A mutant of trehalose synthase and its preparation method and application

A technology of trehalose synthase and mutant, applied in the fields of genetic engineering and enzyme engineering, can solve the problem of high cost of maltose

Active Publication Date: 2017-10-31
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

[0004] Maltose can be obtained by hydrolyzing starch, and a certain amount of glucose will be produced during the production process. The cost of using pure maltose in the industrial production of trehalose is too high

Method used

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  • A mutant of trehalose synthase and its preparation method and application
  • A mutant of trehalose synthase and its preparation method and application
  • A mutant of trehalose synthase and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Recombinant bacteria construction

[0019] The laboratory preserves the previously constructed plasmid TreS / pMD18T 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(+).

[0020] 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 / pET24...

Embodiment 2

[0021] Embodiment 2: the preparation of mutant

[0022] (1) Single mutation

[0023] Mutant enzymes E289G, H295N, M344K, M367L of trehalose synthase derived from Thermobifida fusca YX.

[0024] According to the trehalose synthase gene sequence of Thermobifida fusca YX, primers were designed and synthesized to introduce E289G, H295N, M344K, and M367L mutations, and site-directed mutation was performed on the trehalose synthase gene. The DNA coding sequence was determined, and the 289th position was identified respectively. The Glu codon at position 295 becomes a Gly codon, the 295th His codon becomes an Asn codon, the 344th Met codon becomes a Lys codon, and the 367th Met codon becomes a Leu codon. The mutant gene is placed in an appropriate expression vector and introduced into Escherichia coli for expression to obtain a single mutant trehalose synthase. Site-directed mutation of single mutations E289G, H295N, M344K, M367L: using rapid PCR technology, using the expression ve...

Embodiment 3

[0049] Embodiment 3: HPLC detects the output of trehalose

[0050] In the reactor, add maltose 300g / L (containing glucose 10%), add the concentrated enzyme liquid of the wild enzyme of a certain amount of obtaining in example 2 and mutant, pH is adjusted to 8.0 with 20% sodium hydroxide aqueous solution, at 30 React in a water-bath shaker at 150 rpm for 30-50 hours and take samples regularly. After the reaction is terminated by boiling for 10 minutes, the sample is centrifuged at 12,000 rpm for 10 minutes. The supernatant is diluted appropriately and filtered with a 0.45 μm ultrafiltration membrane for HPLC analysis. 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.

[0051] Table 1 takes industrial grade maltose as the conversion rate of producing trehalose as substrate

[0052] enzyme

Con...

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Abstract

The invention discloses a mutant for trehalose synthase as well as a preparation method and an application of the mutant and belongs to the fields of genetic engineering and enzyme engineering. The mutant is prepared by the following steps: mutating 289th bp glutamate near the active center of the trehalose synthase of Thermobifida fusca YX into glycine to obtain a mutant E289G; mutating 295th bp histidine into an asparagine mutant H295N; mutating 344th bp methionine into a lysine mutant M344K; and mutating 367th bp methionine into a leucine mutant M367L, and carrying out double mutations on the basis of the H295N to obtain mutants H295N / E289G, H295N / M344K, H295N / M367L and H295N / M344K / M367L. According to the mutant disclosed by the invention, although a substrate contains a certain quantity of glucose, the transformation efficiency of preparing trehalose by virtue of the trehalose synthase can not be greatly influenced still, and the mutant has very high industrial value.

Description

technical field [0001] The invention relates to a mutant of trehalose synthase and its preparation method 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 hyg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/12
CPCC12N9/1051C12N9/90C12N15/70C12P19/12C12Y204/01245C12Y504/99016
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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