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Method for synthesizing trehalose under catalysis of co-immobilized double enzymes

A technology of trehalose and trehalose synthase, which is applied in the fields of immobilization on/in organic carriers, chemical industry, sustainable manufacturing/processing, etc., can solve the problems of low utilization rate and high price of catalytic raw materials and substrates, and achieve The effect of reducing raw material costs, shortening production cycle, and broad application prospects

Inactive Publication Date: 2015-02-04
NANJING HAIHE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to overcome the low utilization rate of enzymes (maltooligosaccharide-based trehalose synthase (MTSase) and maltooligosaccharide-based trehalose hydrolase (MTHase)) in the existing dual-enzyme catalytic system (Trehalose synthase (TreS)) Catalyzing the problem of high price of raw materials and substrates, using low-cost starch as the catalytic substrate and using a single-enzyme catalytic system to provide a new method for synthesizing trehalose with immobilized catalytic substrates , to reduce the cost of raw materials, thereby further reducing the production cost of trehalose

Method used

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  • Method for synthesizing trehalose under catalysis of co-immobilized double enzymes
  • Method for synthesizing trehalose under catalysis of co-immobilized double enzymes

Examples

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Embodiment 1

[0038] This example illustrates the source of strains.

[0039] The strain used in this patent is Escherichia coli derived from the trehalose synthase gene of Thermus thermophilus ATCC 33923 (Gene Bank: AQOS01000003) saline-alkali soil metagenomes (Saline-Alkali Soil Metagenomes). Using the document "Ling Jiang, Ming Lin, Yang Zhang et al. Identification and Characterization of a Novel Trehalose Synthase Gene Derived from Saline-Alkali Soil Metagenomes. Pols One 8(10): e77437. doi previously published by the inventor of the present application : 10.1371 / journal.pone.0077437." The method described in, the bacterial strain number that component obtains is BL21-treS.

[0040] At the same time, the applicant hereby declares that within 20 years from the date of patent application, the starting bacterial strain described in the present invention will be provided free of charge to the public.

Embodiment 2

[0042] This example illustrates the specific synthesis steps and synthesis effects of catalytic synthesis of trehalose.

[0043] The BL21-treS strain obtained in Example 1 was used for trehalose synthesis catalysis.

[0044] The high-temperature resistant α-amylase and β-amylase used in this example were all purchased from Aladdin Company with a purity of 93%-96%.

[0045] Firstly, the genetic engineering bacteria of Escherichia coli were cultivated on the solid LB medium with ampicillin by streaking method to obtain single colonies of Escherichia coli. The formula of the solid medium with ampicillin was: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, ampicillin antibiotic 0.1 g / L, agar 20 g / L. The specific operation process is to prepare the medium according to the requirements, sterilize the prepared culture at 121°C for 30 minutes, pour it on the plate after cooling, inoculate with the streaking method, and culture in a constant temperature incubator to obtain...

Embodiment 3

[0060] This example illustrates the effect of using pullulanase instead of β-amylase to catalyze the synthesis of trehalose from starch hydrolyzate after dual enzyme immobilization.

[0061] The pullulanase used in this example was purchased from Aladdin with a purity of 93%-96%.

[0062] According to the method described in Example 2, the method for synthesizing trehalose by co-immobilization of double enzymes to catalyze starch hydrolyzate is different in that:

[0063] Its step (6) replaces β-amylase with pullulanase to catalyze the synthesis of trehalose from starch hydrolyzate after double-enzyme immobilization

[0064] After testing, the initial conversion rate was 64.9%, the conversion rate was 63.2% after the dual-enzyme-immobilized microspheres were reused 8 times, and the conversion rate was 60% after the dual-enzyme-immobilized microspheres were reused 16 times, with an average conversion time of 23h, and the process of repeated production 16 times was not contamin...

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Abstract

The invention relates to a method for synthesizing trehalose under catalysis of co-immobilized double enzymes. According to the method, recombinant escherichia coli capable of synthesizing high-temperature-resisting trehalose synthase in a great quantity by fermentation culture is subjected to permeability treatment, and beta-amylase is added; chitosan and sodium alginate are used for co-immobilizing to form micro-spheres; starch with the weight content of 25%-30% is catalyzed by alpha-amylase and then is subjected to a contact reaction with the micro-spheres to generate the trehalose, wherein after the micro-spheres are repeatedly utilized for 8-16 times, the conversion rate of the starch can be up to 60%-65%. By adopting the method provided by the invention, the high-quality trehalose can be provided in a large-scale, low-cost and high-efficiency manner.

Description

technical field [0001] The invention relates to a method for synthesizing trehalose catalyzed by co-immobilized double enzymes, belonging to the technical field of functional sugar alcohol synthesis. It specifically relates to a method for catalytically synthesizing trehalose by using starch as a raw material. Background technique [0002] Trehalose is a non-reducing disaccharide formed by combining two glucose molecules with α,α-1,1 glycosidic bonds. Trehalose has no hemiacetal hydroxyl group and is chemically stable. Since Wiggers first extracted trehalose from ergot bacteria in 1832, trehalose has been widely found in animals, plants and microorganisms in nature, such as bacteria, archaea, yeast, mushrooms and invertebrates. Because trehalose can form a unique protective film on the cell surface under harsh environmental conditions such as high temperature, high cold, high osmotic pressure, and dehydration, effectively protecting protein molecules from denaturation and i...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04C12P19/12
CPCY02P20/50
Inventor 黄和江凌崔怀言晏晓琴李霜
Owner NANJING HAIHE BIOTECH
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