Trehalose synthetase and application thereof

A technology of trehalose synthase and sequence, applied in the direction of enzymes, using carriers to introduce foreign genetic material, enzymes, etc., can solve the problems of long catalysis time, high substrate concentration, low expression of trehalose synthase, etc., and achieve catalysis time Short, low substrate concentration, high enzyme activity

Inactive Publication Date: 2008-01-09
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

The trehalose synthases reported in these literatures have the disadvant...

Method used

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  • Trehalose synthetase and application thereof
  • Trehalose synthetase and application thereof
  • Trehalose synthetase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, acquisition of trehalose synthase and its coding gene

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

[0052] 1. Extraction of Corynebacterium glutamicum (C.glutamicum) genomic DNA: transfer the activated Corynebacterium glutamicum (C.glutamicum) CGMCC 1.1886 bacterial classification into nutrient gravy liquid medium (peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH 7.0), and cultured at 210rpm on a shaker at 30°C for 24h. Take 100mL of the culture solution and collect the bacteria by centrifugation, add 9.5ml TE to suspend the sediment, add 0.5ml 10% SDS, 50μl 20mg / ml proteinase K, mix well, and incubate at 37°C for 1 hour; add 1.5ml 5mol / L NaCl, mix well Add 1.5ml of 10% CTAB solution (prepared with 0.7mol / L NaCl solution), mix well, and keep warm at 65°C for 20 minutes; extract with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), 5000rpm Centrifuge for 10 minutes, transfer the supernatant to a clean centrifuge...

Embodiment 2

[0089] Example 2. Preparation of trehalose using trehalose synthase expressed by Escherichia coli (E.coli) PlysS cgN-T31-30a27 CGMCC No.2011

[0090] Take 1ml of the trehalose synthase CG-TreS enzyme solution (19.0U) in Example 1, mix it with 1ml of 30% (30g / 100ml) maltose solution, shake and react at 20°C for 20h, and stop the reaction in a boiling water bath for 5min. 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 was DIONEX 2500, the column: CarboPac PA-100, the mobile phase: 100nmol / L NaOH and 500nmol / LNaAC with a volume ratio of 70:30, the flow rate was 1.0mL / min, and the injection volume was 10μL.

[0091] The concentration of the standard sample is known, and the content of trehalose in the sample...

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Abstract

A mycose synthetase and its use are disclosed. It consists of (a) or (b) protein, (a) protein is made of amino-acid residue sequence in sequence 2; (b) protein is derived by (a) and substituted and/or lost and/or added by one or several amino-acid residue and has mycose synthetase activity. It can be used to produce mycose by catalyzed malt dust.

Description

technical field [0001] The invention relates to a trehalose synthase and its application. Background technique [0002] Trehalose (Trehalose) molecule is composed of two glucopyranose linked by 1,1-glycosidic bond, there is no free hemi-condensed hydroxyl group in the molecule, it is a stable non-reducing disaccharide. There are three optical isomers of trehalose, among which αβ type (Neotrehalose, new trehalose) and ββ type (Isotrehalose, different trehalose) rarely exist in nature, and αα type is the most common trehalose (also known as mushroom Sugar), widely present in bacteria, fungi, algae, lower plants and insects. [0003] Trehalose is colorless and odorless, has strong thermal stability, acid stability and chemical stability, and has a strong stabilizing effect on living components such as proteins, nucleic acids, and cell membranes. Trehalose is usually used as a Compatibility medium protects biomacromolecules and even organisms. Studies have shown that trehalos...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N1/21C12P19/12
Inventor 丁宏标李镭乔宇吴秀丽
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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