Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof

A technology of galactosidase and screening method, applied in the field of biochemical industry, can solve the problem of strain separation without clear data, etc., and achieve the effects of excellent thermal stability and good stability

Inactive Publication Date: 2016-03-16
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Similar publication number is CN103614313A, which is called a strain and its screening method for fermenting high-temperature-resistant β-galactosidase, and the publication number is CN102250856A, which is called a heat-resistant β-galactosidase mutant. Construction, proposed the strains and their screening methods for fermentin

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  • Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof
  • Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof
  • Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof

Examples

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Embodiment 1

[0029] Example 1: Screening of strains producing thermostable β-galactosidase.

[0030] A kind of screening method of the bacterial strain that the present invention proposes produces high-temperature resistant β-galactosidase, it is characterized in that comprising following specific steps successively:

[0031] (1) Sample collection: Take some soil from hot springs and dairy farms;

[0032] (2) Primary screening: put 1g of soil sample in 10ml of distilled water, mix it evenly and do gradient dilution, pipette 120μL of bacterial solution and spread it evenly on the screening medium plate, and place it in a 30°C incubator for 24-48h; Pick a single colony with a blue hydrolysis circle on the screening medium plate and streak it, and streak it 3 times repeatedly to obtain a pure culture. Streak 1-3% on a solid medium at 35°C and culture at 35°C for 48 hours , take 3-5 single colonies and culture them in liquid medium at 35°C for 24 hours, take 1-3% of them and inoculate them in s...

Embodiment 2

[0036] Example 2: Morphological and physiological and biochemical characteristics of a strain producing thermostable β-galactosidase.

[0037] After the β-galactosidase producing bacterial strain obtained by the screening method of the present invention was cultivated on beef extract protein medium plate for 24 hours, the colony and the thalline morphology were observed, the surface was rough, the edges were irregular, the colony was dirty white, Gram Positive staining; the shape of the bacteria is short rod rod; the size of the bacteria is 0.65-0.7 μm and 2.5-3 μm; endophytic spores are formed in the center of the bacteria, and the bacteria do not expand after the formation of the spores; the bacteria have no capsule, Peripheral flagella, the bacteria have motility; Gram-positive; grow in liquid medium, form wrinkled ether; cellulolysis test is negative; in sugar fermentation test, the strain can utilize soluble starch, glucose, fructose , lactose, xylose, maltose and sucrose...

Embodiment 3

[0041] Example 3: The enzymatic characteristics of the thermostable β-galactosidase produced by the strain.

[0042] 1. The optimum temperature for β-galactosidase:

[0043] After fermentation, the enzyme liquid was centrifuged to remove bacteria, and the supernatant was taken as crude enzyme liquid for preliminary research on enzymatic properties. The optimal reaction temperature of the enzyme refers to the relative enzyme activity at different temperatures, the reaction pH 7.0, and the highest enzyme activity as 100. The results show that the optimum reaction temperature of the enzyme is 65°C, and at the same time, the enzyme has high catalytic activity and strong adaptability in a relatively high temperature range.

[0044] 2. Thermal stability of β-galactosidase:

[0045] Place the crude enzyme solution of β-galactosidase at different temperatures (30°C, 40°C, 50°C, 60°C, 70°C) and pH 7.0, keep it warm for 180min, and take samples every 30min to measure the enzyme activi...

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Abstract

The invention discloses a bacterial strain producing high-temperature-resistant beta-galactosidase and a screening method thereof and belongs to the technical field of biological chemistry and protein engineering. The screening method includes the specific and sequential steps of sample collection, primary screening, secondary screening of culture and beta-galactosidase activity assay. Mainly, soil of hot spring land is collected and boiled, then bacterial colonies are screened, and the technical problem how to produce high-enzyme-activity beta-galactosidase at high temperature is solved through a high-temperature culturing method. The bacterial strain is short in beta-galactosidase production period, high in beta-galactosidase production speed, good in genetic stability, thermal stability and pH stability, high in enzyme catalytic activity and high in production safety, and culturing conditions are simple. The bacterial strain has broad prospects in the fields of food industry and medical and biochemical analysis, in particular to the dairy industry.

Description

1. Technical field [0001] The invention relates to a strain producing high-temperature-resistant β-galactosidase and a screening method thereof, belonging to the technical field of biochemical engineering and protein engineering. 2. Background technology [0002] β-galactosidase (β-galactosidase), the scientific name is β-D-galactoside galactohydrolase (β-D-galactoside, galactohydrolase, EC3.2.1.23), the trade name is lactase (Lactase) , capable of catalyzing the hydrolysis of β-1,4-galactosidic linkages in lactose or lactose analogues. [0003] β-galactosidase is widely used in the fields of food industry, medicine and biochemical analysis, especially in the dairy industry. It is often used to hydrolyze lactose in milk and other dairy products, and can also be used to generate galacto-oligosaccharides. It is widely used in the production of low-lactose milk and non-crystallized concentrated milk. It degrades lactose into glucose and galactose to produce low-lactose milk. ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12Q1/34C12R1/125
CPCC12N1/02C12N1/20C12Q1/34C12Q2334/70
Inventor 王丹邹环泽包谢茹周小华
Owner CHONGQING UNIV
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