Method for quick preparing glycosyl transferred beta-galactosidase

A technology of galactosidase and transglycosylation, which is applied in the field of rapid preparation of beta-galactosidase, can solve the problems of unfavorable galacto-oligosaccharide separation and purification, cumbersome purification process, low yield of enzyme protein, etc. Prospects for industrial application, simplified purification process, high yield effect

Inactive Publication Date: 2006-02-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The methods can be divided into two categories: one is to use purified enzymes for reaction, that is, to use purified or partially purified enzymes for reaction synthesis in lactose solution. This method has less environmental pollution and less uncertain factors, but the enzyme purification process is more cumbersome. , time-consuming and labor-intensive, and the yield of enzyme protein is low; the second is to use the complete cells of microorganisms to react, there are three methods: use the cells treated with toluene to produce galacto-oligosaccharides, but the residue of toluene will pollute the product; or directly use The cells produce galacto-oligosaccharides, but the reaction time is long (3 days) and the yield is low; or the method of cell fermentation is used, that is, the cells are inoculated in a lactose medium and cultured to obtain galacto-oligosaccharides, but the reaction time is long (3 days) ), and there are a large number of microbial metabolites and residual medium components in the fermentation broth, which is not conducive to the separation and purification of galacto-oligosaccharides

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Preparation of Crude Enzyme Liquid of Enterobacter cloacae B5CGMCC 1401 Transglycosyl β-galactosidase

[0023] Enterobacter cloacae (Enterobacter cloacae) B5CGMCC No.1401 (on June 30, 2005 was preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (abbreviation: CGMCC), and the preservation number is CGMCC No.1401) was inoculated into fresh slant After the medium is activated, connect a ring to 50mL of enzyme-producing fermentation medium, and culture it on a shaker at 37°C for 16 hours at a speed of 180 rpm, or transfer to 500mL of the same enzyme-producing fermentation medium after cultivation for expansion. , after the end of the culture, centrifuge at 12,000 rpm for 3 minutes to harvest Enterobacter cloacae cells.

[0024] The above slant medium formula: lactose 10g / L, peptone 5g / L, yeast powder 10g / L, NaCl 3g / L, agar 20g / L, pH 7.5;

[0025] The formula of the above enzyme-producing fermentation medium: l...

Embodiment 2

[0032]Example 2: Preparation of crude enzyme solution of Bacillus circulans ATCC 31382 transglycosyl β-galactosidase

[0033] Bacillus circulans (Bacillus circulans) ATCC 31382 strains were activated on the slant medium, then put one ring into 50mL enzyme-producing fermentation medium, cultured on a shaker at 37°C for 18 hours, and the shaker speed was 180 rpm, or After cultivation, transfer to 500mL of the same enzyme-producing culture medium to expand the cultivation. After the cultivation, centrifuge at 12,000 rpm for 2 minutes to obtain bacterial cells.

[0034] Above-mentioned slant culture medium formula is the same as embodiment 1.

[0035] The formula of the above enzyme-producing fermentation medium: lactose 10g / L, peptone 5g / L, yeast powder 10g / L, CaCl 2 0.11g / L, MnSO 4 0.001g / L, MgSO 4 ·7H 2 O 0.3g / L, KH 2 PO 4 0.05g / L, FeSO 4 ·7H 2 O 0.03g / L, pH 7.0.

[0036] Take 50 mg of the bacterial cells prepared above, add 250 μL of 50 mmol / L potassium phosphate b...

Embodiment 3

[0041] Example 3: Preparation of Aspergillus oryzae (Aspergillus oryzae) ATCC 20423 transglycosyl β-galactosidase crude enzyme solution

[0042] Aspergillus oryzae (Aspergillus oryzae) ATCC 20423 strain was activated by slant culture medium, then put a ring spore into 50mL enzyme-producing culture medium, and cultured on a shaker at 28°C for 40 hours, the shaker speed was 180 rpm, or after culture Then transfer to 500mL of the same enzyme-producing culture medium to expand the culture. After the culture is over, use filter paper (Shuangquan brand 101 rapid qualitative filter paper) to obtain bacterial cells.

[0043] Above-mentioned slope medium formula: potato juice (potato 200g / L), lactose 10g / L, agar 20g / L, natural pH;

[0044] The formulation of the above enzyme-producing fermentation medium: lactose 10g / L, peptone 5g / L, yeast powder 10g / L, NaCl 3g / L, pH 6.5.

[0045] Take 50 mg of bacterial cells, add 250 μL of 50 mmol / L sodium acetate buffer with pH 5.4 to wash once, re...

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PUM

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Abstract

The invention discloses a method for rapid preparation of transgalactosylation beta-galactosidase, which comprises the steps of (1) bacterial strain selection, (2) bacterial activating, (3) bacterial culturing and enzyme producing, (4) gathering bacterial cell, (5) washing, (6) making crude enzyme liquid of beta-galactosidase, (7) making galacto-oligosaccharides from the crude enzyme liquid, (8) carrying out quantitative analysis to the galacto-oligosaccharides.

Description

technical field [0001] The invention relates to a preparation method of transglycosyl beta-galactosidase, in particular to a rapid preparation method of beta-galactosidase which can be used to produce galactooligosaccharides. technical background [0002] Galactooligosaccharide is one of the natural components in breast milk, and its most important and fundamental physiological function comes from its effect on the proliferation of bifidobacteria. Because galactooligosaccharide has good heat stability and acid resistance, its health care effect has aroused great concern of people. At present, the production of galacto-oligosaccharides is mainly synthesized by microbial enzymatic methods, that is, β-galactosidase or lactase uses lactose as a substrate to transglycosylate synthesis. The methods can be divided into two categories: one is to use purified enzymes for reaction, that is, to use purified or partially purified enzymes for reaction synthesis in lactose solution. This...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38
Inventor 肖敏卢丽丽
Owner SHANDONG UNIV
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