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Method for producing elastase preparation by utilizing liquid-state fermentation of microbes

An elastase and liquid fermentation technology, applied in the field of bioengineering, can solve the problems of poor preservation, complicated process, low enzyme production and the like, and achieve the effects of simple operation process, low production cost and low impurity content

Inactive Publication Date: 2014-03-12
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, elastase is mostly prepared by obtaining it from animal pancreas. This method has low enzyme yield, poor preservation, complicated process and high cost.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Preparation of fermented seed liquid.

[0037] 1. Activation of strains:

[0038] The preserved Bacillus licheniformis was streaked twice on the strain activation medium, and cultured in a 37°C incubator for 18-24h, and the single colony obtained by the first streak was used as the bacterial species for the second streak. Then select a single colony from the second streaked plate and insert it into the liquid strain activation medium or solid strain activation medium, culture temperature 37°C, time 180-200r / min, shake flask culture OD 600 to 1D 600 ;

[0039] 2. Preparation of Bacillus licheniformis seed solution:

[0040] Take 2-4ml of bacterial liquid from the activated liquid test tube bacteria and put it into a 500mL Erlenmeyer flask with 40-80mL of seed medium, 35-37°C, 160-180r / min shaker shaker culture for 18-24h, as the seed liquid.

[0041] Seed medium (g / L): 0.4 beef extract, 0.8 peptone, 1 yeast powder, 0.5NaCl, 0.1KH 2 PO 4 , 0.04MgSO 4...

example 2

[0042] Example 2, shaking flask fermentation culture method.

[0043] Inoculate the cultivated seed culture liquid into a 250ml Erlenmeyer flask containing 30-40mL fermentation medium with an inoculum amount of 2%-8% (v / v), and culture on a shaker at 160-180r / min. The fermentation temperature adopts a staged control method, that is, the temperature is controlled at 35-37°C for 6-10 hours before fermentation, and then switched to 26-30°C after 10 hours and kept until the end of fermentation; The influence of product synthesis rate, the pH two-stage control method was determined through the fermentation optimization test, that is, the pH of the fermentation broth was controlled at 7.0-7.5 at 0-10h, and the pH was kept at 7.5-8.5 from 10h to the end of fermentation; the pH control was dripping acid alkaline solution method. The enzyme activity obtained by the shake flask fermentation method is: 212u / ml.

[0044] Shake flask fermentation medium formula (g / L): 15 bean cake powder...

example 3

[0045] Example 3: deep liquid aeration fermentation

[0046] Seed liquid culture method is the same as embodiment 2.

[0047] Before the fermenter is sterilized, add defoamer at a ratio of 1:1000; heat up to 98°C through the interlayer of the fermenter, then directly pass steam into the fermenter, raise the temperature to 121°C, keep it warm for 30 minutes, and then gradually cool down to 30°C -35°C; feed sterile air, and maintain the pressure of the fermenter at 0.04Mpa during the entire fermentation process; inoculate the cultivated and activated seed culture solution with an inoculation amount of 3-6% (v / v) until the 10L (packed) Liquid coefficient of 0.67) in the fermenter of the fermentation medium, from the beginning of fermentation to 14 hours of fermentation, the ventilation rate is controlled at 0.55vvm; for 14-24 hours of fermentation, the ventilation rate is controlled at 0.75vvm. From 25 hours of fermentation to the end of fermentation, the ventilation control is 0...

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Abstract

The invention relates to a method for producing elastase preparation by utilizing liquid-state fermentation of microbes. The method is characterized in that the elastase preparation is prepared by activating baclicus lincheniformis ELAT4 (CGMCC5700) which serves as a strain of elastase production, preparing a seed solution, culturing a fermentation broth, performing ventilation fermentation on deep liquid, and preparing an elastase preparation finished product. The enzyme activity of the liquid prepared by the method is 352U / ml, and the enzyme activity of the solid enzyme preparation is 2,512u / g. The method has the advantages of improving the efficiency of elastase production, strengthening the activity of the elastase and reducing the production cost, does not have the problem of waste solid treatment in the production process, does not pollute the environment, and is suitable for large-scale production of elastase.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for producing elastase enzyme preparation by using microorganism liquid fermentation. Background technique [0002] Elastase is a proteolytic enzyme characterized by decomposing insoluble elastin. It mainly exists in animal pancreas, and is also distributed in skin, aorta, platelets and white blood cells. At the same time, it is also widely distributed in microbial groups. Bacteria , actinomycetes, and fungi can secrete extracellular elastase. Elastase can digest and decompose elastin in connective tissue proteins, including hydrolysis of peptide-bonded, amide-bound and ester-bound elastin. Elastase has obvious β-lipoproteinase effect, can activate phospholipase A, lower serum cholesterol, improve serum lipids, reduce plasma cholesterol and density lipoprotein, triglyceride, increase density lipoprotein, prevent lipid from Arterial wall deposits and increases th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/66C12R1/10
CPCC12N9/6448C12Y304/21011
Inventor 高兆建张桂英张建萍侯进慧陈尚龙张从省
Owner XUZHOU UNIV OF TECH
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