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A strain fermenting and producing high-temperature-resistant β-galactosidase and its screening method

A technology of galactosidase and high temperature resistance, applied in the field of bioengineering, can solve the problems of high cost, low enzyme activity, difficult to meet the market and production needs, etc., to achieve unchanged synthesis ability, stable enzyme activity, and excellent thermal stability sexual effect

Inactive Publication Date: 2016-03-02
XUZHOU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many literatures have reported the schemes of screening and isolating various β-galactosidase strains, such as yeast, lactic acid bacteria, Bacillus, and Aspergillus, etc., most of the β-galactosidases currently used in production are The optimum temperature is about 30°C, the enzyme activity is very low at high temperature, and the cost is high, it is difficult to meet the needs of the market and production

Method used

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  • A strain fermenting and producing high-temperature-resistant β-galactosidase and its screening method
  • A strain fermenting and producing high-temperature-resistant β-galactosidase and its screening method
  • A strain fermenting and producing high-temperature-resistant β-galactosidase and its screening method

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Effect test

Embodiment 1

[0026] Example 1: Screening of strains producing thermostable β-galactosidase.

[0027] The present invention proposes a method for screening strains that ferment and produce high temperature resistant β-galactosidase, which is characterized in that the following specific steps are included in sequence:

[0028] (1) Enrichment culture: Take 1g of soil sample and grind it into fine powder, add it to a 250ml Erlenmeyer flask with 80ml sterile water, add sterile glass beads, shake it in a shaker at 150r / min for 20min, then shake it in a water bath at 80°C Shake in the bed for 30min, take 1-2ml into 50mL enrichment medium, 40 ℃ constant temperature water bath shaking culture for 36h-48h, the shaking frequency is 150-180r / min; wherein, the enrichment medium is: lactose 2g, yeast powder 0.5 g, peptone 1g, NaCl 0.5g and distilled water 100ml, conditions: pH6.5, sterilization temperature 121℃, sterilization time 20min;

[0029] (2) Preliminary screening: Dilute the enriched culture m...

Embodiment 2

[0034] Example 2: Morphology and physiological and biochemical characteristics of thermostable β-galactosidase-producing strain F69.

[0035] After the β-galactosidase-producing strain obtained by the screening method of the present invention is cultured on a beef extract peptone medium for 18-24 hours, the colony and the shape of the bacterium are observed. The surface is rough, the edges are irregular, the colony is dirty white, and the leather The lantern staining was positive; the shape of the cell was short rod-shaped; the size of the cell was 0.65-0.7μm×2.5-3μm; the endospore formed in the center of the cell, and the cell did not expand after the spore was formed; the cell had no capsule , Peripheral flagella, the cells are motile; Gram-positive; Grow in liquid medium and form wrinkle; Negative in cellulolysis test; In sugar fermentation experiments, the strain can utilize soluble starch, glucose, Fructose, lactose, xylose, maltose and sucrose are the only carbon sources...

Embodiment 3

[0038] Example 3: Sequence analysis of 16S rDNA of β-galactosidase-producing strain F69.

[0039] Extract the genome of the strain F69 of the present invention, and design primers according to the most conserved sequence in bacterial 16SrDNA, wherein the forward primer is F: 5'-AGAGTTTGATCCTGGCTCAG-3'; the reverse primer is R: 5'-AAGGAGGTGATCCAGCCGCA-3'; PCR reaction system 50 μL; thermal cycling parameters: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 2 min, 35 cycles, and extension at 72 °C for 7 min. The measured sequence was compared with the 16S rDNA sequence of similar bacteria obtained from NCBI for multiple sequence alignment using ClustalX1.83, and then the MEGA4.0 biology software was used to construct a phylogenetic tree. The results are as follows figure 2 shown, the 16S rDNA nucleotide sequence of strain F69 is shown to be the same as Bacillussubtilis (AJ276351) has the highest 16SrDNA ...

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Abstract

The present invention relates to a strain for producing high temperature resistance beta-galactosidase through fermentation, and a screening method thereof. The strain F69 has a classification name of Bacillussubtilis, is preserved in the China General Microbiological Culture Collection Center on January 9, 2012, and has a preservation number of CGMCC No.5699. The screening method specifically and sequentially comprises: enrichment culture, primary screening, strain purification, re-screening, strain preservation, and beta-galactosidase activity determination. The strain has characteristics of short beta-galactosidase production fermentation period, rapid beta-galactosidase production, good genetic stability, good pH value stability, strong catalytic activity, simple culture condition and high production safety, and has broad application prospects in industrial fields of food, feeds and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a strain producing high temperature resistant β-galactosidase and a screening method thereof. Background technique [0002] β-galactosidase (β-galactosidase, EC3.2.1.23), scientific name β-D-galactoside galactohydrolase, trade name lactase, can catalyze β- D -Galactopyranoside hydrolysis or transglycosylation, commonly used to hydrolyze lactose in milk and other dairy products, can also be used to generate galactooligosaccharides, widely used in the production of low lactose milk and non-crystalline concentrated milk. The problem of lactose intolerance that exists in more than half of the world's population, β-galactosidase also has a galactosidase effect and is used to produce bifidogenic factor-galacto-oligosaccharides, which is widely used in probiotic food production. The important application value of β-galactosidase in food has attracted extensive attent...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/38C12R1/125
Inventor 高兆建李勇张桂英张建萍张家路司瑞寒田圣英
Owner XUZHOU UNIV OF TECH
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