Immobilization method of beta-galactosidase and application of beta-galactosidase in preparation of low-lactose milk

A technology of galactosidase and immobilized enzyme, applied in the field of immobilized enzyme biocatalysis, can solve the problems of unsuitable food industry, toxicity and the like, and achieve the effects of good industrial application value, high hydrolysis rate and low cost

Pending Publication Date: 2020-10-13
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the current research on immobilized β-galactosidase has made progress, there are defects in varying degrees.
For example, the β-galactosidase immobilized with poly(GMA–MMA) hydrolyzes lactose for 60 hours, and the enzyme activ

Method used

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  • Immobilization method of beta-galactosidase and application of beta-galactosidase in preparation of low-lactose milk
  • Immobilization method of beta-galactosidase and application of beta-galactosidase in preparation of low-lactose milk
  • Immobilization method of beta-galactosidase and application of beta-galactosidase in preparation of low-lactose milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Optimization of the immobilization conditions of β-galactosidase

[0027] Now use phosphate buffer to prepare 23.2mM sodium alginate solution, add 1mL enzyme solution (enzyme concentration 0.04mg / mL) to 1mL sodium alginate solution, put it in an ice bath (0°C), and run at 50-60rpm stirring speed. Use a syringe to draw the mixed solution and add it dropwise to 20mL 90.1mM CaCl at a rate of 2d / s 2 In solution, calcium alginate beads were formed. After obtaining the immobilized enzyme particles, continue to stand at room temperature for 1-2 hours to make them harden, and finally wash the particles with 50mM pH 6.5 phosphate buffer to remove unbound enzyme molecules and obtain the immobilized enzyme of β-galactosidase , stored in the refrigerator at 4°C. Among them, the concentration of sodium alginate, CaCl 2 Concentration and initial enzyme amount need to be optimized, such as figure 1 , figure 2 , image 3 As shown, the results show that when the conce...

Embodiment 2

[0028] Example 2: Determination of immobilized β-galactosidase activity

[0029] β-galactosidase can specifically hydrolyze the β-1,4-glycosidic bond in o-nitrophenyl-β-D glucopyranoside (oNPG), releasing o-nitrophenyl (oNP) and glucose, oNP It is yellow under alkaline conditions. Under a certain range of conditions, the concentration of oNP and its OD 420 There is a certain linear relationship between the absorbance value. With oNPG as the substrate, the enzyme activity unit (U) definition: under the optimal action conditions, the amount of enzyme required to catalyze the hydrolysis of oNPG to generate 1umol oNP per minute is 1 U. The measured oNP curve of o-nitrobenzene is: Y=2.1286X-0.0023, R 2 = 0.9999. Among them, Y is OD 420 The absorbance value of X is the concentration of oNP in the system (mM).

[0030] With 50mM, pH 6.5 citrate-Na 2 HPO 4 Prepare 50mM oNPG solution in the buffer as the substrate, mix 1mL of the substrate solution with 0.12U / mL immobilized enzy...

Embodiment 3

[0031] Embodiment 3: Optimum temperature, pH determination of immobilized enzyme

[0032]Set the temperature gradient (50°C, 55°C, 60°C, 65°C, 70°C, 75°C), set three parallels for each treatment, measure the enzyme activity at each temperature under the condition of pH 6.5, and draw the optimum temperature curve. Such as Figure 4 As shown, the optimum reaction temperature of the immobilized enzyme was found to be 65°C, which was the same as that of the free enzyme.

[0033] Prepare 50mM pH5-7.5 pH series buffers, dissolve the substrate oNPG in the pH series buffers, set up three parallels for each, measure enzyme activities at different pHs at 65°C, and draw the optimum pH curve. Such as Figure 5 As shown, the optimum pH of the immobilized enzyme was measured to be 6.5, which was the same as that of the free enzyme.

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Abstract

The invention discloses an immobilization method of beta-galactosidase and an application of the beta-galactosidase in preparation of low-lactose milk, and the immobilized beta-galactosidase is prepared by taking sodium alginate as an immobilization carrier. The enzyme activity of the obtained immobilized beta-galactosidase is up to 438 U/mg; when the immobilized beta-galactosidase is used for hydrolyzing lactose in milk, 64% of lactose can be hydrolyzed only in 40 min, and after the immobilized beta-galactosidase is recycled four times, the lactose hydrolysis rate can still be kept 60% or above. The preparation method has the advantages of simple process, low cost, high speed, high hydrolysis rate and good industrial application value.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzyme biocatalysis, and in particular relates to a method for immobilizing β-galactosidase and its application in preparing low-lactose milk. Background technique [0002] β-galactosidase (β-galactosidase, EC.3.2.1.23), the full name is β-D-galactoside galactohydrolase, and the trade name is lactase. The enzyme can hydrolyze galactosidic bonds and degrade lactose into glucose and galactose. Therefore, it can be used to produce low-lactose milk and provide nutrition for people with lactose intolerance. [0003] The catalytic activity of most β-galactosidases is low, and the efficiency of hydrolyzing lactose in milk is not high. For example, the free β-galactosidase in Chinese invention patent application 201110170088.8 hydrolyzes lactose for 1 hour at an optimum temperature of 55-60°C, and the hydrolysis rate of lactose is only 32%; the free β-galactosidase in Chinese invention patent applica...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04C12N9/38A23C9/12
CPCC12N11/10C12N11/04C12N9/2471A23C9/1206C12Y302/01023
Inventor 彭惠华珍孔慧慧高毅
Owner ANHUI UNIVERSITY
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