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Trehalose synthetase as well as encoding genes and application thereof

A technology of trehalose synthase and coding gene, which is applied in the field of trehalose synthase and its coding gene and application, can solve the problems of high production cost and achieve the effect of short catalytic time and low substrate concentration

Active Publication Date: 2009-09-02
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] For a long time, the fundamental reason restricting the wide application of trehalose is the high production cost

Method used

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  • Trehalose synthetase as well as encoding genes and application thereof
  • Trehalose synthetase as well as encoding genes and application thereof
  • Trehalose synthetase as well as encoding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, the acquisition of trehalose synthase and its effect verification

[0051] 1. Acquisition of the gene encoding trehalose synthase

[0052] 1. Extraction of Genomic DNA from Arthrobacter aurescences ATCC 13344

[0053] The activated Arthrobacter aurescences ATCC 13344 (purchased from China Common Microorganism Culture Collection Management Center) was transferred to nutrient gravy liquid medium (containing peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH7.0 culture medium), cultured on a shaker at 210rpm at 30°C for 24h. Take 100mL of culture solution to collect the bacteria by centrifugation, add 9.5ml TE (10mmol / L Tris-HCl, 1mmol / L EDTA, pH8.0) to suspend the precipitate, and add 0.5ml 10% (mass content) SDS, 50μl 20mg / ml proteinase K , mix well, keep warm at 37°C for 1 hour; add 1.5ml 5mol / L NaCl, mix well; add 1.5ml 10% (mass content) CTAB solution (prepared with 0.7mol / L NaCl solution), mix well, keep warm at 65°C for 20 minutes; extract wit...

Embodiment 2

[0097] Example 2. Preparation of trehalose by trehalose synthase expressed by Escherichia coli (E.coli) PlysS aaN-TreS-30a

[0098] Take 0.5ml of trehalose synthase AA-TreS enzyme solution purified in Step 2 of Step 2 in Example 1, dilute it to 500 μL with 100 mmol / L pH6.5 phosphate buffer, add maltose to a final concentration of 90 mM, shake at 35 °C Reaction 8h, boiling water bath 5min to terminate the reaction. The content of trehalose in the samples was determined by ion chromatography. The determination method of the ion chromatography is as follows: the supernatant of the reaction liquid is collected by centrifugation, diluted 1000 times with 100 mmol / L phosphate buffer solution of pH 7.0, and injected. The ion chromatography is DIONEX2500, the column: CarboPac PA-100, the mobile phase: 100nmol / L NaOH and 500nmol / L NaAC with a volume ratio of 70:30, the flow rate is 1.0mL / min, and the injection volume is 10μL.

[0099] The concentration of the standard sample is known,...

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Abstract

The invention discloses a trehalose synthetase as well as encoding genes and application thereof. The trehalose synthetase is a protein comprising one of the following amino acid residue sequences: 1) an SEQ ID No. 1 amino acid residue sequence in a sequence table; and 2) a protein which is derived from the SEQ ID No. 1, has trehalose synthetase related functions, and is obtained by replacing and / or deleting and / or adding the SEQ ID No. 1 amino acid residue sequence in the sequence table. Trehalose can be obtained by using maltose as a substrate and performing catalysis with the application of the protein of the trehalose synthetase; besides, the catalytic reaction has the advantages that the catalysis time is short, the concentration of the required substrate is low, a 90mM maltose is used as the substrate, the catalysis time is 8 hours, and the conversion rate reaches 69 percent.

Description

technical field [0001] The invention relates to a trehalose synthase, its encoding gene and application. Background technique [0002] Trehalose is a non-reducing disaccharide composed of two glucopyranose linked by 1,1-glycosidic bonds. There are three optical isomers of trehalose, among which the αβ and ββ types rarely exist in nature, and the αα type is the most common trehalose. The structural formula of the αα type trehalose is shown in Formula I. [0003] (Formula I) [0004] Trehalose is colorless and odorless, with a slightly sweet taste, and its sweetness is equivalent to 45% of that of sucrose. The physical and chemical properties of trehalose are stable, it will not caramelize, it cannot reduce Fehling's reagent, and it cannot be hydrolyzed by α-glucosidase, but it can be hydrolyzed into two glucose molecules by trehalase. [0005] Preparation method of trehalose: Traditional methods include chemical synthesis and microbial extraction, especially yeast extrac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N5/10C12N1/00C12P19/12C12R1/06C12R1/19
Inventor 丁宏标吴秀丽
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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