A method for preparing trehalose by continuous extracellular enzyme biological method

A technology of trehalose and biological method, applied in the field of fermentation engineering, can solve the problems of increasing the preparation cost of chemical reagents, increasing the cost of crushing, and the inability to recover free enzymes, so as to reduce the probability of contamination and the generation of by-products, and avoid fragmentation and separation and purification. Cost and effect of solving the difficulty of recycling

Active Publication Date: 2021-01-22
QILU UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, in view of the low enzyme activity produced by the original enzyme-producing strains after fermentation, the fermentation cycle is very long, the energy consumption is high, the limiting factors of the fermentation cost are high, and the production efficiency is low. By constructing recombinant Escherichia coli, the double enzyme and single enzyme expression, but the enzyme produced by E. coli is expressed intracellularly and needs to be obtained after crushing, and the free enzyme has disadvantages such as non-recyclable, low utilization rate, difficult separation and purification, and high cost of use, which not only increases the cost of crushing, And there are food safety constraints
Immobilized enzyme-producing and permeabilized cells can reduce a large number of broken cells, improve the stability of enzymes, and save the process of extracting and purifying enzymes. However, the addition of chemical reagents increases the cost of preparation, and the utilization rate of equipment in industrialized continuous production Low, the production cycle is too long

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A method for preparing trehalose by a continuous extracellular enzyme biological method, comprising the steps of:

[0058] (1) Activate the extracellularly secreted enzyme-producing strains that synthesize trehalose using soluble dextrin as a substrate in LB medium for 10 hours at 37°C, then inoculate LB medium at a volume percentage of 10%, and expand at 37°C Cultivate for 20 hours to obtain a bacterial solution with a bacterial cell concentration of 50-80 g / L;

[0059] In the step (1), the trehalase synthesized by using soluble dextrin as a substrate is a fusion enzyme of maltooligosaccharide-based trehalose synthase and maltooligosaccharide-based trehalose hydrolase, and its expression strain is Bacillus subtilis that secretes and expresses the fusion enzyme Bacillus (refer to the construction method disclosed in Chinese patent document CN105039374A (application number 201510486544.8) for preparation);

[0060](2) Inoculate the bacterial solution obtained in step (1...

Embodiment 2

[0072] A method for preparing trehalose by a continuous extracellular enzyme biological method, comprising the steps of:

[0073] (1) Activate the extracellularly secreted trehalose-producing enzyme strain using maltose as a substrate in LB medium at 37°C for 10 hours, then inoculate it into LB medium at a volume percentage of 10%, and carry out expansion culture at 37°C for 20 hours , to obtain a bacterial solution with a cell concentration of 50-80 g / L;

[0074] In the step (1), the enzyme that synthesizes trehalose using maltose as a substrate is trehalose synthase, and its expression strain is disclosed in Bacillus subtilis that secretes and expresses trehalose synthase (see Chinese patent document CN105039381A (application number 201510431599.9) The construction method is prepared);

[0075] (2) Inoculate the bacterial solution obtained in step (1) into the fermentation medium, ferment and cultivate at 37°C for 24 hours, induce the expression of extracellular secretion w...

Embodiment 3

[0087] A method for preparing trehalose by a continuous extracellular enzyme biological method, comprising the steps of:

[0088] (1) The strains displaying enzymes on the surface of spores that use maltose as a substrate to synthesize trehalose were activated in LB medium at 37°C for 8 hours, and then inoculated into fermentation medium at a ratio of 10%, at 37°C, at high density After 48 hours of fermentation and cultivation, it enters the nutrient deficient stage, and Bacillus subtilis begins to produce spores. After continuing to ferment for 8 hours, the spores are separated by microfiltration membrane filtration, and the spores displaying trehalose synthase on the surface are obtained;

[0089] In the step (1), the enzyme that synthesizes trehalose using maltose as a substrate is trehalose synthase, and its expression strain is disclosed in Bacillus subtilis that displays trehalose synthase on the surface (see Chinese patent document CN105132450A (application number 201510...

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Abstract

The invention relates to a method for preparing trehalose by a continuous exoenzyme biological process. The complete cell immobilized or secretive trehalose synthase gene engineering bacterium is utilized to continuously and efficiently produce trehalose and perform continuous chromatographic separation, purification and crystallization so as to prepare the trehalose. By establishing the high-continuity enzyme production-trehalose preparation production technique, the technique implements transformation by using the complete cell surface display or exocytosis trehalose synthase, or malt-oligosaccharide-base trehalose synthetase-malt-oligosaccharide-base trehalose hydrolase fused enzyme, thereby avoiding the cost on cell crushing and separation purification. The enzyme solution or immobilized enzyme is convenient to acquire and simple to operate.

Description

technical field [0001] The invention relates to a method for preparing trehalose by a continuous extracellular enzyme biological method, in particular to a method for continuously and efficiently producing trehalose by using whole cell immobilization or secreting trehalose synthase genetic engineering bacteria and preparing seaweed by continuous chromatographic separation, purification and crystallization The sugar method belongs to the technical field of fermentation engineering. Background technique [0002] Trehalose is a non-reducing disaccharide composed of two glucose molecules with α, α-1,1 glycosidic bonds, which widely exists in microorganisms, seaweeds, mushrooms, beans, shrimps and other organisms. Because trehalose has a non-reducing end and has good stability in extreme environments, it is widely used in food, medicine, cosmetics, agriculture and other industries. Exogenous trehalose also has non-specific protective effects on organisms and biological macromolec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/12C12R1/84C12R1/125
CPCC12P19/12
Inventor 王腾飞王瑞明汪俊卿王建彬郭传庄
Owner QILU UNIV OF TECH
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