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Bacillus subtilis gene engineering strain, construction method thereof and application in lactulose production

A technology of Bacillus subtilis and genetically engineered bacteria, applied in the field of bioengineering, can solve problems such as potential safety hazards, and achieve the effects of improving productivity, avoiding loss of enzyme activity, and being convenient to operate.

Active Publication Date: 2016-01-20
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Escherichia coli itself produces endotoxin, which poses a great safety hazard and is not suitable for industrial application in the food field

Method used

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  • Bacillus subtilis gene engineering strain, construction method thereof and application in lactulose production
  • Bacillus subtilis gene engineering strain, construction method thereof and application in lactulose production
  • Bacillus subtilis gene engineering strain, construction method thereof and application in lactulose production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Synthesis of the cellobiose epimerase gene.

[0039] According to the reported gene sequence of cellobiose epimerase, the gene sequence shown in SEQ ID NO.1 was synthesized by the method of total gene synthesis.

Embodiment 2

[0040] Example 2: Construction of recombinant Bacillus subtilis.

[0041]Using the DNA obtained in Example 1 as a template, the cellobiose epimerase gene fragment was amplified. The PCR amplification system is: 2 μL of synthesized DNA, 1 μL of each primer Primer1 and Primer2, 2 μL of dNTP, 2.5 μL of 10×Ex-Tag buffer, 0.5 μL of Ex-Tag polymerase, ddH 2 O14 μL. The PCR reaction program was: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 2 min; then annealing at 60°C for 30 s, extension at 72°C for 1 min, and 35 cycles; extension at 72°C for 1 min.

[0042] The amplified bands were gel-cut and recovered with a column-type gel-slicing recovery kit, and the PCR product was double-digested with BamHI and SmaI. The digestion system is: 50 μL of PCR product or plasmid, 1 μL of XhoI, 1 μL of NdeI, 10 μL of 10× buffer, ddH 2 O38μL, total volume 100μL.

[0043] The digested PCR product was ligated with the pHT-01 plasmid (or pHT-43 plasmid) treated with the same restric...

Embodiment 3

[0045] Example 3: Inducible expression of cellobiose epimerase.

[0046] The constructed recombinant Bacillus subtilis str.168 (pHT-01-CsCE) was inoculated in 5 ml of LB liquid medium supplemented with ampicillin resistance, and cultivated overnight at 37°C; LB medium, fermented for 2 to 4 hours to OD 600 When reaching 0.6, add 0.6mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG) or 10g / L lactose to continue to induce expression for 24h.

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Abstract

The invention discloses a method for producing lactulose by means of a recombinant bacillus subtilis, and belongs to the technical field of bioengineering technology. The recombinant bacillus subtilis is used as the production strain, the recombinant bacillus subtilis includes cellobiose epimerase capable of catalyzing lactose for producing the lactulose, and induction expression is performed through the lactose or isopropyl-beta-D-isopropylthiogalactoside (IPTG) so as to use the lactose or whey as a substrate for a biological catalysis reaction. Bacillus subtilis wet cells or a fermentation solution is used for the catalysis reaction, the method is simple and easy to implement, and enzyme activity losses and cost waste in the enzyme separation and purification process are avoided; the food-grade bacillus subtilis is used as the production strain, the method is safe and reliable, and the effective references are provided for industrial and environment-friendly lactulose production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for producing lactulose by using recombinant bacillus subtilis expressing cellobiose epimerase. Background technique [0002] Lactulose (4-O-β-D-galactopyranosyl-D-fructose), also known as lactulose, is a disaccharide formed by combining galactose and fructose with β-1,4-glycosidic bonds. Pure lactulose is a white irregular powder with a relative density of 1.35 and a melting point of 169°C, easily soluble in water. Commercial lactulose syrup is generally light yellow and slightly transparent, and its sweetness is only 48% to 70% of that of sucrose. It has low calorific value, high safety, good stability, and no Maillard reaction. Lactulose is not only a bifidobacteria proliferation factor, but also promotes the adsorption of minerals, affects bones, reduces glutamate in the blood, lowers cholesterol, reduces the risk of urolithiasis, prevents or redu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/24C12P19/12C12R1/125
Inventor 徐虹徐岑徐铮刘超
Owner NANJING UNIV OF TECH
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