A kind of preparation method and application of β-galactosidase

A technology of galactosidase and galactose, applied in the field of preparation of β-galactosidase, which can solve the problems of long fermentation period of β-galactosidase, inability to use genetically modified strains, restrictions on the use of cross-linking agents, etc. , to achieve the effect of increasing personnel burden and equipment investment, shortening production cycle, and high specific vitality

Active Publication Date: 2022-03-25
量子高科(广东)生物有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of immobilizing enzymes, it is often necessary to use glutaraldehyde as a cross-linking agent for the immobilized enzyme carrier, but the use of cross-linking agents is sometimes limited, for example, in the production process of nutritional enhancer oligosaccharides. Glutaraldehyde use
In addition, the symbiotic fermentation method of lactose and microorganisms is often restricted, especially infant formula food, organic food, etc. cannot use genetically modified strains
In addition, in the prior art, the use batch of immobilized β-galactosidase is only 8 batches, that is, it can only be recycled 8 times (the patent application number is 201480049114.3), and the utilization rate of the enzyme not tall
In the prior art, the fermentation cycle of direct use of microbial fermentation to produce β-galactosidase is long and the fermentation process is complicated

Method used

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  • A kind of preparation method and application of β-galactosidase
  • A kind of preparation method and application of β-galactosidase
  • A kind of preparation method and application of β-galactosidase

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Embodiment 1: Preparation of β-galactosidase

[0066] A preparation method of β-galactosidase, comprising the following steps:

[0067] (1) Inoculate 1 loop of Bacillus circulans (purchased from the American Type Culture Collection (ATCC), No. 31382) into 50 mL of LB medium, and culture it at 37°C for 18 hours to activate it. Bacillus spp. was added to an Erlenmeyer flask containing 500mL of LB medium, shaken and cultivated at 37°C and 180rpm for 6 hours, and then inoculated into a seed tank for cultivation to obtain a seed culture solution;

[0068] (2) the seed culture liquid that step (1) is made adds in the fermentor of 200L that contains 120L fermentation medium and ferments, and the pH in the fermentor is adjusted to be 6.64 with 10% (mass fraction) sodium hydroxide solution, at 37 At ±0.5°C, stirring speed 180rpm, ventilation rate 120L / min, ferment for 8 hours, after 8 hours, adjust the pH value to 7.45±0.5 with hydrochloric acid, after 12 hours, take samples at...

Embodiment 2

[0071] Embodiment 2: Preparation of β-galactosidase

[0072] The difference between Example 2 and Example 1 is that in step (1) Bacillus circulans CCTCC NO: M2015424 was used instead of Bacillus circulans (purchased from the American Type Culture Collection (ATCC), No. 31382), and fermented for 30 h 115L of Bacillus circulans fermentation broth with an enzyme activity of 46.3U / mL was obtained; 46.0L of enzyme fluid with an enzyme activity of 107.6U / mL was obtained after microfiltration to remove bacteria and 50KD membrane concentration.

Embodiment 3

[0073] Embodiment 3: the preparation of the immobilized enzyme of β-galactosidase

[0074] 1. Refining of crude enzyme

[0075] Take 46L of the enzyme solution obtained by concentrating in Example 2, slowly add 6.9kg of ammonium sulfate to it, and after the ammonium sulfate is completely dissolved, let it stand at 6°C for 1 hour, then use a 50KD membrane to clean the enzyme solution to obtain 46.8L of enzyme solution Refined β-galactosidase solution with an activity of 102.1U / mL and a conductivity of 7.84ms / cm (that is, in every 1mL of β-galactosidase solution, the enzyme activity of β-galactosidase is 102.1U).

[0076] 2. Preparation of immobilized enzyme of β-galactosidase

[0077] A method for preparing an immobilized enzyme of β-galactosidase, comprising the following steps:

[0078] Soak 42Kg of macroporous styrene series weakly basic anion exchange resin A103s (this resin is provided by Brilliant (China) Co., Ltd., and the resin aperture is 30-50nm) in deionized water ...

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Abstract

The invention belongs to the technical field of biochemical industry, and discloses a preparation method of β-galactosidase, comprising the following steps: (1) inoculating bacillus circulans into LB medium for cultivation and activation, and then inoculating them into seed tanks for cultivation , to obtain the seed culture liquid; (2) adding the seed culture liquid to ferment in the fermenter containing the fermentation medium to obtain the Bacillus circulans fermentation liquid; (3) filtering or centrifuging the Bacillus circulans fermentation liquid to remove the thalline, A β-galactosidase solution is obtained; the fermentation medium contains lactose, galactose, plant peptone, corn flour, yeast extract, phosphate, carbonate and water. The fermentation time of the method is short, the production cycle is shortened, and the specific activity of the produced enzyme liquid is high. The present invention also provides a method for preparing an immobilized enzyme of β-galactosidase without using a cross-linking agent. The prepared immobilized enzyme has good stability and can be used continuously for more than 264 hours. The cost of preparing galactose with the immobilized enzyme is greatly increased. reduce.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, in particular to a preparation method and application of β-galactosidase. Background technique [0002] Galacto-oligosaccharide (GOS) is a kind of naturally occurring oligosaccharides, and its molecular structure is generally 1-7 galactosyl groups connected to galactose or glucose molecules. Exist in trace amounts in animal milk, but higher in breast milk. Galacto-oligosaccharides have strong stability, even under acidic conditions. It is not digested by human digestive enzymes, and is rarely digested and absorbed by human small intestine. It has good bifidobacteria proliferation activity, inhibits harmful pathogenic bacteria and The growth of spoilage bacteria can promote the absorption of calcium, magnesium and potassium, and because it has the property of water-soluble dietary fiber, it can achieve the effect of laxative. Therefore, galacto-oligosaccharides are widely used in th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/38C12N11/082C12P19/00C12P19/04C12P19/12C12P19/14C12R1/09
CPCC12N9/2471C12Y302/01023C12N11/082C12P19/00C12P19/04C12P19/12C12P19/14C12P21/02C12R2001/09C12N1/205C12N1/20C12P19/02
Inventor 魏远安曾宪维谢志龙杨新球陈子健浣金刚
Owner 量子高科(广东)生物有限公司
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