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Thermostable trehalose synthase as well as expression gene and application thereof

A trehalose synthase and gene expression technology, applied in the fields of application, genetic engineering, glycosyltransferase, etc., can solve the problems of poor thermal stability, long reaction time, low substrate conversion rate, etc., and achieve the effect of increasing expression

Active Publication Date: 2015-09-30
湖南尚道生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The catalytic efficiency and enzymatic properties of trehalose synthase from different microbial sources are different, but they have the same basic characteristics: first, the substrate conversion rate is low, the highest is only about 80%, generally 60%-70%; The thermal stability of the enzyme is poor, and the optimum reaction temperature is about 25°C; the third is that the reaction time is too long, generally 48 hours, and some as long as 72h

Method used

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  • Thermostable trehalose synthase as well as expression gene and application thereof
  • Thermostable trehalose synthase as well as expression gene and application thereof
  • Thermostable trehalose synthase as well as expression gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning the trehalose synthase gene of Pseudomonas putida KT2440 N42-C680 fragment

[0032] Based on the full-length amino acid sequence of Pseudomonas putida KT2440 published on NCBI, the three-dimensional structure simulation was carried out. The simulation results are as follows: figure 1 As shown, it is found that the two ends of the full-length trehalose synthase protein have obvious flexible regions, that is, the N-terminal to the 41st glutamic acid and the 680th isoleucine to the C-terminal. Design primers to remove the flexible regions at both ends, and the primer sequences are as follows:

[0033] Upstream primer: 5'-atc gga tcc ATG GCCCAGCCCCGC-3';

[0034] Downstream primer: 5'-tca ctc gag tta CCGCAGGCACAG-3';

[0035] Using the full-length gene of Pseudomonas putida KT2440 as a template, PCR amplification was performed using the above primers. The PCR reaction system was as follows:

[0036]

[0037] The above-mentioned PCR reaction is carri...

Embodiment 2

[0040] Example 2: Transforming the modified trehalose synthase gene into an expression host to obtain a positive expression strain.

[0041] Double digestion reaction of PCR product and plasmid vector

[0042] Enzyme digestion system for PCR products:

[0043]

[0044] Reaction conditions: react at 37°C for 2 to 3 hours.

[0045] Enzyme digestion system for plasmid vectors:

[0046]

[0047] Reaction conditions: react at 37°C for 6-8 hours.

[0048] The PCR product and the vector digested product were subjected to 1% agarose gel electrophoresis, and were purified and recovered using a DNA gel recovery kit.

[0049] Connection reaction system:

[0050]

[0051] Mix well and centrifuge for a few seconds, collect the drop on the tube wall to the bottom of the tube, and connect overnight at 16°C to obtain the ligated product.

[0052] Transformation of recombinant plasmids

[0053] (1) Preparation of Competent Cells

[0054] ①Pick a single colony of BL21 (or pick a p...

Embodiment 3

[0076] Example 3: Fermentation and culture of positive expression strains, separation and purification of recombinant recombinant trehalose synthase protein

[0077] Seed culture: Pick positive clones in a conventional method and place them in 5 mL of LB liquid medium containing 100 mg / L ampicillin, and shake and culture at 37°C for 5-6 hours;

[0078] Ultrasonic disruption of bacterial cells: ultrasonic 3s, interval 6s, 400W, work 60 times.

[0079] Ultracentrifugation: after sonication, the cell disruption solution was centrifuged at 14000rpm, 4°C for 45min, and the supernatant was collected for the next step of separation and purification.

[0080] Ni-NTA affinity chromatography: pour the collected supernatant containing soluble protein into the regenerated Ni-NTA column; , 15mM imidazole) to wash 10 column volumes to remove non-specifically adsorbed proteins; finally use elution buffer (25mM Tris-HCl, pH8.0, 100mM NaCl, 250mM imidazole) to elute the target protein and col...

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Abstract

The invention relates to a thermostable trehalose synthase as well as an expression gene and application thereof. The nucleotide sequence of the expression gene of the transformed thermostable trehalose synthase is shown by SEQ ID No.1; and the amino acid sequence of the transformed thermostable trehalose synthase is shown by SEQ ID No.2. In the invention, the trehalose synthase with higher stability is obtained for the first time based on the three-dimensional structure of pseudomonas putida trehalose synthase (pseudomonas putida KT2440) and by cutting the flexible area in the structure and retaining the stable structure domain at the activity center. The preparation method of the trehalose synthase is simple, the yield is large, and the purity is high; and experiments prove that the transformed trehalose synthase still keeps relatively high catalytic activity, the thermal stability is greatly improved, the production cost of trehalose can be reduced, and a foundation is laid for the industrial production of trehalose.

Description

technical field [0001] The present invention relates to a high-temperature-resistant trehalose synthase and its expression gene and application, in particular to a high-temperature-resistant trehalose synthase obtained after directional transformation of Pseudomonas putida trehalose synthase and its expression gene and application. It belongs to the technical field of biotechnology. Background technique [0002] Trehalose (Trehalose) is a non-reducing disaccharide composed of two glucopyranose molecules linked by α-1,1-glycosidic bonds, widely present in bacteria, yeast, filamentous fungi, plants, insects, organisms such as vertebrates. Studies have shown that its properties are stable and have very important biological significance to organisms. It is mainly manifested in the fact that it is a reserve of energy and carbon sources for organisms, a stabilizer and protector for proteins and biofilm molecules in harsh environments such as dehydration, high temperature, oxygen...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/18C12P19/12C12R1/19
CPCC12N9/1081C12P19/12C12P19/18C12Y204/99
Inventor 苏静王瑞明李珍珍张云霄
Owner 湖南尚道生物科技有限公司
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