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Method for enhancing adsorption of Bacillus thuringiensis insecticidal proteins on mineral carrier

A technology of Bacillus thuringiensis and insecticidal protein, applied in botany equipment and methods, insecticides, biocides, etc., can solve the problem of low adsorption amount of insecticidal protein, achieve lower free protein concentration, high adsorption percentage, adsorption high volume effect

Inactive Publication Date: 2011-05-04
襄阳新叶生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to overcome the defect that the insecticidal protein has a low adsorption amount on minerals in the preparation process of the existing Bacillus thuringiensis adsorbent type, and provide a method for enhancing the adsorption of Bt insecticidal protein on minerals

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  • Method for enhancing adsorption of Bacillus thuringiensis insecticidal proteins on mineral carrier
  • Method for enhancing adsorption of Bacillus thuringiensis insecticidal proteins on mineral carrier

Examples

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Effect test

Embodiment 1

[0033] Embodiment 1: the insecticidal protein montmorillonite adsorbent formulation enhanced by sodium chloride

[0034] The original powder of Bacillus thuringiensis was made into a suspension with a weight-to-volume ratio of 10%, washed three times with 0.5 mol / L NaCl solution and sterile deionized water, and then extracted with NaOH solution of pH 11-12 for 4-5 h. Centrifuge to remove the precipitate, collect the supernatant and add acetic acid to adjust the pH to 5-6 to obtain the undegraded protoxin protein precipitate with a molecular weight of 130 kDa. Dissolve the protoxin insecticidal protein with Tris buffer at pH 9 to prepare a solution with a concentration of 1.0 g / L.

[0035] Montmorillonite was selected from Anji County, Huzhou City, Zhejiang Province. Make a 2% (w / v) suspension of montmorillonite in distilled water. Add 10 mL of insecticidal protein Tris buffer solution with a concentration of 1.0 mg / mL into a 50 mL plastic test tube (containing 10.0 mg of pur...

Embodiment 2

[0037] Embodiment 2: the insecticidal protein montmorillonite adsorption formulation that potassium chloride strengthens

[0038] Mix Bacillus thuringiensis protoxin insecticidal protein (130 kDa) with montmorillonite at a ratio of 1:1 (mass ratio), and then add 1mol / L KCl solution dropwise to make the concentration of KCl in the adsorption system 2 mmol / L. After shaking and adsorbing at ℃ for 4 h, centrifuge, and pour off the supernatant to obtain the protoxin insecticidal protein montmorillonite adsorption complex. In the presence of 2 mmol / L KCl, the adsorption capacity of montmorillonite to protoxin insecticidal white was 0.483 mg / mg, and the adsorption rate to protoxin insecticidal protein was 48.3%.

Embodiment 3

[0039] Example 3: Calcium Chloride Enhanced Insecticidal Protein Rectorite Adsorbent Form

[0040] Retortite was selected from Zhongxiang, Hubei. Prepare a 3% (w / v) suspension of rectorite with distilled water, dissolve Bacillus thuringiensis protoxin insecticidal protein (130 kDa) in Tris buffer with pH 9, and prepare a solution with a concentration of 1.0 g / L. Add 20 mL of Bacillus thuringiensis protoxin insecticidal protein Tris buffer solution (pH9) at a concentration of 1.0 mg / mL into a 50 mL plastic test tube (containing 20.0 mg of pure protein), and add 335 μL of a 3% rectorite suspension (containing 10.0 mg of topolite), and was shaken and adsorbed at 25°C for 5 h. Put the centrifuge tube into the centrifuge, centrifuge at 15000 r / min for 10 min, pour off the supernatant, and detect the concentration of protoxin insecticidal protein in the supernatant by Folin-phenol method. In the absence of metal ions, the adsorption amount of rectorite to protoxin insecticidal pro...

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Abstract

The invention discloses a method for enhancing adsorption of Bacillus thuringiensis insecticidal proteins on a mineral carrier. 0.1-5mmol / L univalent or bivalent metal cations are added into a mixed system of Bacillus thuringiensis insecticidal proteins and mineral particles, and the adsorption capacity of the mineral particles for the Bacillus thuringiensis insecticidal proteins is improved by 0.2 to 3 times compared with that in the common method through the bridging of chemical bonds. The insecticidal proteins are firmly adsorbed, and are difficultly desorbed by deionized water. The invention provides a simple, convenient and feasible improved method for a Bacillus thuringiensis adsorption formulation, and provides technical guarantee for low-cost production of the Bacillus thuringiensis adsorption formulation. The method can make insecticidal protein content in the Bacillus thuringiensis adsorption formulation improved, and titer of the product greatly improved. Because the adsorption efficiency for the Bacillus thuringiensis insecticidal proteins in an adsorption system is improved, the manufacturing cost is reduced.

Description

【Technical field】 [0001] The invention relates to the technical field of preparation of microbial pesticide Bacillus thuringiensis formulations, in particular to a method for enhancing the adsorption of Bacillus thuringiensis insecticidal protein on a mineral carrier by adding metal cations. 【Background technique】 [0002] Bacillus thuringiensis ( Bacillus thuringiensis , referred to as Bt) is a bacterium that is closely related to Bacillus cereus and produces parasporal crystals and spores. It has attracted worldwide attention due to its high insecticidal activity against Lepidoptera insects and its safety and non-toxicity to humans and animals. 1938 The first commercial preparation of Bacillus thuringiensis appeared in France in 1999, and industrialized production was realized in the middle of the 20th century. Its output accounts for 97% of the total amount of biopesticides, and it has become the largest and most widely used microbial pesticide in the world. Used to cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N63/02A01N25/08A01P7/04
Inventor 周学永董建萍董庆洁马锦明王学利
Owner 襄阳新叶生物科技有限公司
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