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Novel application of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase in mycose production

A technology of trehalose synthase and trehalose hydrolase, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of susceptible bacteria and low double enzyme activity, and achieves improved stability, improved efficiency, high optimum reaction temperature and The effect of thermal stability

Active Publication Date: 2013-07-17
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 application of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase in mycose production
  • Novel application of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase in mycose production
  • Novel application of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase in mycose production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1 bacterial cell culture

[0100] After extensive screening, Arthrobacter oxidans with good activity of MTSase and MTHase ​​( Arthrobacter oxydans ) TL-3, the strain is deposited in the American Type Culture Collection (ATCC), 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.

[0101] 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

[0102] Example 2 MTSase separation and purification and enzymatic characteristics research

Embodiment 2-1

[0103] Embodiment 2-1 MTSase separation and purification

[0104] (1) Bacterial fragmentation

[0105] 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.

[0106] (2) Ammonium sulfate precipitation

[0107] 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 dissolved...

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Abstract

The invention discloses a novel application of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase in mycose production. The malt oligosaccharide based mycose synthetase has an amino acid sequence as shown in SEQ ID NO:1; and the malt oligosaccharide based mycose hydrolase has the amino acid sequence as shown in SEQ ID NO:3. The malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase disclosed in the invention also have higher most-suitable reaction temperature and thermal stabilities, and lower most-suitable pH values, so that contamination risks are lowered, production stability is improved, the mycose production efficiency by acting reducing glucidtemns through the malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase and pullulanase in a combined manner is improved and the mycose cost is lowered, therefore, a solid foundation is laid up for the extensive application of the mycose in medicines, foods and cosmetics.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to maltooligosaccharide-based trehalose synthase and maltooligosaccharide-based trehalose hydrolase in trehalose with higher optimum reaction temperature and thermal stability and lower optimum pH. application in production. Background technique [0002] Trehalose (Trehalose) is a non-reducing sugar composed of two glucose molecules bonded by α-1,1 glycosidic bonds through the 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 shown in formula 1: [0003] [0004] Formula 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 protective effect is mainly reflected in the fact that when ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/24C12P19/14C12P19/12
Inventor 张全景付吉明刘敏陈平平庄祎郑秀宁王乔隆李慧君
Owner SHANDONG TIANLI PHARMA
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