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Bacterium beta-galactosidase as well as encoding gene and application thereof

A technology of galactosidase and coding, which is applied in the field of microorganisms to achieve efficient hydrolysis

Pending Publication Date: 2017-07-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These reported β-galactosidases can only have good effects on lactose hydrolysis or galacto-oligosaccharide synthesis, and there are few reports of β-galactosidases with excellent performance in lactose hydrolysis and galacto-oligosaccharide synthesis

Method used

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  • Bacterium beta-galactosidase as well as encoding gene and application thereof
  • Bacterium beta-galactosidase as well as encoding gene and application thereof
  • Bacterium beta-galactosidase as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the preparation of recombinant β-galactosidase

[0067] 1. Construction of recombinant β-galactosidase-encoding gene expression plasmid

[0068] 1. Extract the genomic DNA of Paenibacillus barengerzia CAU904 and use it as a template to artificially synthesize PbBgal2ANheF: 5'-ctcag GCTAGC GTGGAACTGAACCGTGAATGG-3' (the underline is the restriction endonuclease NheI site) and PbBgal2AXhoR: 5'-gtcat CTCGAG CTATTCTTCTTCCTTAAATATCGGCTG-3' (the underline is the restriction endonuclease XhoI restriction endonuclease cutting site) is used as primer, carries out PCR amplification, obtains DNA fragment.

[0069] The reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 3 min, 35 cycles; extension at 72°C for 5 min.

[0070] 2. Digest the DNA fragment obtained in step 1 with restriction endonucleases NheI and XhoI, and recover the digested product.

[0071] 3. Digest the vector ...

Embodiment 2

[0084] Embodiment 2, the property of recombinant β-galactosidase

[0085] 1. Optimum pH value and pH stability of recombinant β-galactosidase

[0086] Buffer to be tested (both concentrations are 50mmol / L): acetic acid-sodium acetate buffer solution at pH 4.0, acetic acid-sodium acetate buffer solution at pH 4.5, acetic acid-sodium acetate buffer solution at pH 5.0, and acetic acid-sodium acetate buffer solution at pH 5.5 Acetic acid-sodium acetate buffer, acetic acid-sodium acetate buffer at pH6.0, MES buffer at pH5.5, MES buffer at pH6.0, MES buffer at pH6.5, MOPS buffer at pH6.0, MOPS buffer at pH6.5, MOPS buffer at pH7.0, MOPS buffer at pH7.5, MOPS buffer at pH8.0, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH6.0, pH6. 5 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH7.0 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH7.5 sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, pH8.0 Sodium dihydrogen pho...

Embodiment 3

[0092] Embodiment 3, the application of recombinant β-galactosidase

[0093] TLC detection method: Spot the sample on the TLC analysis plate to develop the layer twice, completely soak it with the color developing solution and dry it, and develop the color at 100°C. The developing agent is n-butanol / ethanol / water=5:3:2 (v / v / v), the chromogenic agent is 5% sulfuric acid methanol solution, and the mixture of standard products of glucose, galactose and lactose is used as standard control.

[0094] HPLC detection method 1: used to determine glucose content, galactose content and lactose content in lactose hydrolysis experiment, the chromatographic column used is cation exchange chromatographic column BP-800Pb 2+ (Benson Polymeric, Reno, NE, USA), the column temperature was 80 °C, the flow rate was 0.6 mL / min, the mobile phase was ultrapure water, and the injection volume was 20 μL. Calculated as follows:

[0095] Glucose content (g / L)=[glucose peak area (mAu*s)-21376] / 458.8 / 100...

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Abstract

The invention discloses bacterium beta-galactosidase as well as an encoding gene and an application thereof. A protein provided by the invention is a1) or a2) or a3) or a4), wherein a1) a protein with an amino acid sequence shown as a sequence 2 in a sequence stable; a2) a fused protein obtained by connecting an N end or / and a C end of the protein shown as the sequence 2 in the sequence stable with a label; a3) a protein with an amino acid sequence shown as a sequence 4 in the sequence stable; and a4) a protein with beta-galactosidase activity, which is obtained by carrying out replacement and / or deletion and / or addition of one or more amino acid residues on the protein shown as the a1) or the a2) or the a3). An experiment shows that the protein provided by the invention can be used for efficiently hydrolyzing lactose and can also be used for efficiently synthesizing galacto-oligosaccharides, and has important economic value and practical significance on production of low-lactose milk products and the galacto-oligosaccharides in a food industry.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a bacterial β-galactosidase, its coding gene and application. Background technique [0002] Dairy products are rich in nutrients such as protein and fat, and also contain about 4.5% lactose. Most Asians are lactose intolerant, limiting dairy consumption. β-galactosidase (β-galactosidase, EC 3.2.1.23) scientific name is β-D-galactohydrolase, trade name is lactase, which can hydrolyze lactose in dairy products, so that lactose intolerant people can consume . Some β-galactosidases also have the function of transgalactosyl conversion, that is, using lactose hydrolyzate or lactose as the glycosyl acceptor to synthesize galactooligosaccharides. Galactooligosaccharide is an important prebiotic, which can promote the proliferation of beneficial bacteria in the intestine, thereby regulating the intestinal flora. [0003] Microbial β-galactosidase has the advantages of large e...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12N1/21C12P19/14C12P19/04C12P19/00C12R1/19
CPCC12N9/2471C12P19/00C12P19/04C12P19/14C12Y302/01023
Inventor 江正强刘瑜闫巧娟陈洲张列兵
Owner CHINA AGRI UNIV
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