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Trehalose synthase with increased maltose conversion rate, method for preparing trehalose synthase and application thereof

一种海藻糖合酶、突变体的技术，应用在基因工程和酶工程领域，能够解决麦芽糖成本高等问题

Active Publication Date: 2017-11-24
HUNAN JINDAI TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Recombinant bacteria construction

[0020] 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 NdeI and HindIII double enzyme digestion respectively. After the digestion products were recovered by tapping the rubber, they were ligated with T4 ligase, and the ligated products were transformed into E.coli JM109 competent cells and cultured at 37°C. After culturing for 8 hours, the transformants were picked and cultured with shaking in LB medium containing 30 mg / L kanamycin liquid, the plasmid was extracted, and the expression plasmid TreS / pET24a(+) was obtained for verification by enzyme digestion.

[0021] Transform the plasmid TreS / pET24a(+) into E.coli BL21(DE3) host bacteria, spread the LB plate containing kanamycin (30mg...

Embodiment 2

[0022] Embodiment 2: the preparation of mutant

[0023] (1) Single mutation

[0024] Mutant enzymes E289G, H295N, M344K, M367L of trehalose synthase derived from Thermobifidafusca YX.

[0025] According to the gene sequence of trehalose synthase of Thermobifidafusca YX, primers were designed and synthesized to introduce E289G, H295N, M344K, M367L mutations, 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 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 vector TreS / pET24a(+) as ...

Embodiment 3

[0063] Embodiment 3: HPLC detects the output of trehalose

[0064] 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.

[0065] Table 1 takes industrial grade maltose as the conversion rate of producing trehalose as substrate

[0066] enzyme

Con...

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Abstract

The invention discloses trehalose synthase with an increased maltose conversion rate, a method for preparing the trehalose synthase and application thereof, and belongs to the field of gene engineering and enzyme engineering. The method includes mutating glutamic acid at 289 sites near trehalose synthase active centers of Thermobifida fusca YX into glycine to obtain mutants E289G; mutating histidine at 295 sites into asparagine mutants H295N; mutating methionine at 344 sites into lysine mutants M344K; mutating methionine at 367 sites into leucine mutants M367L and carrying out combined mutation on the basis of the H295N to obtain mutants H295N / E289G, H295N / M344K, H295N / M367L and H295N / M344K / M367L. The trehalose synthase, the method and the application have the advantages that by the aid of the mutants, the conversion efficiency of trehalose prepared from the trehalose synthase still can be prevented from being severely affected even if substrates contain certain glucose, and accordingly the trehalose synthase, the method and the application have high industrial values.

Description

[0001] This application is a divisional application with the application number: 201510210023.X, the application date: April 28, 2015, and the application name: a mutant of trehalose synthase and its preparation method and application. technical field [0002] The invention relates to a trehalose synthase with improved maltose conversion rate and its preparation method and application, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0003] 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 hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/70C12P19/12
CPCC12N9/1051C12N9/90C12N15/70C12P19/12C12Y204/01245C12Y504/99016
Inventor 吴敬宿玲恰张悦
Owner HUNAN JINDAI TECH DEV CO LTD
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