Novel beta-galactosidase and application thereof in degrading lactose in milk

A technology of galactosidase and galactose, which is applied to a new type of beta-galactosidase and its application field in degrading lactose in milk, can solve problems such as being difficult to achieve, achieve large output, good prospects for industrial application, purification simple method effect

Inactive Publication Date: 2020-10-30
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Treatment for lactose intolerance mainly involves reducing or eliminating the amount of lactose in your diet, but this is difficult to achieve because lactose is found in dairy products and is even commonly used as a food additive

Method used

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  • Novel beta-galactosidase and application thereof in degrading lactose in milk
  • Novel beta-galactosidase and application thereof in degrading lactose in milk
  • Novel beta-galactosidase and application thereof in degrading lactose in milk

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Sequence analysis and recombinant expression of β-galactosidase Gal42

[0024] The enzyme-producing gene gal42 of the β-galactosidase Gal42 in the present invention is derived from the marine bacterium Bacillussp.BY02, contains 2010 base sequences, and encodes 669 amino acid sequences. The marine bacterium Bacillus sp.BY02 is derived from sea mud samples, collected from the coast of the Yellow Sea, separated and purified using the lactose screening model, and using blue-white spots. Using the conserved domain analysis of the National Center for Biotechnology Information (NCBI) to analyze the Conserved domain (CDD) and the multiple sequence alignment Basic Local Alignment Search Tool (Blast), it was found that the sequence contained a β-galactose of the polysaccharide hydrolase GH family Conserved region of glycosidase. Among the reported β-galactosidases, the one with the highest amino acid sequence similarity to Gal42 is the β-galactosidase (GenbankWP_053430...

Embodiment 2

[0033] Preparation and purification method of embodiment 2β-galactosidase Gal42

[0034] Culture the recombinant strain BL21(DE3) / pET28a-Gal42 in 100 mL of LB liquid medium (50 μg / mL kanamycin) in a shaker at 37°C at 150 rpm to OD 600 =0.6, add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and induce at 20° C. for 24 hours. The method for measuring β-galactosidase activity is: add 450 μL 10 mM ONPG (200 mM phosphate buffer, pH=8.0) to 50 μL enzyme solution, react at 40 ° C for 10 min, add 500 μL Na 2 CO 3 (1 mM) to terminate the reaction, the mixture was centrifuged at 8000 rpm for 10 minutes, and the absorbance was detected at OD420. Enzyme activity is defined as 1 U is the amount of enzyme required to produce 1 μM ONP per min. After testing, the activity of β-galactosidase in the fermentation broth can reach 306.3U / mL.

[0035] After the fermentation stopped, centrifuge at 12000rpm for 10min, discard the supernatant, collect the bac...

Embodiment 3

[0036] Embodiment 3 Temperature and the influence of pH on β-galactosidase Gal42

[0037] The β-galactosidase Gal42 purified in Example 2 was tested for enzyme activity under different conditions to detect the effects of different temperatures and pH on the enzyme activity. React at different temperatures (0-70° C.) for 10 min, detect the effect of different reaction temperatures on enzyme activity, and calculate the relative enzyme activity of Gal42 at different temperatures with the highest enzyme activity as 100%. like figure 2 As shown in A, the optimal reaction temperature of β-galactosidase Gal42 is 40°C.

[0038] The β-galactosidase Gal42 purified in Example 2 was incubated at different temperatures (0-70°C) for 1 hour, and immediately after taking it out, its enzyme activity was detected at its optimum reaction temperature (40°C) to measure the enzyme activity before incubation. Vitality as 100%, such as figure 2 As shown in B, the temperature stability of β-galac...

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Abstract

The invention relates to a novel beta-galactosidase and application thereof in degrading lactose in milk. The amino acid sequence of the beta-galactosidase disclosed by the invention is as shown in SEQ ID NO. 1. The yield of the beta-galactosidase can reach 306.3 U / mL, and the optimal reaction temperature and pH are 40 DEG C and 7.5 respectively. The beta-galactosidase provided by the invention shows wide pH stability in a pH range of 6.0 to 9.0, and is suitable for hydrolysis of lactose in milk. Moreover, the beta-galactosidase is added into the enzyme-containing milk product, so that a functional milk product beverage suitable for children to drink can be produced. The beta-galactosidase disclosed by the invention is high in yield and is an ideal choice for producing lactose-free dairy products.

Description

technical field [0001] The invention relates to a novel beta-galactosidase and its application in degrading lactose in milk, belonging to the field of biotechnology. Background technique [0002] β-galactosidase (EC 3.2.1.23, lactase) is an important medical enzyme that regulates the hydrolysis of lactose by cleaving the terminal non-reducing β-D-galactose component. β-galactosidase could be a potential treatment option for lactose intolerance. Lactose intolerance is currently defined as a clinical syndrome characterized by pain, bloating, gas, and diarrhea following ingestion of lactose. Lactose is the most common disaccharide in human nutrition today, both in breastfed infants and adults, but its digestion requires a special enzyme, lactase. During adulthood, a genetically programmed reduction in lactase activity affects the majority of adults in the world, with approximately two-thirds of the world's population lactose intolerant due to lactase deficiency. Treatment fo...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12N15/70A23C7/04A23C9/12
CPCA23C7/04A23C9/1206C12N9/2471C12N15/70C12Y302/01023
Inventor 夏玉军
Owner QINGDAO UNIV
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