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High temperature resistant L-arabinose isomerase and application thereof

An arabinose, high temperature-resistant technology, applied in the field of bioengineering, can solve the problems of low optimal reaction temperature, poor thermal stability, unfavorable industrial applications, etc., and achieve good activity and stability, good thermal stability and pH tolerance , the effect of wide application prospects and economic significance

Inactive Publication Date: 2010-09-29
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the araA gene of many species has been identified, there are not many L-AIs used in the research of D-tagatose production, because most L-AI enzymes have poor thermal stability and low optimum reaction temperature. Not good for industrial applications

Method used

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  • High temperature resistant L-arabinose isomerase and application thereof
  • High temperature resistant L-arabinose isomerase and application thereof
  • High temperature resistant L-arabinose isomerase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Extraction of Lactobacillus fermentum NXTag-1 genomic DNA.

[0039] According to the instruction manual provided by the manufacturer, the genomic DNA of Lactobacillus fermentum (Lactobacillus fermentum) NXTag-1CGMCC NO.2921 in the logarithmic growth phase was extracted with the Genomic DNA Purification Kit (Takara, Dalian), and 8 g / L agarose was used to coagulate The obtained bacterial genomes were detected by gel electrophoresis.

Embodiment 2

[0040] Example 2: Cloning of the gene encoding L-arabinose isomerase (araA) and construction of recombinant bacteria.

[0041] 2.1 PCR amplification of araA gene

[0042] According to the sequence of the L-AI gene of Lactobacillus fermentum reported on Gene Bank, use Vector NTI software to design primers Primer1 and Primer2, the primer sequence is:

[0043] Primer1: 5'-AGAGAATTCATGCGTAAGATGCAAGATTAC-3'

[0044] Primer2: 5'-AAGCTCGAGCTACTTGATGTTGATAAAGT-3'

[0045] Using the genomic DNA of Lactobacillus fermentum obtained in Example 1 as a template, the gene fragment of Lactobacillus fermentum was amplified.

[0046] The PCR amplification system is: 2 μL of genomic DNA, 1 μL of each of primers Primer1 and Primer2, 2 μL of dNTP, 2.5 μL of 10×Tag buffer, 0.5 μL of ExTag polymerase, ddH 2 O 14 μL.

[0047] 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...

Embodiment 3

[0066] Example 3: Induced expression of L-arabinose isomerase.

[0067] Recombinant Escherichia coli BL21-AI was inoculated in 5 mL of LB liquid medium supplemented with 25 μg / mL kanamycin, and cultured on a shaker at 37°C overnight; then transferred to 100 mL of LB medium ( In a 500mL shake flask containing 25μg / mL kanamycin), culture on a shaker at 37°C for 2-3 hours, until OD 600 At about 0.6, add IPTG for induction (IPTG final concentration 1mmol / L), or add 1g / L lactose for induction, and then continue to induce expression for 6 hours, and collect the bacteria by centrifugation.

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Abstract

The invention discloses a high temperature resistant L-arabinose isomerase which has an amino acid sequence shown in SEQ ID NO:2. The invention also discloses a gene sequence for encoding the high temperature resistant L-arabinose isomerase, a gene engineering bacterium and construction method thereof, an expression method for the L-arabinose isomerase, and application of the L-arabinose isomerase and the gene engineering bacterium in preparing D-tagatose. The reaction temperature of the high temperature resistant L-arabinose isomerase is 40 to 70 DEG C, and the reaction pH is 5.5 to 7.5. The recombinase shows good heat stability and pH tolerance; the added low-concentration Mn2+, Co2+ ions can greatly improve enzyme activity and heat stability; and the high temperature resistant L-arabinose isomerase has wide application prospect and economic significance for industrial production of the novel functional sweetener D-tagatose.

Description

technical field [0001] The invention relates to a high-temperature-resistant L-arabinose isomerase derived from Lactobacillus fermentum NXTag-1, construction of a high-temperature-resistant L-arabinose isomerase engineering bacterium, and high-temperature-resistant L-arabinose isomerization The application of the enzyme belongs to the technical field of bioengineering. Background technique [0002] Tagatose is a rare natural ketohexose in nature, which is a kind of rare sugar. The International Rare Sugar Association (ISRS) defines rare sugar as "a class of monosaccharides and its derivatives". D-tagatose (D-tagatose) is the "epimer" of fructose, widely present in nature, many foods, such as sterilized milk, milk powder, hot cocoa, various cheeses, some varieties of sour There is a certain amount of tagatose in milk and some plants. D-tagatose is a functional sweetener with special health functions. It is produced by isomerization of D-galactose in industrial production. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/63C12N1/21C12P19/24C12P19/02C12R1/19
Inventor 徐虹徐铮朱宏阳欧阳平凯
Owner NANJING UNIV OF TECH
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