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A cellobiose epimerase mutant and its application in producing lactulose

A technology of epimerase and cellobiose, applied in the field of genetic engineering, can solve problems such as doubtful safety

Active Publication Date: 2020-08-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, Escherichia coli and Pichia pastoris are not food-safe strains. If the recombinant enzymes obtained from these strains are to be further used in the food field, their safety is questionable.

Method used

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  • A cellobiose epimerase mutant and its application in producing lactulose
  • A cellobiose epimerase mutant and its application in producing lactulose
  • A cellobiose epimerase mutant and its application in producing lactulose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Recombinant plasmid pBSMuL3-ce (-)build process

[0035] The cellobiose epimerase (NCBI: WP_012547288.1) gene derived from thermophilic anaerobic bacteria was synthesized, and the gene fragment was connected to the pET-24a vector to obtain the plasmid pET-24a-ce. Using the plasmid pET-24a-ce as a template, the forward and reverse primers were designed as F1 / R1 (sequence information shown in SEQ ID NO.3 and SEQ ID NO.4, respectively) to amplify the CE gene fragment. The target fragment ce obtained by PCR was ligated with the pMD-19T Simple vector and transformed into E.coli JM109, and the single-clonal transformant was selected and cultured in LB liquid medium for 8-10 hours, the plasmid was extracted, and the double-enzyme digestion was verified to perform gene sequencing. The correctly sequenced plasmid was designated as pMD-19T-ce. Plasmid pMD-19T-ce was double digested with restriction endonucleases HindⅢ and BamHI, and the ce fragment was obtained by ge...

Embodiment 2

[0036] Embodiment 2: Recombinant plasmid pBSMuL3-ce (-) transformation

[0037] Plasmid pBSMuL3-ce (-) Transformed into Bacillus subtilis CCTCC NO:M2016536 prepared in advance to obtain genetically engineered bacteria Bacillus subtilis CCTCC NO:M 2016536 / pBSMuL3-ce (-) , spread on LB plates containing kanamycin (100 μg / mL) resistance, and incubate at 37°C for 10-12h. Pick a single colony into 10 mL of liquid LB medium containing kanamycin (100 μg / mL) resistance, culture at 37 ° C for 8 h, save the glycerol tube, and store it in a -80 ° C refrigerator. After the verification is correct, shake flask fermentation is carried out to produce enzyme.

Embodiment 3

[0038] Embodiment 3: Shake flask fermentation produces enzyme

[0039] The recombinant Bacillus subtilis strain obtained in Example 2 was inoculated in LB medium, and after culturing at 37°C for 8 hours, it was transferred to 50mL TB fermentation medium with 5% inoculum size, and then placed at 37°C and 200rpm for constant temperature cultivation 2h, when the cell OD600 is 0.5-1.0, turn to 33°C and 200rpm to carry out shake-flask induction fermentation for 48h. After the fermentation, the fermentation broth was ultrasonically broken and then centrifuged, and the supernatant was the recombinant CE. The measured recombinase activity can reach 8.0U / mL, and the protein electrophoresis results show that there is a band consistent with the theoretical molecular weight at 43kDa ( figure 1 ).

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Abstract

The invention discloses a cellobiose epimerase mutant and application thereof in galactopoiesis fructose, and belongs to the technical field of gene engineering. By connecting a cellobiose epimerase mutant gene shown in SEQ ID NO.2 to a pBSMuL3 carrier, a recombinant plasmid pBSMuL3-ce() is established; by converting bacillus subtilis CCTCC NO:M2016536, recombinant bacillus subtilis is obtained. With the recombinant bacteria as the strain, ellobiose epimerase is generated through fermentation, and the enzyme activity of the obtained recombinase reaches 8 U / mL. The recombinant enzyme is used for preparing lactulose, and the yield of generating the lactulose through CE catalytic lactose is 200 g / L and the converting rate can reach 51% under the reaction conditions of the temperature being 80DEG C, the Ph being 7.5, the enzyme addition amount being 20 U / mL and the substrate concentration being 400 g / L.

Description

technical field [0001] The invention relates to a cellobiose epimerase mutant and its application in producing lactulose, belonging to the technical field of genetic engineering. Background technique [0002] Lactulose is an indigestible disaccharide. Its finished product is a yellow clear liquid. The sweetness is equivalent to lactose, but less than sucrose, about 48%-60% sucrose. It has a cool taste, low viscosity, low calories, and is safe. High performance and good stability. In addition, lactulose can increase the activity of bifidobacteria and is a good bifidobacterium promoting factor. Therefore, lactulose has been widely used in the fields of medicine, food and animal feed. [0003] Commercial lactulose is mainly prepared by chemical methods, but chemical methods require the addition of a large amount of catalysts, the conditions required for the reaction are generally more intense, and the obtained products are more by-products. These mixtures bring great difficult...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/75C12N1/21C12P19/12C12P19/24C12R1/125
CPCC12N9/90C12N15/75C12P19/12C12P19/24C12Y501/03011
Inventor 吴敬陈晟王鑫淼
Owner JIANGNAN UNIV
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