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Bacillus brevis, method for extracting bacillus brevis esterase and application of same

A technology of bacillus and extraction method, applied in the field of extraction of bacillus and its esterase, can solve the problems of unstable enzyme source, high cost, large difference in enzymes, etc. Cost, good repeatability

Inactive Publication Date: 2011-07-27
GUANGZHOU JIJIAZHUANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing domestic esterases used for large-scale production are all extracted from animals, and there are mainly the following disadvantages and problems: (1) The screening of enzyme sources is a lot of work and the cost is high; (2) The source of enzymes is unstable , different batches of enzymes from different individual sources have large differences

Method used

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  • Bacillus brevis, method for extracting bacillus brevis esterase and application of same
  • Bacillus brevis, method for extracting bacillus brevis esterase and application of same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Morphological characteristics

[0025] The colonies of the strains on the plate are round, light yellow, opaque, smooth, and with neat edges. The cells are rod-shaped, with a diameter of about 0.8 μm and a length of about 2-3 μm. Gram staining is positive, and the spores are oval and proximal.

[0026] 2. Physiological and biochemical identification

[0027] The results of antibiotic resistance and contact enzyme tests on the strains showed that the strains were resistant to nalidixic acid and spectinomycin, and the results of contact enzyme tests were positive.

[0028] 3.16SrDNA sequence

[0029] The 16SrDNA sequence of the strain is shown in SEQ ID NO:1.

[0030] Example 2

Embodiment 2

[0032] (1) Slant strains: inoculate the preserved strains on nutrient agar slant for culture, and store at 0-4°C.

[0033] (2) Enlarge the seed liquid culture: inoculate the slant bacterial classification into the 250ml Erlenmeyer flask that 100ml seed culture medium (peptone 1%, yeast extract 0.5%, NaCl 0.5%, pH7.0) is housed, 37 ℃, 200rpm, shake The bed was incubated for 12 hours.

[0034] (3) Fermentation culture: Inoculate 100ml of expanded culture seed solution into 10L fermenter, and cultivate it for 24 hours at 37°C (medium formula: glucose 1%, peptone 2%, K 2 HPO 4 0.1%, NaH 2 PO 4 0.1%, pH7.0).

[0035] (4) Extraction of enzyme liquid: centrifuge the fermented bacterial liquid (4000rpm) for 15min, collect the bacterial cells, wash with 0.02mol / L phosphate buffer (pH7.0), suspend with phosphate buffer again, and extract by ultrasonic wall breaking method. For enzyme, centrifuge (4000rpm) for 15 minutes, and the obtained supernatant is the esterase solution.

[00...

Embodiment 3

[0038] Materials: bacterial esterase, indophenol acetate solution, and phosphate buffer

[0039] Instrument: constant temperature water bath, spectrophotometer

[0040] Determination of esterase activity: add 4.7mL 0.02mol / L, pH7.0 phosphate buffer, 200μL 2% indophenol acetate, mix well, bathe in 37℃ water for 10 minutes, add 100μL Bacillus esterase, react for 5 minutes Then add 5mL of absolute ethanol to stop the reaction (the control tube added bacillus esterase after adding absolute ethanol), measure OD605.

[0041] Inhibition rate calculation: (OD0-OD1) / OD0×100% (OD0: OD value without inhibition; OD1: OD value after inhibition)

[0042] The measurement results:

[0043] (1) Sensitivity of Bacillus esterase to dichlorvos

[0044] When the concentration of dichlorvos is 0.005ppm, the inhibition rate is higher than 50%, lower than the relevant standard [GB / T 5009.199-2003 Rapid detection of organophosphorus and carbamate pesticide residues in vegetables].

[0045] (2) Sen...

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Abstract

The invention discloses bacillus brevis GDXEase8, a method for extracting bacillus brevis esterase and the application of same. The bacillus brevis is preserved in CCTCC (China Center for Type Culture Collection), the preservation date is 9th, Nov. 2010, and the serial number of preservation is CCTCC M2010295. The bacillus brevis has the advantages of low cost, high sensitivity and stable enzyme source.

Description

technical field [0001] The invention relates to an extraction method and application of bacillus and esterase thereof. Background technique [0002] Organophosphorus pesticides play a huge role in protecting crops from diseases and insect pests and increasing food production, but their excessive use often leads to excessive pesticide residues in agricultural products such as vegetables and fruits, which directly endanger people's health. Therefore, effective control of pesticide residues in agricultural products is It is one of the main tasks to solve the food safety problem in our country at present. One of the most effective ways to reduce the hazards of pesticide residues in agricultural products is to establish rapid and effective pesticide residue detection methods. At present, most domestic farmers’ markets are using the enzyme inhibition method to detect organophosphorus pesticide residues in fruits and vegetables. This method involves enzyme reagents, substrates and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/16C12Q1/44C12R1/08
Inventor 肖桂秋隆湘蕾
Owner GUANGZHOU JIJIAZHUANG BIOTECH
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