Heat-resistant beta-galactosidase and application thereof to lactose degradation

A technology of galactosidase and galactose, which is applied in the field of heat-resistant β-galactosidase and its application in lactose degradation, can solve the problems of poor heat resistance and low yield, and achieve simple purification methods and excellent physical and chemical properties , the effect of wide temperature stability

Active Publication Date: 2021-10-29
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a lot of research work has been done on lactase at home and abroad, but it has not been widely used, the main reasons include low yield and poor heat resistance

Method used

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  • Heat-resistant beta-galactosidase and application thereof to lactose degradation
  • Heat-resistant beta-galactosidase and application thereof to lactose degradation
  • Heat-resistant beta-galactosidase and application thereof to lactose degradation

Examples

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Embodiment 1

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

[0024] The enzyme-producing gene Gal-T of the β-galactosidase Gal-T in the present invention is derived from the marine bacterium Baciliiussp.BY57, which contains a sequence of 3750 bases and encodes a sequence of 1250 amino acids. 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-galactoside of the polysaccharide hydrolase GH family Enzyme conserved region. Among the reported β-galactosidases, the one with the highest amino acid sequence similarity to Gal-T is the β-galactosidase (Genbank AJ316559.1) of the polysaccharide hydrolase family 42 (GH42). The amino acid sequence similarity (Identity) is 75.75%. The β-galactosidase Gal-T described in this invention belongs to the polysacch...

Embodiment 2

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

[0034] Culture the recombinant strain BL21(DE3) / pET20b-Gal-T in 100 mL of LB liquid medium (100 μg / mL ampicillin) in a shaker at 37°C at 200 / min 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 the activity of β-galactosidase is: add 450 μL 10 mM o-nitrophenol-β-D-galactoside (ONPG) substrate (20 mM phosphate buffer, pH=8.0) to 50 μL enzyme solution, at 40 ° C After reacting for 10 min, add 500 μL of Na 2 CO 3 The reagent terminates the reaction. The mixture was centrifuged at 10,000 rpm for 10 min, and its 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 chitosanase activity in the fermentation broth can reach 460.4U / mL.

[0035] After the fermentation stopped, centrifuge at 12...

Embodiment 3

[0036] Optimum temperature and pH determination of embodiment 3β-galactosidase Gal-T

[0037] The β-galactosidase Gal-T 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 (0-80° C.) for 10 min, detect the effect of different reaction temperatures on enzyme activity, and calculate the relative enzyme activity of Gal-T at different temperatures with the highest enzyme activity as 100%. like figure 2 As shown in A, the optimal reaction temperature of β-galactosidase Gal-T is 45°C.

[0038] The β-galactosidase Gal-T purified in Example 2 was reacted with the lactose substrate of the Britton-Robinson buffer system (PH5.5-10.5). The buffer consists of phosphoric, boric and acetic acids, to which different amounts of sodium hydroxide can be added to form a buffered solution with a wide pH range. The activity was detected at the optimum temper...

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Abstract

The invention relates to heat-resistant beta-galactosidase and an application thereof to lactose degradation. The amino acid sequence of the beta-galactosidase is as shown in SEQ ID NO. 1. The beta-galactosidase Gal-T is a lactose hydrolase with a novel structure and functions, the similarity between the amino acid sequence of the beta-galactosidase Gal-T and the reported beta-galactosidase sequence is only 75.75%, and the beta-galactosidase Gal-T is a new enzyme. The yield of the beta-galactosidase can reach 460.4 U/mL, the optimum reaction temperature is 45 DEG C, and the optimum reaction PH is 7.5. The beta-galactosidase disclosed by the invention has heat resistance, and shows wide temperature stability in a temperature range of 40 DEG C to 60 DEG C. The beta-galactosidase disclosed by the invention is high in yield and good in thermal stability, and has excellent industrial application potential.

Description

technical field [0001] The invention relates to a heat-resistant β-galactosidase and its application in lactose degradation, belonging to the field of biotechnology. Background technique [0002] Lactose is a common disaccharide in human nutrition at present. It can be absorbed and utilized by the human body after being hydrolyzed into monosaccharide by lactase. Lactose intolerance is a disease caused by indigestion of lactose, mainly due to the lack of lactase in the organism or its low activity. The incidence rate of lactose malabsorption among Chinese adults is as high as 86.7%. [0003] β-galactosidase (EC 3.2.1.23, lactase) is an active enzyme that hydrolyzes β-D-galactosidic bonds to produce free D-galactose. It widely exists in microorganisms, animals and plants, and can be applied in the fields of medicine, dairy products, biology and environmental protection. β-Galactosidase can also be used to make galactooligosaccharides (oligosaccharides with galactose residue...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12P19/14C12P19/00
CPCC12N9/2471C12P19/14C12P19/00C12Y302/01023
Inventor 何宁宁周子涵宋静宜
Owner QINGDAO UNIV
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