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Beta-galactosidase with cellulose adsorption zone and application thereof

A technology of galactosidase and cellulolytic enzyme, which is applied in the field of enzyme protein engineering, can solve the problems of unsatisfactory stability, high cost, and low recovery rate of enzyme activity, and achieve large-scale industrial application prospects, low cost, and high output Effect

Active Publication Date: 2010-12-01
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional immobilization methods can generally be divided into five types, namely adsorption, embedding, covalent bonding, cross-linking and microencapsulation, but the recovery rate of enzyme activity of these methods is generally low, and their stability is not ideal, and some immobilization The cost of the immobilized carrier is high, and these factors restrict the practical application of the immobilized enzyme to a certain extent.

Method used

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  • Beta-galactosidase with cellulose adsorption zone and application thereof
  • Beta-galactosidase with cellulose adsorption zone and application thereof
  • Beta-galactosidase with cellulose adsorption zone and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Obtaining a β-galactosidase gene with a cellulose adsorption domain

[0043] According to the principle of overlap extension PCR, the β-galactosidase gene sequence (GenBank Accession No.NC_008054) sequence of Lactobacillus delbrueckii subsp.bulgaricus ATCC NO.11842 and the sequence of Acetivibrio cellulolyticus (Acetivibrio cellulolyticus) ATCC NO.33288 cellulolytic enzyme CBM sequence (GenBank Accession No.AJ969241.1) and the restriction endonuclease sites BamH I and Xho I (shown underlined) on the plasmid pET-22b (+), design Upstream and downstream primers P1, P4 and linking primers P2, P3, with 10 overlapping regions on the linking primers (shown in bold font): the sequence is as follows:

[0044] P1: 5'-TC GGATCC GATGAGCAATAAGTTAGTAAAAG-3'

[0045] P2: 5'-TGCAAAGCCCTTTTAGTAAAAGGGGCTGAAT-3'

[0046] P3: 5'-TTTACTAAAAGGGCTTTGCACATACTTT-3'

[0047] P4: 5'-CG CTCGAG GTCAAACTCTACATAATATACTCCA-3'

[0048] The total DNA of Lactobacillus delbrueckii subsp...

Embodiment 2

[0049] Example 2: Preparation of β-galactosidase with cellulose adsorption domain

[0050] Transform pBga-CBM into Escherichia coli BL21(DE3), coat the transformed bacteria solution on an ampicillin LB plate, culture overnight at 37°C, select well-growing colonies, and use colony PCR to verify positive transformants, and add ampicillin Cultured in liquid LB medium, the recombinant Escherichia coli that fully meets the requirements is preserved as a strain.

[0051] Inoculate the above-mentioned preserved recombinant Escherichia coli strain into 3 mL of liquid LB culture solution added with ampicillin, culture overnight at 37°C, transfer 300 mL of new culture solution with 1% (v / v) inoculation amount, and reach the cell concentration OD 600 When it reached 0.8, the inducer IPTG was added to a final concentration of 0.5mmol / L, and induced at 30°C for 24 hours. Centrifuge at 10,000 rpm for 10 minutes to collect recombinant E. coli cells that have been induced to express recombin...

Embodiment 3

[0053] Example 3: Immobilizing β-galactosidase by immobilizing Bga-CBM on the surface of microcrystalline cellulose particles

[0054] Dilute the Bga-CBM crude enzyme solution obtained in Example 1 to an enzyme activity of 16U / mL with 50mmol / L pH7.0 potassium phosphate buffer containing 100mmol / L NaCl, adjust pH5.4 with phosphoric acid solution, press 1g Microcrystalline cellulose: 100U enzyme solution Add 6.4g cellulose to 40mL crude enzyme solution, stir and mix at room temperature at 70 rpm for 20 minutes, then centrifuge at 7000 rpm for 6 minutes, discard the supernatant, and use 40mL , 50mmol / L, pH5.4 acetic acid-sodium acetate buffer, resuspend the cellulose and wash twice, each time for 10 minutes, centrifuge at 7000 rpm for 6 minutes, discard the washing solution, and obtain the immobilized β-galactosidase Granules, stored in a refrigerator at 4°C.

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Abstract

The invention relates to beta-galactosidase and application thereof, in particular to a beta-galactosidase with a cellulose adsorption zone and application thereof in the production of galactooligosaccharide, and belongs to the technical field of enzyme protein engineering. The beta-galactosidase gene with the cellulose adsorption zone has a nucleotide sequence shown as SEQ ID No.1; and the beta-galactosidase coded by the gene has an amino acid sequence shown as SEQ ID No.2. The related GOS production method has the advantages of low cost, high yield, simple and reasonable steps, and the like, and has great industrialized application prospect.

Description

technical field [0001] The invention relates to a β-galactosidase and its application, in particular to a β-galactosidase with a cellulose adsorption domain and its application in the production of galactooligosaccharides, belonging to the technical field of enzyme protein engineering . technical background [0002] Galactooligosaccharides (GOS) is a functional oligosaccharide with natural properties. It exists in animals and human milk in nature. Its physiological role is mainly reflected in the promotion of Bifidobacterium and Lactobacillus The proliferation of (Lactobacillus) probiotics (Probiotics) promotes the establishment of a beneficial intestinal microenvironment and has a positive impact on maintaining human health. [0003] At present, commercialized GOS is prepared by microbial enzymatic method, that is, β-galactosidase (β-Galactosidase, Bga) with transglycosylation activity is used to synthesize GOS by transglycosylation with lactose as a substrate. Its molecul...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N9/38C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/70C12N11/12C12P19/14
Inventor 肖敏徐淑泽卢丽丽李正义
Owner SHANDONG UNIV
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