Study On The Hydroxylation And Glycosylation Of Tyrosol

Phenylglycosides(PhGs)are glycosides containing phenolic hydroxyl groups,phenylglycosides and sugar groups.Common phenylethanol glycosides include tyrosol,hydroxytyrosol,salidroside,mullein glycoside,echinoside,etc.Pharmacological studies have shown that these compounds have the effects of protecting cardiovascular system,anti-oxidation,anti-aging,anti-tumor,liver protection enhancing memory,etc.In recent years,due to their extensive pharmacological effects,they have become a research hotspot in the field of biology and medicine,and the demand is increasing year by year.In this experiment,engineering bacteria petduet-1-hpab-c /BL21(DE3)and petduet-1-ugt73b6 /BL21(DE3)were constructed.In the process of culturation,tyrosol was added to hydroxylate and glycosylation,and biotransformation of phenylethyl glycosides was conducted.The experimental research contents and results are as follows:(1)HpaB gene was amplified by PCR to construct hpab-t clonal plasmid.The hpaC gene was synthesized by the company and the recombinant plasmid petduet-1-hpac was independently constructed.To construct the prokaryotic expression engineering bacteria petDuet-1-hpaB-C/BL21(DE3),double-enzyme digestion,purification and recovery,connection and transformation,etc were performed to extract the correct sequencing results of petDuet-T and petDuet-1-hpaC plasmids,respectively.Through the induced expression,the experimental results showed that the engineering bacteria had specific protein bands at 57.2 KD and 18.7KD,which were consistent with the molecular weight of the target protein.(2)Glycosyltransferase gene ugt73b6 was synthesized by Sangong Biotech(Shanghai)co.,Ltd,and engineering bacteria petduet-1-ugt73b6 / BL21(DE3)was independently designed and constructed.Engineering bacteria were cultured and induced to express,and the expression products were analyzed by SDS-PAGE.The results showed that after induction at 16 ? for 24 h,the specific protein bands were produced around 52.8 KD by SDS-PAGE analysis,which was consistent with the molecular weight of the target protein.(3)The engineering strain petDuet-1-hpaB-C/BL21(DE3)was cultured andbiotransformed by adding exogenous precursor tyrosol.The samples were identified by high performance liquid chromatography(HPLC)and mass spectrometry,and the results showed that there was a target product hydroxytyrosol.(4)The biotransformation from tyrosol to salidroside was achieved by adding the precursor tyrosol in the culture engineering strain petDuet-1-ugt73b6/ BL21(DE3,).(5)Engineering bacteria-hpaB petDuet-1-C/BL21(DE3)and engineering bacterium petDuet-1-ugt73b6 / BL21(DE3)were co-cultured,and added the precursor tyrosol,hydroxylation and glycosylation at the same time,The biotransformation of tyrosol to 3,4-dihydroxyphenylethanol glucoside and its isomers was realized.It laid an experimental foundation for the further glycosylation of phlebiflorin and echinacea.(6)To compare the two engineering bacterium of hydroxy butyl alcohol conversion rate,design the related experiments,engineering bacterium hpaB-Pet-a /28 BL21(DE3)in inducing temperature 16 ? condition,the conversion time in 5 h,hydroxy tyrosol high biological conversion of 70%.Under the same conditions,engineering bacteria petDuet-1-hpaB-C/BL21(DE3)were induced,and the maximum bioconversion rate of hydroxytyrosol was 92% when the conversion time was1/2 h.Experimental results showed that petduet-1-hpab-c /BL21(DE3)was more beneficial to the biotransformation of hydroxytyrosol under the same experimental conditions.(7)In order to optimize the bioconversion conditions of hydroxytyrosol by engineering bacteria petduet-1-hpab-c /BL21(DE3),the bioconversion time and induced temperature conditions were optimized.It was found that the conversion rate of hydroxytyrosol would increase with the prolongation of the conversion time,but it was not in direct proportion.It increased within a certain time range,and when it reached the maximum value,it showed a downward trend.The results showed that the highest conversion rate of hydroxytyrosol in the conversion system was up to 92%when the transformation time of engineering strain petDuet-1-hpaB-C/BL21(DE3)was 1/2 h and the induction condition was 16?(8)To improve the bioconversion efficiency of salidroside,the eukaryotic expression system was constructed.The recombinant plasmid ppic9k-ugt73b6 wasconstructed.After linearized,the recombinant plasmid was transferred to pichia pichia GS115 competent cells by electric shock.The high transformants were screened by four YPD plates with increasing concentrations of hereditary mycin(0.5 mg/mL,1.0mg/mL,2.0 mg/mL,4.0 mg/mL).The genome was extracted after single colony culture on a plate with high genetic concentration of mycin,and the genome was used as a template for PCR identification.The engineering bacterium with correct identification results was named ppic9k-ugt73b6 /GS115.Fermentation culture and methanol induction were carried out on the engineered bacteria,and SDS-PAGE analysis was performed on the samples.The results showed that specific protein bands were observed at 48 h and 72 h after induction,and the molecular weight of the protein bands was about 52.8kd,which was consistent with the target product.