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Method for constructing self-assembled expression double-enzyme strain and application

A technology of self-assembly and bacterial strains, which is applied in the fields of genetic engineering and enzyme engineering, and can solve problems such as lower conversion rates

Active Publication Date: 2020-09-11
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the prior art, it has not been reported that the self-assembly system is used to construct MTSase-MTHase ​​multi-enzyme complexes with different ratios and structures in recombinant E. coli, and the effect on the conversion rate of trehalose
[0004] Chinese Patent Document Application No. 2020101068312 discloses the construction and application of a recombinant Bacillus subtilis that secretes MTHase ​​and MTSase synchronously. The ratio of trehalose increases, and the conversion rate of trehalose increases gradually. The conversion rate of the multi-enzyme complex forming MTSase:MTHase=3:1 in Bacillus subtilis is the highest 81.5%. The conversion rate when =3:1 slightly reduces, is because the spatial structure between multiple enzymes influences and the binding ability of substrate causes; Can find out by the conversion rate result of comparative example 1,4,5,6, The ratio of MTSase determines the conversion rate, which is the limiting factor in the process of double-enzyme conversion and production of trehalose; the multi-enzyme complex recombinant bacteria disclosed in this invention is Bacillus subtilis, and the multi-enzyme complex produced is an extracellular enzyme , that is, the conversion rate of the fermentation supernatant to trehalose, but this patent document does not involve the influence of different ratios of multi-enzyme complexes on the conversion rate of trehalose in other recombinant bacteria, and the same ratio of multi-enzyme complexes, and The different binding modes of the domain SpyTag-SpyCatcher affect the conversion rate of trehalose, and it does not involve the effect of the multi-enzyme complex as an intracellular enzyme on the conversion rate of algae

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  • Method for constructing self-assembled expression double-enzyme strain and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] A method for constructing a self-assembled expression dual enzyme strain, comprising the steps of:

[0065] (1) Acquisition of the target gene

[0066] Using the genome of Sulfolobus acidocaldarius ATCC 33909 as a template, primers were designed with seamless cloning software, and PCR amplification was performed to obtain MTSase encoding gene treY and MTHase ​​encoding gene treZ, the sequence of the gene treY is SEQ ID NO. 1. The sequence of the gene treZ is SEQ ID NO.2; the artificially synthesized SpyCatcher gene sequence is used as a template, primers are designed with seamless cloning software, and the SpyCatcher gene is obtained by PCR amplification, and the SpyCatcher gene sequence is SEQ ID NO. 3. The SpyTag gene sequence is directly synthesized in the primer, and the SpyTag gene is obtained by PCR amplification, and the SpyTag gene sequence is SEQ ID NO.4.

[0067] The amplification primer sequence of the treY gene is as follows:

[0068] treY-F: GCGATGCGCATAT...

Embodiment 2

[0128] Three target expression strains prepared in Example 1 and one target expression strain involved in Comparative Example 1 were used to prepare fermentation broth.

[0129] The target expression strain was inoculated in LB liquid medium supplemented with kanamycin resistance (final concentration of kanamycin: 80 μg / mL), and cultured at 37° C. and 200 rpm for 12 hours as a seed solution. The seed solution was inoculated into TB liquid medium with kanamycin resistance (final concentration of kanamycin: 80 μg / mL) at a volume ratio of 1:100, cultured at 37 °C and 200 rpm for 8 h, and lactose was added as Inducer (the final concentration of lactose is 6 mg / L), the temperature was adjusted to 25° C. and the fermentation was continued for 12 hours to obtain the fermentation broth of the target expression strain.

Embodiment 3

[0131] The double-enzyme complex enzyme is used for substrate conversion. Using the fermented liquid of 4 kinds of purpose expression bacterial strains prepared in Example 2, each 100 mL of the fermented liquid is centrifuged at high speed to obtain the bacterial cell precipitation, and the bacterial cell is weighed with 10 mL of 10 mM PBS buffer solution of pH5.5. Suspension, using an ultrasonic breaker to break the cells, the breaking conditions are: power 300w, breaking time 3s, intermittent time 5s, time 15min, to obtain crude enzyme solution, 65 ° C, pH 5.5 conditions of conversion concentration of 15% maltodextrin , and add 5% cyclodextrin glucosyltransferase (CGTase), transform for 12h to complete transformation, after the transformation is completed, boil at 100°C for 10min to inactivate and terminate the reaction, and detect the content of trehalose in the sample by HPLC.

[0132]

[0133] The strain E.coli BL21(DE3) / pET28a-SpyCatcher-treY-SpyCatcher / pETDuet-SpyTag-...

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Abstract

The invention relates to a method for constructing a self-assembled expression double-enzyme strain and application. The method comprises the following steps of constructing MTSase-MTHase multi-enzymecomplexes with different proportions and different structures to obtain an escherichia coli recombinant. According to the invention, the MTSase-MTHase multienzyme complexes with different proportionsare discovered, and the obtained escherichia coli recombinant has a certain influence on the conversion rate of trehalose; through the MTSase-MTHase multienzyme complexs with the same proportion anddifferent structures, the influence of the obtained escherichia coli recombinant on the conversion rate of the trehalose is large; and a theoretical basis is provided for research on production of trehalose by fermentation of a multi-enzyme system.

Description

technical field [0001] This research relates to a method and application for constructing self-assembled and expressing double-enzyme strains, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Trehalose (α, α-Trehalose, Trehalose) was first discovered by Wiggers in rye ergot fungi in 1832. Trehalose in nature is composed of two molecules of glucose connected by α-1,1-glycosidic bonds. It is not easy to be hydrolyzed by acid, and the α-1,1-glycosidic bond cannot be digested by α-glucosidase. Trehalose is known as the "sugar of life" because of its excellent anti-stress protection properties for biological macromolecular life forms, and has a wide range of applications in food transportation and medical preservation. [0003] At present, the production of trehalose by the double-enzyme method has become the first choice for industrial production of domestic and foreign enterprises. This production method has a highe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/54C12N15/56C12N1/21C12P19/14C12P19/12C12R1/19
CPCC12N9/1051C12N9/2402C12N15/70C12P19/14C12P19/12C12Y204/01245C12Y302/01028
Inventor 王腾飞刘洪玲王松
Owner QILU UNIV OF TECH
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