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Enzyme solution with better enzyme activity ratio of beta-mannase to alpha-galactosidase as well as preparation method and application of enzyme solution

A mannanase and galactose technology, applied in the directions of microorganism-based methods, methods using microorganisms, biochemical equipment and methods, etc., can solve the problem of reducing production costs, low β-mannosidase activity, and no good results. effect and other issues, to achieve the effect of reducing production costs and improving yields

Pending Publication Date: 2021-05-07
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Trichoderma reesei also has the ability to synthesize β-mannosidase. Therefore, when synthesizing an enzyme solution with an optimal ratio of β-mannanase and α-galactosidase, it is required that β- The activity of mannosidase should be as low as possible, so that the enzyme liquid obtained by fermentation can be directly used in the production of small molecule galactomannan and galactomannan oligosaccharides without purification, thereby further reducing production costs, and this technology At this stage, it has not achieved good results

Method used

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  • Enzyme solution with better enzyme activity ratio of beta-mannase to alpha-galactosidase as well as preparation method and application of enzyme solution
  • Enzyme solution with better enzyme activity ratio of beta-mannase to alpha-galactosidase as well as preparation method and application of enzyme solution
  • Enzyme solution with better enzyme activity ratio of beta-mannase to alpha-galactosidase as well as preparation method and application of enzyme solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Trichoderma reesei uses microcrystalline cellulose and melibiose as carbon sources to ferment and produce enzymes, including the following steps:

[0043] (1) Enzyme production medium composition (g / L): glucose 1.0, microcrystalline cellulose 25.0 or melibiose 25.0, ammonium sulfate 4.72, urea 2.15, potassium dihydrogen phosphate 2.0, anhydrous calcium chloride 0.3, heptahydrate Magnesium sulfate 0.3, ferrous sulfate heptahydrate 0.005, manganese sulfate heptahydrate 0.0016, zinc sulfate heptahydrate 0.0014, cobalt chloride 0.002. Add 50 mL of 1 mol / L sodium citrate buffer to adjust the pH of the medium to 4.8.

[0044] (2) Enzyme production by fermentation

[0045] Put the above 50mL medium into a 250mL Erlenmeyer flask with a cotton plug, inoculate Trichoderma reesei spores according to 10% inoculum size, and place them in a constant temperature shaker at 28-30°C and 170 rpm for 4 days. After the cultivation, the culture solution was centrifuged at 3000 rpm for 10 m...

Embodiment 2

[0048] Compound enzymatic hydrolysis of mannanase and galactosidase to prepare small molecule galactomannans and galactomannoligosaccharides, the steps are as follows:

[0049] (1) Directed enzymatic hydrolysis of galactomannan

[0050] The leguminous seeds containing galactomannan (Tianjing) were mechanically crushed to 20-100 mesh, added distilled water at a solid-to-liquid ratio of 1:50, extracted at 50°C for 24 hours, and then centrifuged at 10,000 rpm for 10 minutes The supernatant was obtained, and absolute ethanol was added to the supernatant, and the obtained precipitate was vacuum-dried to obtain galactomannan powdery solid.

[0051] The enzyme liquid 1 and enzyme liquid 2 obtained in Example 1 were compounded so that the enzyme activity ratios of β-mannanase and α-galactosidase were 2, 4, 6, 8, 10, 12, 15, 20, 30, 40. Then weigh 20.0 g of the above-mentioned galactomannan in a 2L enzyme reaction tank, add distilled water, enzyme solution, and 1mol / L citric acid buf...

Embodiment 3

[0058] The compound production enzyme of microcrystalline cellulose and melibiose, its steps are as follows:

[0059] (1) The enzyme-producing medium was the same as in Example 1, wherein the substrate was replaced by a mixture of microcrystalline cellulose and melibiose in different proportions, and the sum of the concentrations of the two substrates was 20.0, 25.0, 30.0, and 35.0 g / L, respectively.

[0060] (2) The method for producing enzyme by fermentation is the same as in Example 1.

[0061] The result is as figure 2 As shown, when the total substrate concentration was 20g / L, with the continuous increase of melibiose, the activity of β-mannanase decreased continuously. But when the total substrate concentration was 25, 30, 35g / L, with the continuous increase of melibiose, the enzyme activity of β-mannanase showed a trend of first increasing and then decreasing. When the total substrate concentration was 25g / L, microcrystalline cellulose was 20g / L and melibiose was 5g / ...

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Abstract

The invention discloses an enzyme solution with a better enzyme activity ratio of beta-mannase to alpha-galactosidase as well as a preparation method and application of the enzyme solution, and belongs to the technical field of microbial culture. According to the preparation method, trichoderma reesei is taken as an enzyme-producing strain, microcrystalline cellulose and / or melibiose is taken as a carbon source, the enzyme solution is produced by adopting a fermentation method, the enzyme activity of the beta-mannase in the enzyme solution is not higher than 0.05 U / mL, and the enzyme solution has a better enzyme activity ratio of beta-mannase to the alpha-galactosidase. The enzyme solution can directly hydrolyze the galactomannan to prepare micromolecular galactomannan and galactomannan-oligosaccharide without purification, the yield of the micromolecular galactomannan and the galactomannan-oligosaccharide can be effectively increased, the production cost is reduced, and the enzyme solution has a very good application prospect.

Description

technical field [0001] The invention belongs to the technical field of microorganism cultivation, and in particular relates to an enzyme liquid with a better enzyme activity ratio of β-mannanase and α-galactosidase, and a preparation method and application thereof. Background technique [0002] At present, the aging of our population and the rejuvenation of diseases have aroused people's attention to health issues. In 2016, the state put forward the "Healthy China 2030" planning outline, which aims to develop China's health industry and improve people's health. Dietary fiber has become one of the research hotspots under the background of "Healthy China" because of its unique physiological functions. Dietary fiber includes natural polysaccharides, oligosaccharides and small molecular polysaccharides, among which oligosaccharides and small molecular polysaccharides have been more and more effective due to their outstanding effects of proliferating intestinal beneficial bacter...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N9/40C12N1/14C12N1/22C12P19/14C12R1/885
CPCC12N9/2494C12N9/2465C12N1/14C12N1/22C12P19/14C12Y302/01022C12Y302/01078C12N9/2491C12P19/04C12Y302/01025
Inventor 黄曹兴陶昱恒勇强张晓彤陈雨姝廖吉丽董昊路李鑫赖晨欢
Owner NANJING FORESTRY UNIV
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