Method for fermentation production of rhodioloside
A technology of salidroside and fermentation broth, which is applied in the fields of genetic engineering and microbial fermentation, can solve the problems of low yield of secondary metabolites, questioned economic value, etc., and achieves simple reaction method, good activity and stability, and thermal stability. good effect
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Embodiment 1
[0021] The acquisition of embodiment 1 high-yielding apple β-glucosidase genetically engineered bacteria
[0022] (1) According to the apple β-glucosidase gene sequence, base optimization and artificial synthesis of its gene mutant MBGLM (its amino acid sequence is shown in SEQ ID NO.1, the highest enzyme activity of this mutant is 198IU / ml, is wild 9 times the enzyme activity of the type strain), and loaded into the vector pUC57.
[0023] (2) The target gene was double-digested with NdeI and EcoRI and then recovered by tapping the gel, ligated with the pET-24d(+) vector digested with NdeI and EcoRI, and transformed into Escherichia coli BL21 (DE 3 );
[0024] Spread the transformation mixture on a 50 mg / L karamycin-resistant LB plate and pick the transformant, culture it at 37°C for 6-8 hours, pick a single colony, and obtain MBGLM-pET-24d(+) / E.coli BL21 (DE 3 ).
[0025] After culturing in LB / Kan liquid medium for 8-10 hours, the strains were preserved.
Embodiment 2
[0026] Embodiment 2 fermentation produces enzyme
[0027] Inoculate the genetically engineered bacteria MBGLM-pET-24d(+) / E.coli BL21(DE3) in the LB liquid medium containing Kan, and cultivate it at 37°C for 8-10 hours; Base, cultured at 37°C for 3h; induced with 0.4mM / LIPTG, cooled to 25°C and cultured at constant temperature for 40-48h to induce enzyme production. After fermentation, the bacteria were collected by centrifugation and ultrasonically disrupted. Centrifuge to obtain the supernatant after induction, which is the crude enzyme solution.
Embodiment 3
[0028] Embodiment 3 enzyme catalyzes the synthesis of salidroside
[0029] Weigh 250mM tyrosol, 50mM glucose, and 1.25U / ml crude enzyme solution and mix them in the buffer / n-hexane (1 / 1, V / V) mixed system. The reaction was shaken at 50° C. and 180 r / min for 24 hours. After the reaction, extract a certain amount of aqueous phase reaction solution, dilute it with 2 times the volume of distilled water, filter through a microporous membrane, concentrate the filtrate to dryness, and obtain a white solid powder. mp 156-159℃, IR(KBr)vcm - : 268, 1630, 1609; 13 CNMR (CDOD) 6: 35.0 (Ar-CH 2 -)69.0 (-CH 2 -O); 61.1(6), 70.7(4), 71.2(3), 73.6(2), 75.2(5), 103.6(Gal-1); 114.8(2), 129.5(3), 155.3(Ar) ,MSm / z:306.8(M + ), 324.8(M+H2O) 287.4(M-H 2 O+H + ). It is consistent with the data in the literature (Duan Jingyun, Zhang Dianzeng, Fan Yinke, etc. Pharmacodynamics Research of Compound Rhodiola Tablets [J]. Chinese Patent Medicine, 1999, 21(11):588-591).
[0030] Take the product ...
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