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Beta-galactosidase mutant and related biological material and application thereof

A technology of galactosidase and biomaterials, applied in β-galactosidase mutants and related biomaterials and application fields, can solve problems such as narrow application range, poor heat resistance, and inability to meet the needs of the food industry, and achieve cost Inexpensive, short incubation time, good industrial application prospects

Inactive Publication Date: 2016-07-13
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the lactase sold on the market is mainly obtained through the expression of yeast system. Due to poor heat resistance and narrow application range, it cannot meet the various needs of the food industry.

Method used

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  • Beta-galactosidase mutant and related biological material and application thereof
  • Beta-galactosidase mutant and related biological material and application thereof
  • Beta-galactosidase mutant and related biological material and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, the acquisition of β-galactosidase mutant and its coding gene

[0053] In order to improve the heat resistance of wild-type β-galactosidase (named β-Gal-1), the following positions of the amino acid sequence of β-Gal-1 were mutated: the amino group at position 523 was mutated from serine to Arginine (S523R); deletion of amino acid residues 512-533 (Del(512-533)); mutation of amino acid 537 from lysine to asparagine (K537N); amino acid 414 from glycine Mutation to cysteine ​​(G414C). Only amino acid 523 (S253R) was selectively mutated. The amino acid residues at position 512-533 were selectively deleted (Del(512-533)) and the amino acid at position 537 was mutated (K537N). Amino acid residues 512-533 were selectively deleted (Del(512-533)) and amino acids 537 and 414 were mutated (K537N and G414C). The specific wild-type β-galactosidase mutants are β-Gal-2, β-Gal-3 and β-Gal-4, respectively, and the specific protein mutation sites are shown in Table 1. ...

Embodiment 2

[0061] The preparation of embodiment 2, β-galactosidase

[0062] 1. Construction of β-galactosidase recombinant expression vector

[0063] Use NcoI and KpnI to double-enzyme digest the DNA molecule shown in SEQIDNo.8, the DNA molecule shown in SEQIDNo.2, the DNA molecule shown in SEQIDNo.4 and the DNA molecule shown in SEQIDNo.6 respectively, and obtain the DNA molecules containing β-Gal respectively - DNA fragments of genes encoding 1, β-Gal-2, β-Gal-3 and β-Gal-4, will contain β-Gal-1, β-Gal-2, β-Gal-3 and β-Gal The DNA fragment of the coding gene of -4 was recovered and purified.

[0064] The purified DNA fragments containing the coding genes of β-Gal-1, β-Gal-2, β-Gal-3 and β-Gal-4 were combined with the lactic acid bacteria vector pNZ8149 obtained by NcoI and KpnI double enzyme digestion of lactic acid bacteria vector pNZ8149 The large fragments were ligated, transformed into DH5α competent cells, and positive clones were selected by restriction enzyme digestion. The r...

Embodiment 3

[0070] Embodiment 3, the biological characteristic detection of β-galactosidase

[0071] 1. Determination of enzyme activity of β-galactosidase

[0072] The enzyme activities of the four β-galactosidases in Example 2 were measured by measuring the amount of nitrophenol released by hydrolyzing the substrate ONPG. The four purified β-galactosidases obtained in Example 2 were diluted 100 times. Take 100 μL of the diluted β-galactosidase dilution and 1.8 mL of 50 mM citric acid-disodium hydrogen phosphate buffer (pH6.5), preheat at 37 ° C for 5 min, and then add 100 μL of 20 mM ONPG for reaction. After 10 min of reaction, add 1 mL of Na 2 CO 3 solution (1mol / L), the reaction terminated.

[0073] The release of nitrophenol was quantitatively detected by measuring the A420 light absorption value. The experiment was repeated 3 times. The unit of β-galactosidase activity is defined as the amount of enzyme required to decompose the substrate to generate 1 μmol of nitrophenol per ...

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Abstract

The invention discloses a beta-galactosidase mutant and a related biological material and application thereof.The provided beta-galactosidase mutant is called beta-Gal-2 and shown as A1) or A2), A1) represents protein with the amino acid sequence shown as SEQ ID No.1, and A2) represents protein with beta-galactosidase activity, wherein one or more amino acid residues are substituted and / or deleted and / or added into the amino acid sequence of the protein A1).The mutant has the advantages of being high in adaptability and heat resistance and good in hydrolysis capacity, and influence on enzymatic activity by metal ions is small.The characteristic of heat resistance is extremely prominent, and enzymatic activity of the mutant at the temperature of 60 DEG C can be kept at 80% or more.An extra poisonous inductive agent is not needed when lactobacilli are used as a host to express the mutant, cost is low, expression quantity is large, and the beta-galactosidase mutant has good industrial application prospects.

Description

technical field [0001] The invention relates to a beta-galactosidase mutant in the field of biotechnology and related biological materials and applications. Background technique [0002] β-galactosidase (β-galactosidase), commonly known as lactase, exists widely in various animals, plants and microorganisms. This enzyme can hydrolyze lactose into glucose and galactose, and also has the transfer effect of galactoside. The main component of milk and whey is lactose, which accounts for about 30% of the dry matter of milk, but its solubility is low, only 20% in water at 20°C, and its sweetness is only 16% of that of sucrose. Due to the precipitation of lactose, dairy products have a sandy feeling and affect the flavor. In addition, many adults, especially infants (2-8% in Western Europe, 60-90% in Asia and Africa) lack lactase in their bodies, so it is difficult for them to digest milk. After drinking milk, lactose enters the intestinal tract. It is decomposed and fermented b...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12P19/14C12P19/02
CPCC12N9/2471C12P19/02C12P19/14C12Y302/01023
Inventor 李相阳陈湘宁张红星
Owner BEIJING UNIV OF AGRI
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