pre-treatment of fermenation raw liquid:

Centrifuging: purpose is to remove large granular impurities such as soybean cake powder through centrifuging.After the bacillus thuringiensis fermentation finishes, with centrifugal 1 min of fermented liquid 1500-3000 r/min;

Washing impurity-removing: the filtered liq after the centrifuging is centrifugal through 1000r/min, 4 ℃, 2min; Remove deposition, through 10000r/min, 4 ℃, 15min filtration, the deposition of collecting is dissolved in 4 times of amounts contains in the saline water of 0.1%TritonX-100 then; Continue under 10000r/min, 4 ℃, 15min centrifugal then; To precipitate and adopt saline water washing three times, and for the last time centrifuge tube will be inverted 3min and make current do collecting precipitation.

(2) extract protein crystal

By following system: take by weighing deposition, PEG, potassium primary phosphate, potassium hydrogenphosphate and mix; With water dissolution, ultimate density is respectively: deposition 24g/L, PEG4000 120 g/L, potassium hydrogenphosphate 90 g/L, potassium primary phosphate 30g/L; Mix the back transfer PH to 8.5. at 1440r/min; 15 ℃, the superiors are removed in centrifugal layering under the 2min condition; Supply volume with water, and then adding accounts for the potassium hydrogenphosphate of TV 9% and 3% potassium primary phosphate (being the potassium primary phosphate that every 100ml volume adds 9g potassium hydrogenphosphate, 3g respectively) mixing carrying out reextraction.At 4500r/min, 4 ℃, centrifugal collecting precipitation part under the 15min, the saline water that adds 4 times of amounts washs, and collects protein crystal.Be dissolved in the saline water-20 ℃ frozen.

Claims (2)

1. one kind is extracted Bt insecticidal proteins crystalline method, and it is characterized in that: the concrete steps of said method are following:

(1) pre-treatment of fermenation raw liquid:

After the bacillus thuringiensis fermentation finishes, with centrifugal 1 min of fermented liquid 1500-3000 r/min; Filtrating is removed deposition through 1000 r/min, 4 ℃ of centrifugal 1.5-2min; Supernatant is through 10000 r/min, 4 ℃ of centrifugal 15min; The deposition of collecting is dissolved in the saline water that contains 0.1% (v/v) TritonX-100; Continue the centrifugal 15min down 10000r/min, 4 ℃ then, deposition is with the saline water washing, get rid of excessive moisture after collecting precipitation subsequent use;

(2) extract protein crystal

The deposition that step (1) is collected is mixed with PEG, potassium primary phosphate, potassium hydrogenphosphate, and the formation mixed solution that is dissolved in water is respectively ultimate density: deposition 24g/L, PEG 120 g/L, potassium hydrogenphosphate 90 g/L; Potassium primary phosphate 30g/L regulates pH of mixed to 8.5, while stirring at 1440r/min; Centrifugal 2min under 15 ℃ removes supernatant, adds water to complement to and above-mentioned mixed solution equal-volume; And then the potassium primary phosphate that adds the potassium hydrogenphosphate and 3% (w/v) account for mixed solution TV 9% (w/v) carries out reextraction, at 4500r/min, and 4 ℃ of centrifugal 15-20 min down; The collecting precipitation part adds the saline water washing, collects the Bt crystal proteins; It is dissolved in the saline water, frozen down in-20 ℃.

2. extraction Bt insecticidal proteins crystalline method according to claim 1, it is characterized in that: the said PEG of step (2) is PEG4000.

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