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Mutant of Beta-galactosidase as well as preparation method and application thereof

A galactosidase and mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of only 19% yield, limited application, poor probiotic effect, etc., and achieves high industrial production value and strong transglycosidic activity.

Active Publication Date: 2018-04-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the yield of B.circulans (circulans) is higher, which is 48.3%, the product of its production of GOS is mainly 4'GalLac, and its prebiotic effect is poor
As a food-safe strain of A.oryzae, the main product of GOS produced is 6'GalLac, and 6'GalLac has better prebiotic effect than 4'GalLac, but its yield is only about 19%, which greatly limits its application

Method used

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  • Mutant of Beta-galactosidase as well as preparation method and application thereof
  • Mutant of Beta-galactosidase as well as preparation method and application thereof
  • Mutant of Beta-galactosidase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: the preparation of single mutant and wild enzyme

[0043]The β-galactosidase gene was connected with pMD19-T simple to obtain the AorE / pMD19-T simple plasmid, which was used as a template and Sac1m-F and Sac1m-R were used as primers for mutation. The above PCR product was digested with Dpn1, and the resulting plasmid was transformed into Escherichia coli JM109 competent cells to obtain the AorE-M / pMD19-T simple plasmid.

[0044] Sac1m-F: TCCACAAGATC AGG GCTCTTGGTTTCAAC (the mutation site is underlined)

[0045] Sac1m-R: GTTGAAACCAAGAGC CCT GATCTTGTGGA (the mutation site is underlined)

[0046] 1. Preparation of wild enzyme: AorE-M / pMD19-T simple plasmid and pPIC9k vector were digested with Snab1 and Not1, the digested product was ligated with T4 ligase, the ligated product was transferred into Escherichia coli JM109 competent cells, and the bacteria were picked The plasmid was extracted, and the plasmid was named AorE-M / pPIC9k.

[0047] Preparation ...

Embodiment 2

[0060] Example 2: Construction of double mutants.

[0061] Using the plasmid of the mutant N140C constructed in Example 1 as a template for double mutation, and according to the primers for site-directed mutation designed in Example 1, using rapid PCR technology, the plasmid carrying the gene encoding the mutant N140C was subjected to site-directed mutation to construct Double mutants N140C / F264W and N140C / W806F. Sequence separately to confirm whether the coding gene of the β-galactosidase double mutant is correct, and carry out Snab1 and Not1 double digestion of the plasmid with the correct sequencing result and the pPIC9k vector, and connect the digested product with T4 ligase, and transfer the ligated product into the large intestine Bacillus JM109 competent cells were picked to extract the correct plasmid and transformed into yeast KM71 cells to obtain recombinant bacteria AorE-M / pPIC9k-N140C / F264W and AorE-M / pPIC9k-N140C / W806F capable of expressing double mutant genes.

Embodiment 3

[0062] Example 3: Preparation of Wild Bacteria and Mutant β-galactosidase Enzyme Solution

[0063] The recombinant bacteria AorE-M / pPIC9k / KM71, AorE-M / pPIC9k-N140C, AorE-M / pPIC9k-F264W, AorE-M / pPIC9k-F304Q, AorE-M / pPIC9k-W806F, AorE-M / pPIC9k-N140C / F264W and AorE-M / pPIC9k-N140C / W806F were respectively transferred to BMGY liquid medium, and cultured at 30°C for 24 hours to obtain the inoculum solution. The above bacterial liquid was centrifuged, all the bacterial cells were transferred into 50 mL BMMY liquid medium, cultured at 30° C. for 5 days, and the final concentration of 0.75% methanol was added every 24 hours to induce enzyme production. The fermented liquid is centrifuged, and the supernatant is crude enzyme liquid.

[0064] The enzymatic activity of the crude enzyme solution was determined, and the ONPG hydrolase activity of wild-type β-galactosidase (WT) and mutants is listed in Table 1.

[0065] Table 1 Enzyme activity of wild-type β-galactosidase and mutant enzyme...

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Abstract

The invention discloses a mutant of Beta-galactosidase as well as a preparation method and application thereof, and belongs to the fields of gene engineering and enzyme engineering. Through mutating an amino acid at a specific locus in the Beta-galactosidase, transferring into a recombinant bacterium and then carrying out enzymatic conversion in an optimized condition, a yield of galactooligosaccharide generated by the mutant reaches 59.8 percent, and is increased by approximately 70 percent compared with that of a wild enzyme; the improvement of the yield of the galactooligosaccharide is realized, and the mutant has extremely high industrialized application.

Description

technical field [0001] The invention relates to a beta-galactosidase mutant and its preparation method and application, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Galacto-oligosaccharide (GOS) is a functional oligosaccharide, which is widely used in the food industry. As a new type of functional food additive, GOS has attracted worldwide attention due to its unique physiological functions and excellent physicochemical properties. With the continuous deepening of GOS development and research, coupled with the abundance of raw materials in my country and the unlimited potential of the consumer market, the production of GOS has set off a strong momentum in the country. At present, the production of oligosaccharides in my country is still an emerging industry, and the development of GOS has not yet reached a large scale. The main constraint on the production of GOS in my country is the lack of industrial enzymes with exc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12P19/14
CPCC12N9/2471C12P19/14C12Y302/01023A23L33/21A23C9/1206C12N15/66C12N15/81
Inventor 吴敬吴丹高鑫
Owner JIANGNAN UNIV
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