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Preparation method capable of improving fermentation yield of tautomycin

An allosteric and yield technology is applied in the field of preparation for improving the fermentative yield of allostericin, can solve problems such as cell dissolution and death, and achieves the effects of convenient operation, increased yield, and improvement of low yield of allostericin

Active Publication Date: 2010-10-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

If the speed of supplementation is too fast or the concentration is too high, the pH of the fermentation broth will become more acidic, and the bacteria will eventually dissolve and die.

Method used

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  • Preparation method capable of improving fermentation yield of tautomycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Strain: Streptomyces spiroverticillatus, the preservation number is CGMCC-0092.

[0029] The culture medium formula of the embodiment of the invention:

[0030] Slant medium: the slant medium is Gao's No. 1 medium;

[0031] Seed medium: glucose 10g / L, beef extract 5g / L, yeast powder 10g / L, NaCl 2g / L, K 2 HPO 4 0.02g / L, initial pH 6.5; solvent is water.

[0032] Fermentation medium: glucose 20g / L, soybean powder 25g / L, starch 10g / L, yeast extract 5g / L, NaCl 2g / L, K 2 HPO 4 0.05g / L Initial pH 6.5; solvent is water.

[0033] experiment procedure:

[0034] Step 1: Seed Culture

[0035] The S. spiroverticillatus strain was inserted into a 500ml Erlenmeyer flask containing 100ml of seed medium from a slant, and cultured in a rotary shaker at 28°C for 24h with a rotation speed of 150rpm.

[0036] Step 2: Effect of maleic anhydride at different pH on fed-fed fermentation

[0037] Transfer 3ml of seed solution into a conical flask containing 50ml of fermentation medium...

Embodiment 2

[0043] Bacterial classification, substratum and culture condition are with example 1

[0044] experiment procedure:

[0045] Step 1: Seed cultivation is the same as example 1

[0046] Step 2: Add maleic anhydride at different times

[0047] Transfer 6ml of seed solution to a conical flask containing 100ml of fermentation medium, and carry out fermentation at a temperature of 28°C and a rotation speed of 150rpm. After it was fermented for a certain period of time, precursor substances were added. There were eight experiments in total. Each experiment consisted of three parallel experiments. The average value of the three parallel experiments was taken as the following results.

[0048] In order to determine the best time for maleic anhydride to add, add 0.2% (g / mL) maleic anhydride in different fermentation time periods respectively, the concentration is 40g / L, add maleic anhydride The amount of the aqueous solution was 5 mL, and the control group was distilled water, and 5 ...

Embodiment 3

[0055] It has been clarified in Example 1 that the addition of maleic anhydride must be added in the form of its anhydride, and its pH must not be adjusted to be neutral or alkaline. Therefore, the added maleic anhydride is an acidic aqueous solution, which greatly affects the pH of the fermentation broth itself. Adding too much concentration or too fast will cause the pH of the fermentation broth to drop sharply, which will eventually affect the production of mutamycin. In order to further clarify the degree of influence of pH on mycelia and the optimum supplementary time and supplementary speed of maleic anhydride, we added before and after (B) maleic anhydride in Example 1 and under different pH conditions The mycelia were observed microscopically. The result is as figure 1 shown.

[0056] Experimental results

[0057] Before adding maleic anhydride, the pH of the fermented liquid was 6.5. At this time, the growth of mycelia was long and strong, and the fermented liquid...

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Abstract

The invention provides a preparation method of an allosteric acid, which comprises: dissolving the tautomycin in 0.1 to 0.5M buffer solution A to prepare solution of the tautomycin, and dissolving lipase in buffer solution B to prepare solution of the lipase; adding the solution of the lipase into the solution of the tautomycin to perform an reaction at 25 to 40 DEG C for 0.5 to 5.0 hours, adjusting the pH value of the reaction solution to 4.0 at the end of the reaction, and extracting with an extracting solvent A in the same volume; taking a water layer, adjusting the pH value of the water layer to 2.0, extracting with an extracting solvent B in the same volume, taking an organic layer, washing the organic layer with saturated saline water, drying the organic layer with Na2SO4, performing distillation under vacuum to concentrate the solvent to obtain a coarse product of the allosteric acid; and performing the gradient elution of the coarse product of the allosteric acid by using an eluent containing acetic acid and a dichloromethane solvent, and recrystallizing the product of the column chromatography of the allosteric acid in acetone to obtain the product of the allosteric acid.The method uses the lipase to hydrolyze the fermented tautomycin to obtain the allosteric acid and has the advantages of mild reaction conditions, single reaction, high efficiency and the like.

Description

(1) Technical field [0001] The invention relates to a preparation method for improving the fermentation yield of allostericin, in particular to a process method for increasing the fermentation yield of allostericin by supplementing a precursor substance when producing allostericin by fermenting and culturing actinomycetes. (2) Background technology [0002] Tautomycin is an antibiotic discovered in the late 1960s by Shen Yinchu, a well-known biochemical scientist and academician of the Chinese Academy of Engineering. In 1987, its structure was initially identified at the Institute of Physics and Chemistry in Japan, and in 1990, allosteric was fully identified, and its molecular formula is C. 41 H 66 O 13 . [0003] Tautomycin is a specific protein phosphatase inhibitor produced by Streptomyces spiroverticillatus [5], especially for protein phosphatase type 1 (PP1) and protein phosphatase type 2A (PP2A), and its IC 50 are 22nM and 32nM, respectively; it is also an effecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16C12N1/20C12R1/465
Inventor 陈小龙徐玉华范永仙嘉晓勤沈寅初
Owner ZHEJIANG UNIV OF TECH
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