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A kind of Klebsiella producing β-galactosidase and its application

A technology of Klebsiella and galactosidase, which is applied in the field of microorganism isolation and cultivation, can solve the problems of lack of resources of excellent natural strains and low enzyme activity of β-galactosidase, and achieve the same ability and strong transformation The effect of glycoside activity and simple operation

Inactive Publication Date: 2018-04-10
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme activity of β-galactosidase produced by the current natural enzyme-producing strains is generally low, and the resources of excellent natural strains are scarce, so it is of great significance to screen new excellent strains with high enzyme production

Method used

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  • A kind of Klebsiella producing β-galactosidase and its application
  • A kind of Klebsiella producing β-galactosidase and its application
  • A kind of Klebsiella producing β-galactosidase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Klebsiella B5582Y screening isolation and identification

[0054] 1. Klebsiella ( Klebsiella michiganensis ) B5582Y screening isolation

[0055] Weigh 0.1g of the soil sample collected near Jinlier Dairy Factory in Xinxiang City, Henan Province, put it into a 1.5mL PE small tube filled with sterile water and shake it evenly; then dilute it with sterile water to the volume concentration 1×10 -4 , 1×10 -5 Two gradient dilutions; use sterilized pipette tips to pipette 100μL of different gradient dilutions and spread them on the pre-prepared, sterilized and coated with 40mL 20mg / mL x-gal screening plate medium, at 37℃ Cultivate for 24-36h. Pick a single colony with a blue hydrolysis circle from the screening plate medium, separate the single colony by streaking, and check the part of the single colony in the microscopic examination to determine the purity of the strain (microscopic observation of the bacteria shows the same morphological characteristics) . ...

Embodiment 2

[0072] Example 2 Preparation and fermentation optimization of fermentation broth containing β-galactosidase and Klebsiella B5582Y

[0073] 1. Incline cultivation

[0074] Klebsiella B5582Y was inoculated on the slant medium and cultured at 37°C for 24 hours to obtain the slant strain.

[0075] 2. Liquid Proliferation Culture and Fermentation

[0076] Pick 2 loops of the obtained slant strain and insert them into the proliferation medium, and cultivate them at 37°C and 180rpm for 24 hours to obtain the proliferation bacteria liquid; the proliferation bacteria liquid is inserted into a 50mL basic fermentation tank with an inoculation amount of 2% (v / v). culture medium in a 250 mL Erlenmeyer flask at 37°C and 180 rpm for 24 hours to obtain a fermentation broth.

[0077] 3. Optimization of fermentation medium and fermentation conditions

[0078] On the basis of the basic fermentation medium, the type and concentration of carbon source, the type and concentration of nitrogen sou...

Embodiment 3

[0079] Example 3 Confirmation of β-galactosidase and measurement of enzyme activity

[0080] 1. Preparation of crude enzyme solution

[0081] Centrifuge the fermentation broth obtained in Example 2 at 4°C and 8000 rpm for 10 min, wash and centrifuge the cell pellet with PBS buffer solution (sodium salt) of pH 6.7, and then add the same volume of pH 6.7 PBS buffer solution, sonicate in an ice-water bath (500W, ultrasonic 1.5s, intermittent 2s) for 10min, then centrifuge at 4°C, 8000rpm for 10min, and take the supernatant as β-galactosidase crude enzyme solution.

[0082] 2. Determination of β-galactosidase hydrolase activity

[0083] Accurately weigh a certain amount o NPG powder, prepared 2 mg / mL with 0.05 mol / L, pH 6.7 PBS buffer o NPG solution; take 2mL o The NPG solution was placed in a test tube, incubated at 50°C for 10 min, and then added with 0.5 mL for proper dilution so that after the final reaction, the OD value of the solution to be tested was between 0.2 and 0....

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Abstract

The invention relates to a Klebsiella bacterium producing β-galactosidase and an application thereof, belonging to the technical field of microorganism separation and cultivation. The β-galactosidase-producing Klebsiella of the present invention is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, the preservation number is CGMCCNo.13035, and the preservation date is September 23, 2016. Klebsiella bacterium of the present invention is cultured on the screening plate culture medium plate at 37 DEG C for 24h, and the bacterial colony is circular, with a diameter of 2-5mm, convex in the middle, smooth surface, sticky, difficult to pick; after Gram staining Red, microscopically observed as Gram-negative bacteria Brevibacterium, no spore formation. The Klebsiella of the present invention can produce β-galactosidase; the produced β-galactosidase is an intracellular enzyme; the produced β-galactosidase has two activities of hydrolysis and transglycosidation. The Klebsiella bacterium of the present invention is continuously passed on for more than 10 generations, and the ability to produce β-galactosidase remains unchanged.

Description

technical field [0001] The invention relates to a Klebsiella bacterium producing β-galactosidase and its application, and belongs to the technical field of microorganism separation and cultivation. technical background [0002] β-galactosidase (β-galactosidase), full name β-D-galactohydrolase (β-D-galactosidegalcagalcato-hydrolase, EC.3.2.1.23), referred to as lactase. β-Galactosidase is widely used in food processing, medicine, immunity, and environmental monitoring, but it is mainly used in food. On the one hand, it can be applied to the production of dairy products to hydrolyze lactose, increase the sweetness of dairy products, improve the flavor, reduce the lactose content in dairy products, and then meet the needs of patients with lactose intolerance; due to the poor solubility of lactose at low temperature , so when the lactose content is reduced, it can effectively prevent crystallization of dairy products when frozen and improve the taste. On the other hand, when t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/38C12R1/22
CPCC12N9/2471C12Y302/01023C12N1/205C12R2001/22
Inventor 朱运平李秀婷郦金龙滕超范光森许丽亚黎芳
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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