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Method for producing trehalose

A production method, the technology of trehalose, is applied in the field of converting maltose into trehalose by using genetically engineered bacteria, which can solve the problems of difficult extraction, difficult mass production of trehalose synthase, difficult immobilization and repeated utilization, etc., and achieves the goal of reducing production costs Effect

Inactive Publication Date: 2014-09-17
CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to construct a trehalose synthase genetically engineered bacterium that exhibits activity on the surface of yeast, thereby overcoming the technical problems that trehalose synthase is difficult to produce in large quantities, difficult to extract, and difficult to immobilize and reuse

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  • Method for producing trehalose

Examples

Experimental program
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Effect test

Embodiment 1

[0011] Example 1 Pichia pastoris displays trehalose synthase from Streptomyces coelicolor

[0012] 1. The synthesis of the trehalose synthase gene (Trehalose synthase, TS) of Streptomyces coelicolor was entrusted to a biological company (in this case, Nanjing GenScript Biotechnology Co., Ltd.), and the gene was cloned on the pUC57 vector (pUC57-TS), and the DNA sequence As shown in SEQ NO1.

[0013] 2. TS subcloning into pPIC9K vector

[0014] The pUC57-TS and pPIC9K vectors were digested with Eco RI and Not I respectively, and the enzyme digestion reaction systems were as follows:

[0015] pUC57-TS plasmid 34ul

[0016] 10* buffer 4 ul

[0017] Eco RI 1 ul

[0018] Not I 1 ul

[0019] A total of 40ul system,

[0020] pPIC9K plasmid 6ul

[0021] 10* buffer 2 ul

[0022] Eco RI 1 ul

[0023] Not I 1 ul

[0024] wxya 2 O 10 ul

[0025] A total of 20 ul system,

[0026] After mixing, they were placed in a water bath at 37°C for 4 h.

[0027] 3. Recover the target ge...

Embodiment 2

[0065] Example 2 Amplification and cultivation of genetically engineered bacteria

[0066] 1. Pick a single colony from the plate, inoculate it in YPD medium, and culture overnight at 30°C and 250 rpm;

[0067] 2. Transfer 1% of the inoculum to fresh BMGY medium, culture at 30°C, 250 rpm for 5 days, add methanol every 24 hours to induce, collect the fermented bacteria, wash 3 times with pH8.0 buffer, and wash with A small amount of buffer solution is resuspended to obtain the recombinant trehalose synthase genetically engineered bacteria liquid, which is centrifuged for dehydration or freeze-dried for later use.

[0068]

Embodiment 3

[0069] Example 3 Production of Trehalose Using Maltose Syrup

[0070] Use pure maltose, pH7.0 disodium hydrogen phosphate and sodium dihydrogen phosphate buffer to make a reaction substrate containing 30% maltose, pH7.0, take 1 liter of prepared reaction substrate and put it into stainless steel In the reaction tank, the temperature is controlled at about 30°C, and the genetically engineered bacterial agent exhibiting trehalose synthase in Example 2 is added according to the weight of 6% sugar solution (about 0.03 liters in volume), and reacted for 4 hours, and then used The HPLC analysis of the various sugar content of the reaction product showed that trehalose was 23.2%, maltose was 4.9%, and glucose was 1.4%. According to 1 liter of 30% maltose + 0.03 liter of enzyme solution, the estimated substrate concentration was 29.1%, and the total conversion rate It is 23.2 / 29.1=79.7%, that is, the content of trehalose in the product of maltose conversion can reach 80%; after the re...

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Abstract

The invention relates to a method for converting maltose into trehalose by using genetically engineered bacteria. According to the technical scheme, the method comprises the following steps: demonstrating a Streptomycescoelicolor, AS4.1061 trehalose synthase gene on the surface of pichia pastoris to establish a genetically engineered bacterium with enzyme activity, performing multiplication culture on the genetically engineered bacterium to greatly increase the yield of the trehalose synthase, adding the genetically engineered bacterium with the demonstrated trehalose synthase into maltose or starch modified maltose, reacting for 1-8 hours at 25-45 DEG C so as to convert maltose into trehalose, wherein the content of trehalose in a maltose converted product can be 80%, and moreover the genetically engineered bacterium can be recycled and used as a solvent after being separated, washed and dehydrated. Therefore, the technical difficult that the trehalose synthase cannot be massively produced, is not easy to extract and is hard to recycle and repeatedly use in an immobilization manner are overcome.

Description

technical field [0001] The invention belongs to the field of food processing and biotechnology, in particular to a method for converting maltose into trehalose by using genetically engineered bacteria. Background technique [0002] Trehalose (Trehalose) and the reducing disaccharide maltose are isomers, which are non-reducing disaccharides formed by connecting two molecules of glucose with α, α-1, 1-glycosidic bonds. It is known as "the sugar of life" and "the new type of sugar in the 21st century" because of its excellent anti-stress protection effect and other excellent properties. Its large-scale production method has attracted widespread attention; the Chinese patent with the patent publication number CN 102154326 A Reported a production method of cloning the Trehalose synthase (TS) gene of Streptomyces coelicolor (AS 4.1061) into Escherichia coli, amplifying and culturing it, and then breaking the wall to extract the enzyme to convert maltose into trehalose. Although t...

Claims

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Application Information

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IPC IPC(8): C12P19/12C12N15/81C12N1/19C12R1/84
Inventor 谢定盛敏谢易真俞健盛灿梅
Owner CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
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