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Preparation and application of maltooligosyltrehalose synthase mutant

A technology based on trehalose synthase and maltooligosaccharides, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low soluble expression, unfavorable industrial application, and low enzyme activity

Active Publication Date: 2017-10-03
湖南金代科技发展有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Double-enzyme production of trehalose takes starch as the substrate, and the conversion rate is as high as 80%, which has the advantage of low cost, but the soluble expression of the maltooligosaccharide-based trehalose synthase gene in the host bacteria is very low, resulting in more inclusions Body, low enzyme activity, not conducive to its industrial application

Method used

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  • Preparation and application of maltooligosyltrehalose synthase mutant
  • Preparation and application of maltooligosyltrehalose synthase mutant
  • Preparation and application of maltooligosyltrehalose synthase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of wild maltooligosaccharide-based trehalose synthase

[0028] (1) Construction of maltooligosaccharide-based trehalose synthase recombinant bacteria

[0029] According to the treY amino acid sequence on NCBI (NCBI number: WP_015385613.1), the treY gene sequence on NCBI (NCBI number: NC_020247.1) was codon-optimized, and maltooligosaccharide-based trehalose synthase was synthesized by chemical total synthesis The gene sequence treY. The plasmid used to construct the expression vector in E. coli is pET24a(+). The pET24a(+) plasmid and the plasmid with the treY gene were subjected to NdeI and HindIII double enzyme digestion respectively, and the digested products were recovered by gel, ligated overnight with T4 ligase, and the ligated products were transformed into Escherichia coli JM109 competent cells, and the transformed products Spread on LB plates containing 100 mg / L kanamycin, culture overnight at 37°C, pick 3 single colonies on the plate, in...

Embodiment 2

[0032] Example 2 Preparation and expression of maltooligosaccharyl trehalose synthase mutant

[0033] (1) Preparation of mutants

[0034]Eight mutant enzymes L96T, L96H, M106K, M106H, P374E, P374H, I520H, I520R derived from Sulfolobus acidocaldarius ATCC 33909 maltooligosaccharide-based trehalose synthase: gene sequence, respectively designed and synthesized primers for introducing L96T, L96H, M106K, M106H, P374E, P374H, I520H, and I520R mutations, performed site-directed mutation on the maltooligosaccharide-based trehalose synthase gene, determined the DNA coding sequence, and identified the first The Leu codon at position 96 becomes Thr codon and His codon, the Met codon at position 106 becomes Lys codon and His codon, the Pro codon at position 374 becomes Glu codon and His codon, The Ile codon at position 520 was changed to a His codon and an Arg codon. The mutant gene is placed in an appropriate expression vector and introduced into Escherichia coli for expression to obt...

Embodiment 3

[0064] Example 3 Enzyme activity analysis of maltooligosaccharide-based trehalose synthase

[0065] (1) Definition of enzyme activity unit

[0066] When using 3,5-dinitrosalicylic acid method (DNS method) to measure the enzyme activity of maltooligosaccharide-based trehalose synthase, the amount of enzyme needed to convert 1 μmol of maltopentaose into maltotriosyl trehalose per minute is taken as a vitality unit.

[0067] (2) Enzyme Activity Determination Steps

[0068] Preheating: Take 0.5mL of 1% maltopentose solution (50mM pH5.5 phosphate buffer) in a test tube and place it in a water bath at 60°C for 10min to preheat.

[0069] Reaction: Add 0.05mL fermented intracellular crude enzyme solution, oscillate evenly, accurately time 10min, boil in a boiling water bath for 10min to terminate the reaction, and cool down. Add 1mL DNS and shake evenly, boil in a boiling water bath for 7min, and cool.

[0070] Measurement: add distilled water to the above reaction system and adju...

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Abstract

The invention discloses preparation and application of a maltooligosyltrehalose synthase mutant and belongs to the field of gene engineering and enzyme engineering. The maltooligosyltrehalose synthase mutant is prepared by substituting an amino acid residue relative to a hydrophobic region on the surface of Sulfolobus acidocaldarius ATCC 33909 maltooligosyltrehalose synthase. Increasements, to different extents, of maltooligosyltrehalose synthase soluble expression quantities are realized by the obtained mutant, the maltooligosyltrehalose synthase soluble expression quantities of mutants M106K and M106H are increased by 145.32 percent and 92.38 percent respectively; and increasement of mutant soluble expression is beneficial to industrial application of the mutant.

Description

technical field [0001] The invention relates to the preparation and application of a mutant of maltooligosaccharide-based trehalose synthase and belongs to the fields of genetic engineering and enzyme engineering. Background technique [0002] Trehalose (Trehalose) is composed of two glucopyranose linked by 1,1-glycosidic bonds. It is a stable non-reducing disaccharide. It has three optical isomers, namely αα type and αβ type. and ββ-type. [0003] Trehalose is a safe non-reducing disaccharide that widely exists in nature. It has special biological functions such as moisture retention, anti-freeze and anti-drying properties, and thermal-acid stability. It has non-specific protective effects on biological macromolecules , so it has great application potential in medicine, agriculture, cosmetics, food and other industries. Since the 1980s, countries have successively carried out studies on the physiology, biochemistry and molecular biology of trehalose, and the sugar has now...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12P19/12
CPCC12N9/1051C12P19/12C12Y204/01245
Inventor 吴敬宿玲恰吴世雄
Owner 湖南金代科技发展有限公司
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