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Trehalose synthase mutant and preparation method and application thereof

A trehalose synthase and mutant technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve problems such as difficulty in popularization, complicated production process, etc., and achieve the effect of increasing yield

Active Publication Date: 2015-09-02
湖南金代科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The double-enzyme method uses starch as raw material, and generates trehalose under the action of maltooligosaccharide-based trehalose hydrolase and maltooligosaccharide-based trehalose synthetase. Production

Method used

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  • Trehalose synthase mutant and preparation method and application thereof
  • Trehalose synthase mutant and preparation method and application thereof
  • Trehalose synthase mutant and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: Recombinant bacteria construction

[0018] The laboratory has preserved the previously constructed plasmid TreS / pMD 18T containing the gene encoding trehalose synthase. The plasmid used for the construction of E. coli was pET24a(+) with T7 promoter. The pET24a(+) plasmid and the plasmid containing the TreS gene were subjected to Nde Ⅰ and Hind Ⅲ double-enzyme digestion, respectively, and the digested products were recovered by tapping the rubber, and then ligated with T4 ligase, and the ligated products were transformed into E.coli JM109 competent cells. Cultivate at ℃ for 8 hours, pick the transformants and culture them with shaking in LB medium containing 30 mg / L kanamycin liquid, extract the plasmids, and verify by enzyme digestion to obtain the expression plasmid TreS / pET24a(+).

[0019] Transform the plasmid TreS / pET24a(+) into E.coli BL21(DE3) host bacteria, spread the LB plate containing kanamycin (30mg / L), culture at 37°C for 8h, and name it TreS / ...

Embodiment 2

[0020] Embodiment 2: the preparation of mutant

[0021] (1) Single mutation

[0022] Six mutant enzymes Q271M, I220V, V228I, C290V, F294Y, I332F of trehalose synthase derived from Thermobifida fusca YX:

[0023] According to the gene sequence of trehalose synthase of Thermobifida fusca YX, primers for introducing Q271M, I220V, V228I, C290V, F294Y, and I332F mutations were designed and synthesized, and the trehalose synthase gene was subjected to site-directed mutation, and the DNA coding sequence was determined to identify Place the mutant gene in an appropriate expression vector and introduce it into Escherichia coli for expression to obtain a single mutant trehalose synthase. Site-directed mutation of single mutations Q271M, I220V, V228I, C290V, F294Y, and I332F: using rapid PCR technology, using the expression vector TreS / pET24a(+) as a template,

[0024] The site-directed mutagenesis primers for introducing the Q271M mutation are:

[0025] Forward primer: 5'-CTCAGCGAAGC...

Embodiment 3

[0067] Embodiment 3: HPLC detects the output of trehalose

[0068] Add 300g / L of maltose in the reactor, add a certain amount of wild enzyme obtained in Example 2 and the concentrated enzyme liquid of the mutant, adjust the pH to 8.0 with 20% aqueous sodium hydroxide solution, at 30°C, in a water bath of 150rpm After reacting in a shaking table for 30-50 hours, samples were taken regularly, and the reaction was terminated by boiling for 10 minutes. After the reaction, the samples were centrifuged at 12000 rpm for 10 minutes. The supernatant was diluted appropriately, filtered with a 0.45 μm ultrafiltration membrane, and analyzed by HPLC. The chromatographic conditions are as follows: differential refractive index detector, NH2 column (APS-2HYPERSIL, Thermo Scientific), mobile phase (water:acetonitrile=1:4), flow rate: 0.8mL min -1 , Column temperature: 40°C.

[0069] Table 1 The conversion rate of wild enzyme and mutants to produce trehalose

[0070] enzyme

Conv...

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Abstract

The invention discloses a trehalose synthase mutant and a preparation method and application thereof and belongs to the field of genetic engineering and enzyme engineering. The 271st Gln codon near the active center of trehalose synthase of trehalose synthase of Thermobifida fusca YX is converted into the Met codon, the 220th Ile codon is converted into the Val codon, the 228th Val codon is converted into the Ile codon, the 290th Cys codon is converted into Val codon, the 294th Phe codon is converted into the Tyr codon, and the 332nd Ile codon is converted into the Phe codon, so as to respectively obtain mutants Q271M, I220V, V228I, C290V, F294T and I332F. Based on the I220V, amphimutation is carried out to obtain mutants I220V / Q271M, I220V / V228I, I220V / C290V, I220V / F294Y and I220V / I332F. The mutants can be used for improving the trehalose conversion rate prepared by trehalose synthase and have relatively high industrial value.

Description

technical field [0001] The invention relates to a trehalose synthase mutant and its preparation and application, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Trehalose is a safe and non-toxic non-reducing disaccharide composed of 1,1-glycosidic bonds. There are three isomers namely (α, α), isotrehalose (β, β), neotrehalose (α, β), generally exists in the form of dihydrate. Co-heating with protein or amino acid will not produce Maillard reaction, and it can maintain certain stability in acidic, alkaline, high temperature and ultra-low temperature environments. Its unique biological activity makes trehalose widely used. A large number of studies have shown that trehalose is a protective agent for single-celled organisms, animal tissues and organs, proteins, biofilms, and pharmaceutical preparations. It can inhibit lipid acidification, starch aging, and protein denaturation. Transformation temperature, low hygroscopicit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12P19/12C12N1/15C12N1/21C12R1/19
CPCC12N9/90C12P19/12C12Y504/99016
Inventor 吴敬宿玲恰张悦
Owner 湖南金代科技发展有限公司
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