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Method for producing trehalose through multienzyme coupling and application thereof

A technology of trehalose and trehalose synthase, which is applied in the direction of fermentation, etc., can solve the problems of reduced utilization of starch substrates, difficulty in utilizing small molecule maltose, and increased production costs

Active Publication Date: 2018-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The dual-enzyme method uses starch as the substrate to produce trehalose, which has the advantage of low cost, but the affinity of maltooligosaccharide-based trehalose synthase to maltotetraose and maltotriose in the double-enzyme method is low, resulting in Small molecule maltose is difficult to utilize, which reduces the utilization rate of starch substrate in industrial production, thus increasing the production cost

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  • Method for producing trehalose through multienzyme coupling and application thereof
  • Method for producing trehalose through multienzyme coupling and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Comparison of multi-enzyme coupling catalysis and common double-enzyme synthesis of trehalose

[0069] 1) Ordinary double-enzyme synthesis of trehalose: add rice starch to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer to prepare a 15wt% rice starch solution, boil the rice starch solution in a boiling water bath, add 10U / mL source Stir with the α-amylase of Bacillus stearothermophilus for 25-30 minutes to liquefy the starch solution into a maltodextrin solution; after cooling the obtained maltodextrin solution to 60°C, add 5U / g Pullulanase from Bacillusderamificans, 2.5U / mL MTSase from Sulfolobus acidocaldariusATCC 33909, 2.5U / mL MTHase ​​from Sulfolobus acidocaldariusATCC 33909, pH5. 5. On a constant temperature water bath shaker at 60°C, 150r / min, stop the reaction after 35h of reaction and add 5U / g glucoamylase (purchased from Novozymes Biotechnology Co., Ltd.) to adjust the pH to 4.5. / min, saccharification for 24h and boiling.

[0...

Embodiment 2

[0079] Example 2: Effect of the amount of cyclodextrin glucosyltransferase added on the yield of trehalose

[0080] 1) Add rice starch to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer to prepare a 15wt% rice starch solution, boil the rice starch solution in a boiling water bath, add 10U / mL derived from Bacillus stearothermophilus ) α-amylase was stirred for 25-30 minutes to liquefy the starch solution into a maltodextrin solution; after the obtained maltodextrin solution was cooled to 60°C, 5U / g of pullulanase derived from Bacillus deramificans was added , 2.5U / mL MTSase from Sulfolobus acidocaldarius ATCC 33909, 2.5U / mL MTHase ​​from Sulfolobus acidocaldarius ATCC 33909, add 0.12, 0.18, 0.24 , 0.3, 0.36U / mL α-CGTase from Paenibacillus macerans, 0.5U / mL 4-αglycosyltransferase from Thermus aquaticus ATCC 33923, pH5.5, 60°C constant temperature water bath shaker, 150r / min, after 30 hours of reaction, stop the reaction and add glucoamylase to adjust pH 4.5,...

Embodiment 3

[0088] Embodiment 3: the influence of reaction temperature on trehalose yield

[0089] Add rice starch to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer to prepare a 15wt% rice starch solution, boil the rice starch solution in a boiling water bath, add 10U / mL derived from Bacillus stearothermophilus (Bacillus stearothermophilus) Stir α-amylase for 25-30 minutes to liquefy the starch solution into a maltodextrin solution; after cooling the obtained maltodextrin solution to 60°C, add 5 U / g of pullulanase from Bacillus deramificans, 2.5 U / mL MTSase from Sulfolobus acidocaldarius ATCC 33909, 2.5U / mL MTHase ​​from Sulfolobus acidocaldarius ATCC 33909, 0.24U / mL from Bacillus softening (Paenibacillus macerans) α-CGTase, 0.5U / mL 4-αglycosyltransferase from Thermus aquaticus ATCC 33923, pH 5.5, placed at 45, 50, 60, 65°C Water-bath shaker, 150r / min, after 30h of reaction, stop the reaction and add glucoamylase to adjust pH4.5, saccharify at 60°C constant temperatu...

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Abstract

The invention discloses a method for producing trehalose through multienzyme coupling and application thereof, and belongs to the technical field of enzymes. According to the method and the application, rice starch is used as a substrate, the disproportionate activity of 4-alpha glycosyl transferase and cyclodextrin glycosyltransferase are used, and pullulanase, maltooligosyl trehalose synthase (MTSase), maltooligosyl trehalose hydrolase (MTHase), the 4-alpha glycosyl transferase (TaAM) and the cyclodextrin glycanotransferase (CGTase) are adopted for multienyme coupling to improve a substrateuse ratio, so that the yield of the trehalose is improved.

Description

technical field [0001] The invention relates to a method for producing trehalose by multi-enzyme coupling and its application, belonging to the field of enzyme technology. Background technique [0002] Trehalose (Trehalose) is a safe non-reducing disaccharide connected by α,α-1,1-glucosidic bonds, which widely exists in nature and has a non-specific protective effect on biological macromolecules. Flavoring agent can better improve food flavor, so it is widely used in food, medicine, agriculture, cosmetics and other industries. [0003] Since the 1980s, countries have successively carried out research on the effect of trehalose on physiological functions, and now it has become one of the main oligosaccharides developed and researched internationally. [0004] The synthesis of trehalose mainly includes three methods: phosphorylase method, trehalose synthase method and double enzyme method. Among them, the conversion rate of trehalose produced by the dual-enzyme method is as ...

Claims

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Application Information

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IPC IPC(8): C12P19/18C12P19/14C12P19/12
CPCC12P19/12C12P19/14C12P19/18
Inventor 吴敬宿玲恰封金云陈春
Owner JIANGNAN UNIV
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