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Trehalose synthase and application thereof in trehalose production

A technology of trehalose synthase and mutants, applied in the field of enzyme engineering, can solve the problems of low enzyme activity and trehalose conversion rate, difficult large-scale industrial production, influence of trehalose purity, difficult large-scale industrial production, etc. The effect of improving the conversion rate of living and trehalose, low requirements for production conditions and low production cost

Active Publication Date: 2019-04-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the use of maltooligosaccharide-based trehalose synthase to produce trehalose will eventually accumulate a large amount of oligosaccharides such as maltotriose, which will affect the purity of trehalose; trehalose synthase is due to low enzyme activity and trehalose conversion rate Difficult for large-scale industrial production
[0008] Therefore, it is urgent to find a trehalose synthase with high enzymatic activity and trehalose conversion rate to solve the problem that it is difficult to carry out large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Extraction of the gene encoding trehalose synthase and construction of recombinant bacteria containing the gene encoding trehalose synthase

[0055] Specific steps are as follows:

[0056] (1) Extraction of Streptomyces_coelicolor (GDM 4.65) genomic DNA

[0057] The bacterial DNA genome extraction kit purchased by Shanghai Jierui Bioengineering Co., Ltd. was used to extract Streptomyces_coelicolor (GDM 4.65) genomic DNA;

[0058] Pick a single colony of Streptomyces_coelicolor (GDM 4.65) and inoculate it into Gaoshi Synthetic No. 1 liquid medium, shake it at 30°C and 180rpm for 5 days, then centrifuge at 8000rpm for 2 minutes to collect the bacteria; use deionized water After washing the bacteria, centrifuge again to collect the bacteria and suspend the collected bacteria in 150 μL TE buffer to obtain a resuspension; first add 20 μL lysozyme to the resuspension, incubate at 37°C for 30 minutes, and then add 300 μL Digestion Solution, mix well, add 4 μL RNas...

Embodiment 2

[0074] Example 2: Expression of trehalose synthase in Escherichia coli host

[0075] The recombinant bacteria E.coli BL21 pET28a-ScTreS obtained in Example 1 and the recombinant bacteria BL21 / pET28a-CgTreS and BL21 / pET28a-PsTreS obtained in Comparative Example 1 were added to 10 mL of LB medium respectively, under the conditions of 37 ° C and 180 rpm After culturing for 10 hours, transfer to 50mL LB liquid medium with 1% inoculum amount, culture at 37°C and 180rpm for 2-3 hours, then add IPTG with a final concentration of 0.5mM and continue to induce culture at 16°C 12h, to obtain the fermentation broth.

[0076] The fermentation broth was centrifuged to collect the bacteria, washed with pH 7.0 50mM sodium phosphate buffer and suspended, ultrasonically broken and centrifuged to obtain the supernatant to obtain a crude enzyme solution, and then the activity of trehalose synthase in the crude enzyme solution was detected.

[0077] The test results are: the specific enzyme activit...

Embodiment 3

[0078] Embodiment 3: the expression of trehalose synthase in Corynebacterium glutamicum host

[0079] Add the recombinant bacterium C. glutamicum pXMJ19-ScTreS obtained in Example 1 into 10 mL of BHI liquid medium, culture it at 30°C and 180 rpm for 16 hours, and then transfer it into In the 50mL BHI liquid culture medium, continue to cultivate at 30°C and 180rpm for 5-8h, then add IPTG with a final concentration of 0.5mM, and continue to induce culture at 16°C for 12h to obtain a fermentation broth.

[0080] Centrifuge the fermentation broth to collect the bacteria, wash and suspend it with pH 7.0 50mM sodium phosphate buffer, add 20 μL of 0.2 mg / mL lysozyme and let it stand on ice for 2 hours, ultrasonically break and centrifuge to get the supernatant to obtain a crude enzyme solution, and then detect the crude enzyme Trehalose synthase enzyme activity in the solution.

[0081] The detection result is: the specific enzyme activity of trehalose synthase in the crude enzyme l...

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Abstract

The invention discloses a trehalose synthase and application thereof in trehalose production, and belongs to the technical field of enzyme engineering. The trehalose synthase of the invention has highenzyme activity, the specific enzyme activity of the trehalose synthase in a crude enzyme solution is as high as 35.2 U / mg when Escherichia coli carrying the trehalose synthase undergoes induction culture for 12 hours, and is as high as 33.5 U / mg when corynebacterium glutamate carrying the trehalose synthase undergoes induction culture for 12 hours. The specific enzyme activity and the trehaloseconversion rate of a trehalose synthase mutant of the present invention are significantly improved than those of wild-type trehalose synthase, wherein the specific enzyme activity of the trehalose synthase mutant K246A is improved by 1.43 times than that of the wild-type trehalose synthase, and the trehalose conversion rate of the trehalose synthase mutant K246A is improved by about 15% than thatof the wild-type trehalose synthase.

Description

technical field [0001] The invention relates to a trehalose synthase and its application in the production of trehalose, belonging to the technical field of enzyme engineering. Background technique [0002] Trehalose (Trehalose) is a disaccharide that exists widely in nature. It is composed of two glucose linked by α,α-1,1-glycosidic bonds. Bacteria, fungi, yeast, lower ferns, algae, insects and invertebrates. [0003] In addition to serving as a structural component and providing energy in the organism, trehalose plays the most important role as a typical stress metabolite, protecting the proteins and lipids in the cells of the organism under many environmental conditions such as dryness, low temperature, and hypertonicity. Therefore, trehalose has become an important protective agent for the preservation of biological activity of vaccines, enzymes, living tissues and cells; at the same time, trehalose is resistant to acid and It has high thermal stability, can prevent st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12P19/24C12P19/12
CPCC12P19/12C12N15/70C12N15/77C12N9/90C12Y504/99016C12P19/24
Inventor 饶志明吴傲张显徐美娟杨套伟邵明龙
Owner JIANGNAN UNIV
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