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Method for preparing beta-D-galactoside galactose hydrolase preparation

The technology of enzyme preparation and hydrolase is applied in the field of preparation of β-D-galactoside galactoside hydrolase enzyme preparation, which can solve the problems of microbial contamination and difficulties, achieve high enzyme extraction rate, improve production efficiency and shorten technological process. Effect

Active Publication Date: 2010-09-29
中诺生物科技发展江苏有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is still difficult to produce food-grade enzyme preparations at present, mainly because the production process is easily polluted by microorganisms in the environment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, prepare the enzyme preparation of β-D-galactoside galactohydrolase

[0045] 1. Fermentation enzyme production

[0046] In this experiment, β-D-galactoside galactohydrolase was produced by high-density fermentation of recombinant yeast in a 50-liter fermenter.

[0047] Plasmid pPIC9 was purchased from the Institute of Biotechnology, Academy of Agricultural Sciences. According to the method described in the patent (Application No. 02108141.7), the recombinant yeast was constructed, the coding gene of lactase was introduced into the vector pPIC9, and then transformed into yeast strain GS115, and the recombinant yeast was screened. Among them, the strain with better enzyme production effect is recorded as Pichia pastoris (Pichia pastoris) LACO Y12; General Microbiology Center of the Management Committee (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code...

Embodiment 2

[0115] Embodiment 2, prepare the enzyme preparation of β-D-galactoside galactohydrolase

[0116] 1. Fermentation enzyme production

[0117] The method is consistent with that described in Example 1.

[0118] Two, filter

[0119] The fermentation broth obtained in Experiment 1 was subjected to the following filtration steps.

[0120] 1. Elimination of impurities and bacteria in the fermented liquid: the fermented liquid is filtered through an inflatable pressure filter to filter out bacteria and other solids, and the filtrate is collected, and the solid content in the filtrate is greatly reduced;

[0121] 2. Further sterilization: The filtrate obtained in step 1 is subjected to circulating cross-flow filtration with a microporous ceramic membrane of 0.1-0.5 μm, and steam condensed water is continuously replenished, and the enzyme is gradually driven out to obtain a sterile enzyme-containing clarified liquid; The enzyme activity in the bacterial enzyme-containing clarified li...

Embodiment 3

[0158] Embodiment 3, prepare the enzyme preparation of β-D-galactoside galactohydrolase

[0159] 1. Fermentation enzyme production

[0160] The method is consistent with that described in Example 1.

[0161] Two, filter

[0162] The fermentation broth obtained in Experiment 1 was subjected to the following filtration steps.

[0163] 1. Elimination of impurities and bacteria in the fermented liquid: the fermented liquid is filtered through an inflatable pressure filter to filter out bacteria and other solids, and the filtrate is collected, and the solid content in the filtrate is greatly reduced;

[0164] 2. Further sterilization: The filtrate obtained in step 1 is subjected to circulating cross-flow filtration with a microporous ceramic membrane of 0.1-0.5 μm, and steam condensed water is continuously replenished, and the enzyme is gradually driven out to obtain a sterile enzyme-containing clarified liquid; The enzyme activity in the bacterial enzyme-containing clarified li...

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Abstract

The invention discloses a method for preparing a beta-D-galactoside galactose hydrolase preparation. The method comprises the following steps of: 1) performing microporous membrane filtration on fermentation solution containing target enzyme to obtain clarified solution containing the target enzyme; 2) introducing the clarified solution containing the target enzyme into an ultra-filtration columnarray 1 and an ultra-filtration column array 2 in turn, and collecting the target enzyme solution; and 3) adding an aid into the target enzyme solution, and drying the mixture to obtain a target enzyme preparation, wherein the step 1) and the step 2) are performed under closed conditions. In the method, the sterile enzyme-containing classified solution filtered by a microporous membrane only needs to flow through an ultra-filtration membrane passage at one time during ultra-filtration and a circulating step is saved; therefore, compared with the prior art, the method has the advantages of avoiding the contact of the liquid and the environment, thereby ensuring that the product is not polluted during extracting and saving a re-sterilizing step. Experiments prove that the number of bacteriain the enzyme preparation finished product obtained by the method is less than 100 in every gram, and the number of bacteria in the enzyme preparation produced by the prior art is generally 1,000 in every gram.

Description

technical field [0001] The invention relates to a method for preparing β-D-galactoside galactohydrolase enzyme preparation. Background technique [0002] β-galactosidase (β-D-galactosido-galatohydrolase, β-D-galactoside galactohydrolase, EC 3.2.2.23) is generally called lactase (Lactase). This enzyme can hydrolyze lactose into galactose and glucose, and also has the transfer effect of galactoside (Zhang Shuzheng et al., Enzyme Industry, Science Press, 1984, p818~819). Lactase exists in plants (especially in apricots, peaches, apples), bacteria (lactic acid bacteria, E. Yeast) and actinomycetes (Streptomyces coelicolor), animal intestines (especially infants). Lactose is the main component of milk whey. Lactose accounts for about 30% of the dry matter of milk, but its solubility is low. Its solubility in water at 20°C is only 20%, and its sweetness is only 16% of that of sucrose. Due to the precipitation of lactose, dairy products stored for a long time have a sandy feelin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12R1/84
Inventor 谷海先张伟许建立毕建成
Owner 中诺生物科技发展江苏有限公司
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