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Application of high-glycosyl-transfer-activity bacillus amyloliquefaciens in biosynthesis of salidroside in non-aqueous phase

A technology for dissolving starch spores and salidroside, which is applied in microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of loss of catalytic activity, stability and easy loss of plasmids, and achieves low cost and prevents impurities. The effect of bacterial interference and reduction of bacterial infection efficiency

Active Publication Date: 2019-02-12
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the method for the synthesis of salidroside with glycosyltransferase (or genetically engineered strains containing glycosyltransferase) as a catalyst has made remarkable progress and has ideal industrial application prospects. However, glycosyltransferase is still The rate-limiting step in the sedroside biosynthesis system, and the stability of genetically engineered strains during large-scale fermentation (genetically engineered bacteria are easily lost during large-scale fermentation of exogenously inserted plasmids, thereby losing catalytic activity), and the ability of substrate molecules The adaptability and tolerance of salidroside are still the bottleneck restricting the biosynthesis of salidroside

Method used

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  • Application of high-glycosyl-transfer-activity bacillus amyloliquefaciens in biosynthesis of salidroside in non-aqueous phase
  • Application of high-glycosyl-transfer-activity bacillus amyloliquefaciens in biosynthesis of salidroside in non-aqueous phase
  • Application of high-glycosyl-transfer-activity bacillus amyloliquefaciens in biosynthesis of salidroside in non-aqueous phase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Screening of Salidroside Efficient Synthesis Strains

[0032] Rescreening was carried out from the bacterial library with glycosylation activity independently developed by our laboratory (the bacterial library contains hundreds of strains with glycosylation activity, such as Pseudomonas XT02 and Bacillus amyloliquefaciens FJ18), to obtain energy A strain that efficiently converts tyrosol to salidroside in a non-aqueous phase.

[0033] LB plate medium: yeast extract 5.0g / L, peptone 10.0g / L, NaCl 10.0g / L, agar powder 20g / L.

[0034] The composition of the screening medium is: tyrosol 1g / L, peptone 10g / L, yeast extract 5g / L, NaCl 10.0g / L, glucose 20g / L, MgSO 4 ·7H 2 O 0.5g / L, DMSO 10% (v / v), initial pH 7.0.

[0035] The strains with transglycosylation activity frozen in the bacterial bank were recovered in LB plate medium, cultured at 30°C for 24 hours, and then inoculated into the screening medium (50mL culture solution / 250mL Erlenmeyer flask), at 30°C, Cultivate at 2...

Embodiment 2

[0037] Fermentation of Bacillus amyloliquefaciens FJ18 and preparation of resting cells

[0038] Fermentation of Bacillus amyloliquefaciens FJ18 was inoculated into the seed medium: yeast extract 5.0g / L, peptone 10.0g / L, NaCl 10.0g / L, pH 7.0, cultured at 30°C, 200rpm for 12 hours. Expansion medium, fermentation medium (using a self-developed medium conducive to glycosyltransferase enzyme activity), its components and contents are: glucose 20g / L, peptone 15g / L.KH 2 PO 4 1.0g / L, CaCl 2 0.8g / L, MgSO 4 ·7H 2O 0.5g / L. The pH was adjusted to 7.5 with NaOH. The seed solution was inoculated into the expansion medium and the fermentation medium at 0.5% (v / v), and cultivated at 30° C. and 200 rpm for 12 hours. After centrifuging at 10000rpm for 15 minutes, the bacterial cells were collected, washed 1-2 times with physiological saline, and the resting cells of Bacillus amyloliquefaciens FJ18 were obtained.

Embodiment 3

[0040] The thalline cell fermentation broth in Example 2 was filtered to obtain wet thalline. The raw material solution, that is, the reaction solution, is prepared with dimethyl sulfoxide, tyrosol, glucose, and phosphate buffer. The ratio of organic solvent dimethyl sulfoxide in the reaction solution is 20% (v / v), tyrosol 10g / L, the molar concentration of phosphate buffer is 150mmol / L, the pH of phosphate buffer is 8.0, and the glucose concentration is 50g / L. Disperse the above obtained wet bacteria in the reaction solution, add it to the reactor, cultivate it at 30°C and 200rpm for 24h, then centrifuge at 10000rpm for 10 minutes to obtain the supernatant of the reaction solution, which is detected by HPLC and has a high conversion rate of tyrosol at 96.5%.

[0041] The product is separated with a macroporous resin, and an appropriate amount of resin is taken, soaked in ethanol for 24 hours, and then resin fragments and sundries are removed. Wet Packing Rinse with 1L of e...

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Abstract

The invention relates to application of high-glycosyl-transfer-activity bacillus amyloliquefaciens in biosynthesis of salidroside in a non-aqueous phase, and belongs to the technical field of biocatalysis. The strain is bacillus amyloliquefaciens, named as bacillus amyloliquefaciens FJ18, and is preserved at China Center for Type Culture Collection with the preservation number of CCTCC NO: M2016272. By the method, a cheap glycosyl donor can be used for efficiently catalyzing tyrosol in the non-aqueous phase to prepare the salidroside, and the problem of the scarcity of the salidroside is solved.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and relates to the application of Bacillus amyloliquefaciens FJ18 with high glycosyl transfer activity in the biosynthesis of salidroside. Background technique [0002] Salidroside is the main active ingredient of the sedum plant Rhodiola rosea, which has significant anti-oxidation, anti-inflammation, anti-fatigue, anti-aging, anti-radiation, and immune regulation effects. As a typical representative of phenolic glycoside natural products, salidroside has been widely used in the industrial production of pharmaceuticals, health products, cosmetics, etc. (Ju Xiulian et al. ZL 201510099303.8). [0003] The traditional method is to obtain salidroside by plant extraction, but the content of this compound in plants is low, usually only 0.5%-0.8%, so the cost of separation and extraction is relatively high. At present, with the industrial application of salidroside becoming more and more extensive...

Claims

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Application Information

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IPC IPC(8): C12P19/44C12R1/07
CPCC12P19/44
Inventor 张森段金廒刘培郭盛宿树兰赵明
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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