Novel bacillus arabinose isomerase gene cloning and expression method and use

A technology of arabinose and bacillus, applied in the field of bioengineering, can solve problems such as unapproved use, and achieve the effect of increasing production

Inactive Publication Date: 2015-12-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tagatose products have been approved for use by the food hygiene departments of the United States, Australia, Japan, and South Korea, but have not yet been approved for use in my country

Method used

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  • Novel bacillus arabinose isomerase gene cloning and expression method and use
  • Novel bacillus arabinose isomerase gene cloning and expression method and use
  • Novel bacillus arabinose isomerase gene cloning and expression method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: PCR amplification of Bacillussp.SYBChb4L-arabinose isomerase gene L-AI, protein sequence functional region prediction

[0033] The bacterial genomic DNA extraction kit (TaKaRa Company) was used to extract the total genomic DNA of Bacillussp.SYBChb4 bacteria according to its operation, its quality and purity were identified by agarose gel electrophoresis, and its concentration was determined by an ultraviolet spectrophotometer. Design upstream and downstream primers according to the whole gene sequencing: h1 (SEQ ID NO: 3, 5'-ATGTTAACAAGTCAGAAG-3') and h2 (SEQ ID NO: 4, 5'-TCACAGTAGAATGCATTTC-3'), using the PCR method, using the above-mentioned Bacillussp.SYBChb4 genome as Template, with the above-mentioned h1 and h2 as specific primers, amplify the full-length coding frame sequence (1446bp) of the Bacillussp.SYBChb4L-arabinose isomerase gene (such as figure 1 shown). The PCR reaction conditions are: using genomic DNA as a template, in a 50 μL reaction syste...

Embodiment 3

[0039] Embodiment 3: the fermentation experiment of recombinant bacterial strain

[0040] The recombinant Escherichia coli constructed as in Example 2 was cultured in shake flasks. Pick a single colony of recombinant Escherichia coli on the Amp resistant plate and inoculate it into LB liquid medium containing ampicillin antibiotic. See Example 2 for the recipe of the culture medium. Cultivate at 37°C and 200rpm for about 12 hours. When the thalline grows to the logarithmic phase, insert it into 50ml of fermentation medium (diluted 10 times by lactose refining wastewater) according to the inoculum size of 2%. Cultivate for about 24 hours at 200 rpm.

Embodiment 4

[0041] Embodiment 4: Quantitative detection of tagatose

[0042] D-tagatose and D-galactose in each eluted sample can be detected qualitatively and quantitatively by high performance liquid chromatography (HPLC). HPLC conditions: chromatographic column: Sugarpark 16.5mm×30mm; mobile phase: pure water; detector: Waters2410 differential refractive index detector; column temperature: 85°C; flow rate: 0.4mL / min;

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Abstract

The invention relates to an L-arabinose isomerase gene L-AI separated from Bacillus sp. SYBC hb4 and its cloning and expression method and use, and belongs to the field of bioengineering. The method comprises separating L-AI from Bacillus sp. SYBC hb4, constructing an expression vector pCold II-ai, and transferring the expression vector pCold II-ai into an escherichia coli expression strain BL21(DE3) so that high-efficiency expression is realized. The recombinase arabinose isomerase can utilize lactose produced by industrial production under the action of lactase to refine waste water and produce D-tagatose. The D-tagatose is a novel low-energy sweetener and can reduce obesity. The D-tagatose has effects of reducing blood sugar, is suitable for diabetic patients, can improve intestinal tract flora and can inhibit intestinal tract pathogenic bacteria. D-tagatose has a great application prospect in fields of medicines and industry.

Description

technical field [0001] The invention relates to an L-arabinose isomerase gene, in particular to the cloning, expression and application of a Bacillus sp.SYBChb4 L-arabinose isomerase gene, which belongs to the technical field of bioengineering. Background technique [0002] L-arabinose isomerase (EC5.3.1.4, L-arabinose isomerase, L-AI), can convert L-arabinose into L-ribulose, and can also convert D-galactose into D-tagatose. The joint action of L-arabinose isomerase, L-ribulokinase and L-ribulose 5-phosphate 4-epimerase will also have an important application in the hemicellulosic ethanol industry. Studies have found that L-arabinose isomerase widely exists in various microorganisms, such as normal temperature microorganisms. Thermophilic microorganisms, acidophilic microorganisms, and even some extremophiles. However, the genes contained in natural strains are generally single copy, and the yield of the enzyme is very little, so the second purpose of the present inventio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/70C12N1/21C02F3/34C02F103/36
Inventor 廖祥儒刘群徐君孙萌蔡宇杰管政兵王爽李韵雅
Owner JIANGNAN UNIV
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