Trehalose synthase and coding gene and application thereof

A technology of trehalose synthase and trehalose, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of poor thermal stability, low substrate conversion rate, long reaction time, etc., and achieve good thermal stability and preparation The method is simple and the reaction time is shortened

Active Publication Date: 2015-07-29
武汉肌赛雪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The catalytic efficiency and enzymatic properties of trehalose synthase from different microbial sources are different, but they have the same basic characteristics: first, the substrate conversion rate is low, the highest is only about 80%, generally 60%-70%; The thermal stability of the enzyme is poor, and the optimum reaction temperature is about 25°C; the third is that the reaction time is too long, generally 48 hours, and some as long as 72h

Method used

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  • Trehalose synthase and coding gene and application thereof
  • Trehalose synthase and coding gene and application thereof
  • Trehalose synthase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Cloning obtains Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) trehalose synthase gene

[0033] Degenerate primers were designed based on the full-length nucleotide sequence of Pseudomonas stutzeri trehalose synthase published on NCBI, and the primer sequences were as follows:

[0034] Upstream primer: 5'-atc gga tcc atg agc ahn cca gac aah anc tat atc-3';

[0035] Downstream primer: 5'-tca ctc gag tta ran cac cgg hgr-3';

[0036] With the genome of Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) as a template, the above primers were used for PCR amplification, and the PCR reaction system was as follows:

[0037]

[0038] The above-mentioned PCR reaction is carried out according to the following procedures:

[0039] Pre-denaturation at 95°C for 5min; denaturation at 94°C for 30s, annealing at 54°C for 30s, extension at 72°C for 4min, 28 cycles; final extension at 72°C for 10min.

[0040] After the PCR, the length of the fragment was analyzed by 1%...

Embodiment 2

[0041] Example 2: Transforming the trehalose synthase gene into an expression host to obtain a positive expression strain.

[0042] Double digestion reaction of PCR product and plasmid vector

[0043] Enzyme digestion system for PCR products:

[0044]

[0045] Reaction conditions: react at 37°C for 2 to 3 hours.

[0046] Enzyme digestion system for plasmid vectors:

[0047]

[0048]

[0049] Reaction conditions: react at 37°C for 6-8 hours.

[0050] The PCR product and the vector digested product were subjected to 1% agarose gel electrophoresis, and were purified and recovered using a DNA gel recovery kit.

[0051] Connection reaction system:

[0052]

[0053] Mix well and centrifuge for a few seconds, collect the drop on the tube wall to the bottom of the tube, and connect overnight at 16°C to obtain the ligated product.

[0054] Transformation of recombinant plasmids

[0055] (1) Preparation of Competent Cells

[0056] ①Pick a single colony of BL21 (or pick ...

Embodiment 3

[0078] Example 3: Fermentation and culture of positive expression strains, isolation and purification of trehalose synthase recombinant protein

[0079] Seed culture: Pick positive clones in a conventional method and place them in 5 mL of LB liquid medium containing 100 mg / L ampicillin, and shake and culture at 37°C for 5-6 hours;

[0080] Bacterial cell expansion culture: Inoculate the seeds into 1L liquid medium containing 100mg / L ampicillin, culture with shaking at 37°C until the bacterial concentration OD600 is 1.0, then cool down to 15°C; add IPTG with a final concentration of 0.6mM after 1 hour , to induce expression overnight.

[0081] Collect bacteria: 4200rpm, centrifuge at 4°C for 15min, discard the supernatant, and harvest the bacteria; add resuspension solution (25mM Tris-HCl, pH8.0, 100mM NaCl), shake and precipitate the bacteria cells; add protease inhibitor PMSF ( Phenylmethyl sulfonyl fluoride, benzoic acid sulfonyl fluoride) to a final concentration of 2 mM. ...

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Abstract

The invention relates to a trehalose synthase and a coding gene and application thereof. A nucleotide sequence of an expression gene of the trehalose synthase is shown in SEQ ID No. 1; the amino acid sequence of the trehalose synthase is shown in SEQ ID No. 2. According to the trehalose synthase and the coding gene and application thereof, the trehalose synthase is obtained from pseudomonas stutzeri for the first time, and a preparation method of the trehalose synthase is simple and convenient and is high in yield and high in purity; proved by experiments, the thermal stability of the trehalose synthase is good, the trehalose conversion ratio can reach 70% in 1-hour reaction, and the reaction time is greatly shortened compared with that of the existing trehalose synthase, so that the production cost of trehalose can be reduced, and a foundation is laid for the industrial production of trehalose.

Description

technical field [0001] The invention relates to a trehalose synthase and its encoding gene and application, belonging to the field of biotechnology. Background technique [0002] Trehalose (Trehalose) is a non-reducing disaccharide composed of two glucopyranose molecules linked by α-1,1-glycosidic bonds, widely present in bacteria, yeast, filamentous fungi, plants, insects, organisms such as vertebrates. Studies have shown that its properties are stable and have very important biological significance to organisms. It is mainly manifested in the fact that it is a reserve of energy and carbon sources for organisms, a stabilizer and protector for proteins and biofilm molecules in harsh environments such as dehydration, high temperature, oxygen free radicals, and low temperature, and a signal-sensing complex and growth agent. It is not only a regulatory factor, but also one of the components of some bacterial cell walls, so trehalose has the reputation of "sugar of life" in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/70C12N1/21C12P19/24C12P19/12
Inventor 苏静王瑞明李珍珍张云霄
Owner 武汉肌赛雪生物科技有限公司
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