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Bacillus subtilis ZJB-063 and its application

A technology of Bacillus subtilis and ZJB-063, applied in the application field of preparing p-hydroxyphenylacetic acid, can solve the problems of lack of vitality, increasing the difficulty of separation, affecting the yield and the like

Active Publication Date: 2007-09-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this strain can produce nitrilase and nitrile hydratase at the same time, so there will be more p-hydroxyphenylacetamide in the product, which increases the difficulty of separation and also affects the yield.
[0005] Another bacterium that can catalyze this reaction reported by the University of Tokyo in 1990 is Alcaligenes faecalis JM3. This bacterium has good activity on phenylacetonitriles with substituents on the benzene ring, but it is basically inactive on other fatty nitriles [European Journal of Biochemistry, 1990, 194:765-772]

Method used

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  • Bacillus subtilis ZJB-063 and its application
  • Bacillus subtilis ZJB-063 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Seed medium formula (weight / volume percentage, the same below): glucose: 1.0%, yeast extract: 0.3%, NaCl: 0.1%, K 2 HPO 4 : 0.02%, KH 2 PO 4 ·3H 2 O: 0.02%, MgSO 4 : 0.02%, prepared with deionized water, pH natural.

[0046] Fermentation medium formula: glucose: 1.8%, yeast extract: 0.5%, K 2 HPO 4 : 0.05%, KH 2 PO 4 ·3H 2 O: 0.05%, MgSO 4 : 0.1%, monosodium glutamate: 0.076%, urea: 0.75%, prepared with deionized water, pH natural.

[0047] Take 100mL of the above-mentioned seed culture medium, evenly distribute it in two 250mL Erlenmeyer flasks, and sterilize. Insert the slant strain Bacillus subtilis ZJB-063, cultivate the bacteria, shake the rotating speed of 150r / min, and cultivate at 28°C for 48 hours as the seed liquid, and set aside.

[0048] Take 1L of the above-mentioned fermentation medium, divide it into ten 500mL shake flasks, and sterilize. The seed liquid was inserted, the inoculation amount was 2% (v / v), and culture was carried out, the cultu...

Embodiment 2

[0051] Fermentation enzyme production medium formula Glucose: 2.0%, yeast powder: 0.7% (NH 4 ) 2 SO 4 : 0.5%, KCl: 1.0%, K 2 HPO 4 12H 2 O: 0.05%, NaH 2 PO 4 2H 2 O: 0.05%, MgSO 4 : 0.01%, prepared with tap water, adjusted to pH 6.5 with HCl.

[0052] Take 1000mL of culture medium, evenly distribute it in ten 500mL Erlenmeyer flasks, and sterilize. Insert the slant strain Bacillus subtilis ZJB-063, cultivate the thallus for 50 hours, and then collect the thallines by centrifugation. These thallines are added to 500 mL of p-hydroxybenzonitrile aqueous solution, and the final substrate concentration is 3 g / L. The cells hydrolyze p-hydroxyphenylacetonitrile; reaction conditions: temperature 33°C, initial pH 6.8, transformation time 4h, shaker speed 100r / min. Detected by high performance liquid chromatography, the conversion rate of p-hydroxyphenylacetonitrile was 100%. The transformation solution was centrifuged to remove cells, filtered, and the transformation solutio...

Embodiment 3

[0054] Liquid seed culture medium is the same as embodiment 1.

[0055] Fermentation enzyme production medium: glucose: 0.5%, yeast powder: 2%, urea: 0.4g%, sodium glutamate: 0.1%, K 2 HPO 4 : 0.01%, KH 2 PO 4 : 0.01%, (NH 4 ) 2 SO 4 : 1.0%, MgSO 4 : 0.01%, pH natural.

[0056] Take 100mL of liquid seed culture medium, evenly distribute it in two 250mL Erlenmeyer flasks, and sterilize. Insert the slant strain Bacillus subtilis ZJB-063, cultivate the bacterial body, shake the rotating speed of 150r / min, and cultivate it at 28°C for 36h as the seed liquid, and set aside.

[0057] Take 2L of the above-mentioned fermentation medium, put it into 20 500mL shake flasks equally, and sterilize. The seed solution was inserted, the inoculum amount was 4% (v / v), and culture was carried out, the culture temperature was 28° C., the culture time was 60 h, and the shaker speed was 150 r / min.

[0058] Collect the thalline by centrifugation at 9000r / min, wash twice with distilled wate...

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Abstract

The invention provides a new stem of microbe-Bacillus subtilis ZJB-063 selected from soil, and application of preparing hydroxy-Benzeneacetic acid. The useful effects of the invention including: sythetising hydroxy-Benzeneacetic acid by Bio-treatment using new selected stem to replace chemical synthesis method. The method has advatages of high conversion ratio, friendly environment and green process. The stem can hydrolyze nitrile compounds with high toxicity to acid compounds with little danger, Bio-treatment is the most econnomic, most effective method for processing nitrile compounds with high toxicity of industrial wastewater so far, so the invention has important actual sense in environment protection.

Description

(1) Technical field [0001] The invention relates to a new microorganism strain Bacillus subtilis ZJB-063 screened from soil and its application in preparing p-hydroxyphenylacetic acid. (2) Background technology [0002] Bacillus subtilis, a species of the genus Bacillus. A single cell is 0.7-0.8×2-3 microns, evenly colored. Uncapsulated, with periosteum flagella, able to move. Gram-positive bacteria, spores 0.6-0.9×1.0-1.5 microns, elliptical to columnar, located in the center of the bacterium or slightly biased, the bacterium does not expand after spore formation. The surface of the colony is rough and opaque, stained white or yellowish, and often forms wrinkles when growing in liquid medium. Aerobic bacteria. It can use protein, various sugars and starch to decompose tryptophan to form indole. Some strains are important producers of α-amylase and neutral protease; some strains have enzymes that strongly degrade nucleotides, so they are often used as parent strains of ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/31C12P7/42C12R1/125
Inventor 郑裕国吴明火柳志强沈寅初
Owner ZHEJIANG UNIV OF TECH
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