A kind of preparation method of α-galactosidase with low content of β-mannosidase
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A technology of mannosidase and galactosidase, applied in the field of preparation of α-galactosidase, to achieve the effect of reducing production costs
Active Publication Date: 2019-05-31
NANJING FORESTRY UNIV
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There are two main methods for producing α-galactosidase by using microorganisms. One is to use genetic engineering, that is, to prepare α-galactosidase by cloning and expressing the α-galactosidase gene, but the method of genetic engineering There are problems such as biological safety, and it is not the best solution for the preparation of food and feed additive products
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Embodiment 1
[0024] The preparation method of galactomannooligosaccharide comprises the following steps:
[0025] (1) Add 20-100 mesh S. japonica seeds into the enzymolysis bottle, add β-mannanase according to the enzyme dosage of 20U / g galactomannan, add 1M citric acid buffer, distilled water, and mix well , the concentration of galactomannan in the reaction system is 20g / L, the initial pH value is 4.80, and it is placed in a constant temperature shaker at 50°C and 150 rpm for enzymatic hydrolysis for 12h. Under treatment for 10 min, centrifuged at 10,000 rpm for 10 min, the supernatant is the enzymatic hydrolyzed supernatant containing galactomannooligosaccharide.
[0026] (2) Take the galactomanno-oligosaccharide-containing enzymatic hydrolysis clear liquid obtained in step (1), add absolute ethanol under stirring conditions, make the ethanol concentration in the system be 65% (v / v), at 10000 rpm / Centrifuge under different conditions for 10 min to obtain supernatant and precipitate. ...
Embodiment 2
[0030] Trichoderma reesei uses galactomannooligosaccharide as carbon source to ferment and produce enzymes, including the following steps:
[0031] (1) Enzyme production medium (g / L): galactomannooligosaccharide 10.0 (prepared in Example 1), glucose 1.0, potassium dihydrogen phosphate 2.0, magnesium sulfate heptahydrate 0.08, ferrous sulfate heptahydrate 0.005, a Manganese sulfate water 0.0016, zinc sulfate heptahydrate 0.0014, cobalt chloride 0.0037. The medium was adjusted to pH 4.8 with 1 M citrate buffer.
[0032] (2) Fermentation to produce enzymes
[0033] 50 mL of culture medium was placed in a 250 mL conical flask with a cotton stopper, inoculated with Trichoderma reesei seeds at 10% of the inoculation amount, and placed in a constant temperature shaker at 28-30°C and 170 rpm for 4 days. After the incubation, the culture medium was centrifuged at 3000 rpm for 10 min. The supernatant was taken to measure the activity of α-galactosidase and β-mannosidase, respectively...
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Abstract
The invention discloses a preparation method of alpha-galactosidase with the low beta-mannosidase content. Alpha-galactosidase liquid containing beta-mannosidase with the enzyme activity not higher than 0.05 U / mL is produced by taking trichoderma reesei as an enzyme producing strain and taking galacto-mannan-oligosaccharides as a carbon source through a fermentation method. According to the method, the alpha-galactosidase is produced by adopting the trichoderma reesei and taking the galacto-mannan-oligosaccharides as the carbon source and an inducer through fermentation, and the content (activity) of the beta-mannosidase in the enzyme liquid is very low while the high activity of the alpha-galactosidase with is achieved; the enzyme liquid can directly cooperate with the beta-mannosidase to hydrolyze galactomannan to prepare micromolecular galactomannan and the galacto-mannan-oligosaccharides without being purified to remove the beta-mannosidase, and therefore the production cost that the micromolecular galactomannan and the galacto-mannan-oligosaccharides are prepared by directionally degrading the galactomannan can be effectively reduced.
Description
technical field [0001] The invention belongs to the technical field of microorganism culture in biochemistry, and in particular relates to a preparation method of α-galactosidase with low β-mannosidase content. Background technique [0002] Functional oligosaccharides and functional small-molecule polysaccharides have been paid more and more attention because of their special biological functions as additives in the production of functional food and feed. The biological functions of functional oligosaccharides and functional small-molecule polysaccharides are mainly manifested in the selective proliferation of beneficial bacteria such as bifidobacteria in the intestinal tract of humans or animals, blocking the colonization of pathogenic bacteria in the digestive tract and the function of soluble dietary fiber. Wait. Generally speaking, the lower the degree of polymerization of small molecular glycans, the stronger its biological function, especially the selective proliferat...
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