Recombinant bacillus subtilis for expressing cellobiose-2-epimerase based on D-alanine defective screening, and construction method of recombinant bacillus subtilis

A technology of Bacillus subtilis and epimerase, applied in the field of genetic engineering of enzymes, can solve the problem of high cost

Inactive Publication Date: 2016-03-02
JIANGNAN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Epilactose can also be synthesized from p-nitrophenylgalactose and mannose as substrates, catalyzed by β-galactosidase, but the reaction also has β-1,3 and β-1,6 by-products , and the cost is high

Method used

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  • Recombinant bacillus subtilis for expressing cellobiose-2-epimerase based on D-alanine defective screening, and construction method of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for expressing cellobiose-2-epimerase based on D-alanine defective screening, and construction method of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for expressing cellobiose-2-epimerase based on D-alanine defective screening, and construction method of recombinant bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: D-alanine-deficient Bacillus subtilis 1A751 ( dal - ) construction

[0025] Table 1D - Alanine-deficient Bacillus subtilis 1A751 ( dal - ) for the construction of primer pairs

[0026]

[0027] Using the plasmid p7S6 as a template, the primer pair P3 / P4 will lox 71- spc - lox 66 antibiotic resistance gene fragments were amplified and recovered.

[0028] D-alanine racemase gene on chromosome of Bacillus subtilis 1A751 dal The fragments with a length of 800-900bp on both sides were selected as homologous regions, and the homologous regions at both ends were amplified and recovered with primer pairs P1 / P2 and P5 / P6 respectively.

[0029] Use the primer pair P1 / P6 to combine the homology arm fragments at both ends with antibiotics lox 71- spc - lox 66 were fused together by PCR technology.

[0030] PCR reaction system: Add the following reagents in order in 0.2mL PCR tube: 1.5μL each of upstream and downstream primers (P1 / P6); 5μL HusionHFbu...

Embodiment 2

[0035] Example 2: Construction of food-grade safe plasmid pUB-P43-TsCE-dal

[0036] Table 2 Primer pairs required for the construction of food-grade safe plasmid pUB-P43-TsCE-dal

[0037]

[0038] To enhance the expression, a strong promoter P43 was fused upstream of the CE enzyme gene to form a P43-TsCE expression unit. The P43-TsCE fragment was amplified using primer pair P7 / P9. Using pUB110 (NCBI-GeneID: 9507338) as the starting vector, the vector backbone pUB was amplified using the primer pair P8 / P10. The P43-TsCE and pUB fragments were then subjected to PCR to form multimers.

[0039] PCR reaction system: Add the following reagents in sequence to a 0.2 mL PCR tube: 5 μL HusionHFbuffer (5×); 2 μL 10 mMdNTPmix (2.5 mMeach); 2 μL P43-TsCE; 2 μL pUB; 0.5 μL Husion high-fidelity DNA polymerase;

[0040] PCR amplification conditions: pre-denaturation at 98°C for 30s; denaturation at 98°C for 30s, annealing at 55°C for 15s, extension at 72°C for 1min (30 cycles); extensio...

Embodiment 3

[0043] Embodiment 3: the fermentation of recombinant bacterium

[0044] 1. CE enzyme activity assay method: Add 800 μL of 250 mM lactose dissolved in phosphate buffer (50 mM, pH 7.0) to 1 mL of reaction system, 200 μL of fermentation broth, incubate at 65°C for 15 minutes, then add HCl to the final concentration 200mM to stop the enzyme reaction.

[0045] 2. Detect the amount of ipilactose produced by HPLC, and calculate the enzyme activity. Enzyme activity unit (U): The amount of enzyme required to catalyze the production of 1 μmol of pilactose per minute.

[0046] 3. Use an inoculation loop to pick fresh colonies from the plate and inoculate them into the seed medium, culture at 37°C, 200rpm, for 12-14 hours. Inoculate into the fermentation medium with 3% inoculum amount, 37°C, 200rpm, and take samples regularly to measure OD 600 , enzyme activity data ( image 3 ).

[0047] Seed medium: tryptone (10g / L), yeast extract (5g / L), NaCl (10g / L), prepared with double distille...

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Abstract

The invention relates to a recombinant bacillus subtilis for expressing cellobiose-2-epimerase based on a D-alanine defective screening marker, as well as a construction method and an application of the recombinant bacillus subtilis, belonging to the technical field of genetic engineering of enzyme. According to the invention, bacillus subtilis 1A751 is taken as an original strain, D-alanine racemase genes (dal) on the chromosome of the bacillus subtilis 1A751 strain are knocked out, so that D-alanine defective bacillus subtilis 1A751 (dal<->) is obtained; by taking cellobiose-2-epimerase (CE enzyme) from thermoanaerobacter tengcongensis as a parent, fusing a P43 promoter on the upstream of the parent to establish P43-TsCE, establishing P43-TsCE to plasmid pUB110 to obtain pUB-P43-TsCE, and replacing antibiotics resistance genes kanamycin and bleomycin on the plasmid pUB-P43-TsCE with (dal), so as to construct pUB-P43-TsCE-dal; and transforming pUB-P43-TsCE-dal into host 1A751 (dal<->), and thus obtaining the recombinant bacillus subtilis for expressing cellobiose-2-epimerase, wherein the preservation number is CCTCC NO: M 2015582. The total enzyme activity of fermentation liquor reaches 7U/mL, and thus the recombinant bacillus subtilis has an important industrial application value.

Description

technical field [0001] The invention relates to a recombinant Bacillus subtilis expressing cellobiose-2-epimerase based on a D-alanine-deficient selection marker and its construction method and application, in particular to a CE enzyme in Bacillus subtilis The food-grade expression in the invention belongs to the technical field of genetic engineering of enzymes. Background technique [0002] Cellobiose-2-epimerase (CE enzyme) can catalyze the epimerization and isomerization reactions of various aldose C2 positions, and is a good biocatalyst for the production of rare sugars, and can synthesize a variety of carbohydrates compound. At present, CE enzyme can catalyze the epimerization reaction of lactose to produce ipilactose, and utilize a single substrate lactose to produce ipilactose. [0003] With the outbreak of a series of chronic diseases caused by excessive high-energy food intake in the world, dietary structure has attracted more and more attention. The development...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/24C12R1/125
Inventor 沐万孟江波陈秋铭张涛
Owner JIANGNAN UNIV
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