Trehalose synthase mutant

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

Active Publication Date: 2018-12-11
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

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: Recombinant bacteria construction

[0015] The plasmid pET24a(+) with the T7 promoter was selected as the expression vector, and the pET24a(+) plasmid and the plasmid TreS / pMD 18T containing the TreS gene were subjected to double enzyme digestion with NdeI and HindIII, respectively, and the digested products were recovered by tapping the gel, and then used Ligated by T4 ligase, the ligated product was transformed into E.coli JM109 competent cells, cultured at 37°C for 8 hours, picked and cultured in LB medium containing 30mg / L kanamycin liquid with shaking, extracted the plasmid, and verified by enzyme digestion The expression plasmid TreS / pET24a(+) was obtained.

[0016] 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 / pET24a(+) / E .coli BL21(DE3). Pick a single colony into liquid LB medium containing 30mg / L kanamycin, culture overnight at...

Embodiment 2

[0017] Embodiment 2: the preparation of mutant

[0018] (1) Single mutation G52H

[0019] According to the gene sequence of the trehalose synthase of Thermobifida fusca YX, the primers for introducing the G52H mutation were designed and synthesized respectively, using the rapid PCR technology and using the expression vector TreS / pET24a(+) as a template,

[0020] The site-directed mutagenesis primers for introducing the G52H mutation are:

[0021] Forward primer: 5’-TAC GAT AGC AAT GGC GAT CAC ACCGGCGATT-3' (the underline is the mutated base)

[0022] Reverse primer: 5'-A ATC GCC GGT GTG ATC GCC ATT GCT ATC GTA-3'(The underline is the mutated base)

[0023] The PCR reaction system is: 5×PS buffer 10 μL, dNTPs Mix (2.5mM) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, PrimerStar HS (5U / μL) 0.5 μL, Add double distilled water to 50 μL.

[0024] The PCR amplification conditions were: pre-denaturation at 94°C for 4 min; followed by 30 cycl...

Embodiment 3

[0036] Embodiment 3: HPLC detects the output of trehalose

[0037] In the reactor, add maltose 300g / L (containing glucose 10%), add the wild enzyme that obtains in a certain amount of embodiment 2 and the crude enzyme liquid of mutant, pH is adjusted to 8.0 with 20% sodium hydroxide aqueous solution, in React in a water-bath shaker at 30°C and 150rpm for 30-50 hours and take samples regularly. After the reaction is terminated by boiling for 10 minutes, the sample is centrifuged at 12000rpm for 10 minutes. The supernatant is diluted appropriately and filtered with a 0.45μm 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.

[0038] According to the yield of trehalose, calculate the conversion rate of maltose (mass ratio of trehalose and maltose), the results are ...

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Abstract

The invention discloses trehalose synthase mutant, and belongs to the field of gene engineering and enzyme engineering. According to the trehalose synthase mutant, a trehalose synthase derived from Thermobifida fusca YX is modified, so that the inhibition effect of the glucose on the activity of the trehalose synthase in the catalytic synthesis of trehalose is weakened when the synthase takes industrial-grade maltose (containing 10% of glucose) as a substrate, when the industrial-grade maltose (containing 10% of glucose) is taken as the substrate, the conversion rate of the trehalose producedby a wild enzyme is 62.2%, and the conversion rate of the trehalose produced by the mutant G52H and the mutant G52H/H134N can reach 74.8% and 82.3% correspondingly. The trehalose synthase mutant has the advantages that even if certain glucose is contained in the substrate, the conversion efficiency of the trehalose prepared by the trehalose synthase is still not greatly influenced, and a high industrial value is achieved.

Description

technical field [0001] The invention relates to a trehalose synthase mutant, 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 hygroscopicity, low sweetness and other propertie...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/10C12N15/70C12P19/24C12P19/12
CPCC12N9/90C12N15/1031C12N15/70C12P19/12C12P19/24C12Y504/99016C12Q2531/113
Inventor 何球山钟红霞黄海军邓希
Owner 湖南金代科技发展有限公司
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