Methyltransferase gene of glycosides
A technology of methyltransferase and compound, which is applied in the direction of transferase, plant gene improvement, microorganism-based methods, etc., can solve the problem of activity reduction and achieve the effect of enhancing stability and enhancing stability
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Embodiment 1
[0086] Example 1 Synthesis of O-methyltransferase gene BbFkbM, and construction of yeast expression vector to obtain yeast transformants
[0087] Sequencing the genome of a strain of Beauveria bassiana obtained, and obtained the genome sequence of the strain. During the process of analyzing glycosyltransferases through relevant bioinformatics software, an O-methyltransferase gene was found through comparison. We named it BbFkbM. The nucleotide sequence of BbFkbM was predicted and analyzed by various software to remove intron splicing to obtain the coding region sequence of BbFkbM. Extract the RNA of Beauveria bassiana and obtain cDNA by reverse transcription, use the designed primers to amplify the coding gene of BbFkbM, recover the target fragment and send it to the sequencing company for sequencing and comparison, and obtain the glycosyltransferase shown in SEQ ID NO.1 Gene BbFkbM.
[0088] Restriction sites are introduced during synthesis. An NdeI restriction site was ad...
Embodiment 2
[0092] Example 2 Using Glucose O-methyltransferase Recombinant Vector PXW06F-BbFkbM to Realize the Modification of Desmethyl-Lasiodiplodin Glycosylated Derivatives of Polyketides
[0093] 1. Purpose of the experiment
[0094] The metabolites of modified O-methyltransferase and glucosyltransferase were separated by HPLC and their molecular structures were analyzed.
[0095] 2. Experimental method:
[0096] 1) Fermentation culture
[0097] Adopt two-step fermentation technology, first inoculate an appropriate amount of yeast transformant cells into the corresponding 25ml - Leu / - In Trp liquid deficient medium, culture at 30°C and 200r min-1 for about 16 hours, then add 25ml of YPD low-sugar medium, and add 5mg of pure Desmethyl-Lasiodiplodin at the same time to continue culturing for 48 hours; extract the fermentation product with ethyl acetate, ethyl acetate The ratio of ester to fermentation broth is 1:1, that is, 50ml of ethyl acetate is used to extract the fermentation ...
Embodiment 3
[0111] Example 3 Application of Glucose O-methyltransferase Recombinant Vector PXW06F-BbFkbM to Realize the Modification of Flavonoids
[0112] 1. Purpose of the experiment
[0113] The metabolites of modified O-methyltransferase and glucosyltransferase were separated by HPLC and their molecular structures were analyzed.
[0114] 2. Experimental method:
[0115] 1) Fermentation culture
[0116] Adopt two-step fermentation technology, first inoculate an appropriate amount of yeast transformant cells into the corresponding 25ml - Leu / - In Trp liquid deficient medium, culture at 30°C and 200r min-1 for about 16 hours, then add 25ml of YPD low-sugar medium, and at the same time add 5mg of pure product to continue culturing for 48 hours. A total of 20 flavonoid samples were used; extracted with ethyl acetate For the fermentation product, the ratio of ethyl acetate to fermentation broth is 1:1, that is, the fermentation product is extracted with 50ml of ethyl acetate; the dry e...
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