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Novel maltooligosyl trehalose synthase, gene of synthase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of synthase

A trehalose synthase and malto-oligosaccharide-based technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low double-enzyme activity and easy contamination, and achieves the advantages of reduced enzyme cost, simple bacterial culture, and difficult bacterial contamination. Effect

Active Publication Date: 2013-07-10
SHANDONG TIANLI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Double-enzyme conversion at 50°C is prone to bacterial contamination, and starch hydrolyzate needs pullulanase debranching treatment
Although this double enzyme has higher optimum reaction temperature and lower optimum pH, but this double enzyme activity of literature report is all very low

Method used

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  • Novel maltooligosyl trehalose synthase, gene of synthase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of synthase
  • Novel maltooligosyl trehalose synthase, gene of synthase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of synthase
  • Novel maltooligosyl trehalose synthase, gene of synthase, recombinant expression vector containing gene, and recombinant bacterium, and preparation of synthase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Embodiment 1 bacterial cell culture

[0082] After extensive screening, Arthrobacter oxydans TL-3 with good activity of MTSase and MTHase ​​was selected, and the strain was deposited in the American Type Culture Collection (ATCC) with the deposit number: 14358. The medium composition of the strain is: peptone 10 g / L, yeast extract 30 g / L, glucose 10 g / L, MgSO 4 0.06 g / L, KH 2 PO 4 2.13 g / L, K 2 HPO 4 ·3H 2 O 16.43 g / L, pH 7.0. Heat sterilization at 121°C for 15 min.

[0083] The process of cell culture is as follows: After the culture medium is cooled, two rings are taken from the slant bacteria. A . oxydans TL-3 cells were inoculated into a 1000 mL Erlenmeyer flask filled with 200 mL of the above-mentioned medium, and cultured with shaking at 220 rpm and 38°C for 24 h.

Embodiment 2

[0084] Example 2 MTSase separation and purification and enzymatic characteristics research

Embodiment 2-1

[0085] Embodiment 2-1 MTSase separation and purification

[0086] (1) Bacterial fragmentation

[0087] Carry out bacterial cell culture according to the method in Example 1, obtain 10 liters of culture, centrifuge at 8000 rpm for 20 min, harvest 150 g of wet bacterial cells, resuspend in buffer, and sonicate the bacterial cells in an ice bath. The buffer composition is 0.2 Mol / L citrate buffer, pH5.5. Centrifuge (10000 r / min, 20 min) after sonication, and take the supernatant to obtain the crude enzyme solution.

[0088] (2) Ammonium sulfate precipitation

[0089] Both ammonium sulfate precipitation and dialysis were carried out in an ice bath. Ammonium sulfate was slowly added to the crude enzyme solution to make the saturation reach 40%, and left overnight at 4°C, then centrifuged at 12000 r / min for 20 min, and sulfuric acid was slowly added to the supernatant Ammonium was used to make the saturation to 60%, centrifuged again, the precipitate was collected and dissolve...

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Abstract

The invention discloses maltooligosyl trehalose synthase, a gene of the synthase, a recombinant expression vector containing the gene, and a recombinant bacterium, and preparation of the synthase. The synthetase has an amino acid sequence shown in SEQ ID NO:1, and can be obtained from the gene including the amino acid sequence shown in SEQ ID NO:2 or a mutant of the gene or a complementary sequence thereof by expression. The synthetase MTsase disclosed by the invention is high in optimal reaction temperature and thermal stability and low in optimal pH, reduces the contamination risk, improves the production stability, obviously improves the efficiency for producing trehalose from reducing starch hydrolysate by joint action with pullulanase, and obviously improves the production efficiency of the trehalose. The gene of expressing the synthase is obtained by the method; the MTSase is produced by a gene recombination technology; the enzyme expression quantity is high; the preparation efficiency is obviously improved; the cost is obviously reduced; and the production cost of the trehalose is also reduced.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to maltooligosaccharide-based trehalose synthetase and its expression gene, a recombinant expression vector containing the gene, a recombinant engineering bacterium, and a method for preparing the enzyme by utilizing the recombinant engineering bacterium containing the gene method. Background technique [0002] Trehalose (Trehalose) is a non-reducing sugar composed of two glucose molecules bonded by an α-1,1 glycosidic bond through a hemiacetal hydroxyl group. The molecular formula is C 12 h 22 o 11 , the relative molecular weight is 378.33. Its structural formula is as figure 1 Shown: [0003] [0004] figure 1 Molecular structure of trehalose [0005] Trehalose widely exists in bacteria, yeast, fungi, algae, insects and plants. It has a non-specific protective effect on organisms and biological macromolecules. This non-specific prot...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N1/21C12R1/19
Inventor 张全景刘敏付吉明陈平平庄祎王乔隆李慧君郑秀宁
Owner SHANDONG TIANLI PHARMA
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