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Total synthetic culture medium and method for producing rhizomucor miehei lipase by fermentation method

A technology of Rhizomucor miehei and culture medium, which is applied in the field of fully synthetic culture medium and fermentation method to produce Rhizomucor miehei lipase, which can solve the problem of complex components, unfavorable quantitative analysis and research of cell metabolic characteristics, and Rhizomucor miehei lipase. Problems such as low activity, to achieve significant effects

Active Publication Date: 2021-07-30
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, Aspergillus oryzae is mainly cultivated by using compound medium, but its composition is complex, which is not conducive to the quantitative analysis and research of cell metabolism characteristics.
Although the conventional filamentous fungal synthetic medium is basically suitable for Aspergillus oryzae, the lipase activity of Rhizomucor miehei is very low, so it is difficult to use in the actual fermentation process

Method used

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  • Total synthetic culture medium and method for producing rhizomucor miehei lipase by fermentation method
  • Total synthetic culture medium and method for producing rhizomucor miehei lipase by fermentation method
  • Total synthetic culture medium and method for producing rhizomucor miehei lipase by fermentation method

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Under sterile conditions, pipette 30 μL of Aspergillus oryzae spore suspension onto the solid plate medium, evenly spread it, and incubate it upside down in a constant temperature incubator at 30°C for 168 hours until dark green spores appear. Wash each plate with 10 mL of sterile water and disperse the spores evenly with glass beads, then count the number of spores with a hemocytometer and dilute to 10 per mL 7 The spore suspension of order of magnitude spore number, spare;

[0052] Under sterile conditions, put the pre-prepared sterilized seed culture medium in the seed shake flask with a liquid content of 20%, inoculate 400 μL of spore suspension in each bottle, and then place it in a shaker for cultivation. The cultivation temperature is 30°C and the rotation speed The temperature is 150 rpm, cultivated for about 28 hours or until the bacteria in the shake flask form agglomerates and hang on the wall to obtain seed liquid, which is set aside.

[0053] The plate cul...

Embodiment 2

[0057] This embodiment provides a fully synthetic medium, including raw materials: sucrose 19.60g / L, alanine 4.02g / L, manganese sulfate 0.86g / L, potassium dihydrogen phosphate 1.31g / L, sodium chloride 0.75g / L L. Magnesium sulfate heptahydrate 1g / L, calcium chloride anhydrous 0.13g / L, disodium hydrogen phosphate dodecahydrate 10.5g / L, zinc sulfate heptahydrate 0.0126g / L, copper sulfate pentahydrate 0.000925g / L, Nickel chloride hexahydrate 0.000225g / L, ferrous sulfate heptahydrate 0.00517g / L and defoamer 1g / L. The pH value of the fully synthetic medium is about 5.

[0058] The seed solution in Example 1 was inoculated into the fully synthetic medium in this example with a 10% inoculation amount, and fermented and cultured in a shaker at 30°C and 150 rpm, and the amount of liquid in the shaker was 20%. After 120 hours, the RML activity of the fermentation supernatant was measured to reach 60.42 U / ml, which was 9.76 times higher than that of Comparative Example 1.

Embodiment 3

[0060] The present embodiment provides a kind of method that fermentation produces Rhizomucor miehei lipase, comprises the steps:

[0061] The seed solution in Example 1 was inoculated in the described total synthetic medium with an inoculum amount of 10%, and the liquid loading amount was 20%, and the shaker fermentation culture was carried out at 30 ± 2° C., 150 ± 50 rpm, and the fermentation time was 120 ±20 hours.

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Abstract

The invention discloses a fully synthetic culture medium and a method for producing rhizomucor miehei lipase by a fermentation method. The molar weight ratio of the carbon element to the nitrogen element in the total synthesis culture medium is 11-18; the total synthesis culture medium comprises a carbon source and a nitrogen source, and the carbon source is cane sugar, trehalose, lactose or fructose; the nitrogen source is alanine. According to the total synthesis culture medium for producing rhizomucor miehei lipase through aspergillus oryzae fermentation, sucrose, trehalose and the like are used as a carbon source, alanine is used as a nitrogen source, the carbon source and the nitrogen source are cooperatively added and used according to a scientific carbon-nitrogen ratio, the carbon source and the nitrogen source have an obvious interaction effect, and the aspergillus oryzae can be effectively promoted to synthesize the rhizomucor miehei lipase. The activity of the rhizomucor miehei lipase obtained through fermentation culture of the fully-synthetic culture medium reaches up to 60.42 U / ml, and the effect is remarkable.

Description

technical field [0001] The application relates to the technical field of industrial microbes, in particular to a fully synthetic medium and a method for producing Rhizomucor miehei lipase by fermentation. Background technique [0002] Rhizomucor miehei Lipase (RML) is the first enzyme to obtain crystal structure analysis and 3D structure analysis, and it has become one of the most representative fungal lipases due to its huge application potential in industrialization. Rhizomucor miehei lipase (RML) has good stability and stereoselectivity, and can participate in various reactions, such as hydrolysis, alcoholysis and esterification of esters, and can be widely used in food, chemical, and feed production , detergents and biodiesel. Naturally occurring Rhizomucor miehei lipase is extremely rare and unstable, and cannot be used on a large scale in an industrialized manner. Common lipase exogenous expression hosts include yeast, Escherichia coli and filamentous fungi, and fila...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N1/14C12R1/69
CPCC12N9/20C12N1/14Y02E50/10
Inventor 储炬孙磊田锡炜庄英萍
Owner EAST CHINA UNIV OF SCI & TECH
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