CN118222658B - Method for biosynthesis of salidroside - Google Patents
Method for biosynthesis of salidroside Download PDFInfo
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- CN118222658B CN118222658B CN202410638087.9A CN202410638087A CN118222658B CN 118222658 B CN118222658 B CN 118222658B CN 202410638087 A CN202410638087 A CN 202410638087A CN 118222658 B CN118222658 B CN 118222658B
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- salidroside
- glycosyltransferase
- fermentation
- inducer
- hydroxyphenylethanol
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- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 title claims abstract description 75
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 37
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 18
- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 claims abstract description 58
- 239000000758 substrate Substances 0.000 claims abstract description 11
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for biosynthesis of salidroside. The method comprises the following steps: under the action of a glycosyl donor, a glycosyl donor having the sequence of SEQ ID NO:1 to carry out catalytic reaction on a substrate 4-hydroxy phenethyl alcohol to obtain salidroside. The method relates to the field of biotechnology, and can solve the problem of lack of a method for efficiently synthesizing salidroside in the prior art. The salidroside produced by the method has high yield and is expected to be applied to the industrial production of the salidroside.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for biosynthesis of salidroside.
Background
Rhodiola rosea glycoside (Salidroside) with molecular formula of C 14H20O7 and structural formulaIs a phenylethanoid glycoside compound of plant source. The salidroside is a main active ingredient of medicinal plants of rhodiola, has strong antioxidant activity, can reduce inflammation, protect cells from oxidative stress, and has the effects of relieving depression, fatigue and stress, relieving altitude stress, and the like, and is widely applied to the pharmaceutical and cosmetic industries. Wherein, the medicine is the main application field of salidroside, and in the field, the salidroside is mainly used for preparing medicines for resisting gout, reducing blood sugar, resisting tumor and liver injury, exciting nerve agents and the like.
The salidroside from natural sources is mainly extracted from the plant rhodiola rosea, but the rhodiola rosea grows in a special high-altitude environment, the growth period is slow, and the rhodiola rosea resources are scarce, so that the yield of the rhodiola rosea is very limited. In addition, the rhodiola rosea has low rhodiola rosea glycoside content, high extraction and purification cost and low efficiency, and further deepens the rarity of the rhodiola rosea glycoside. The chemical synthesis of salidroside requires complex hydroxy protection and deprotection steps and a relatively expensive catalyst, has high cost, is not friendly to the environment, has harsh operating conditions, and has low or trace amount of other toxic chemicals remained in the product, thereby having poor safety. Therefore, the development condition is mild and the high-efficiency biological method for synthesizing the salidroside is beneficial to reducing the production cost and expanding the market value.
Glycosylation refers to the process of attaching carbohydrates to proteins or lipids under the control of enzymes. Glycosylation is an important modification to proteins, and has the effects of regulating the functions of proteins and helping protein folding, and further affects biological processes such as cell signaling, immune response, cell recognition and the like. The last step in the biosynthesis of salidroside is the glycosyltransferase-catalyzed glycosylation reaction, which is a critical rate-limiting step. Although various glycosyltransferases have been reported to catalyze the synthesis of salidroside from 4-hydroxyphenylethanol (tyrosol), further improvements in catalytic activity and regioselectivity are still needed.
In view of the above, developing a high-efficiency glycosyltransferase has important significance for realizing the industrial production of salidroside by using a microbial fermentation method, and is a technical problem to be solved urgently.
Disclosure of Invention
The invention mainly aims to provide a method for biosynthesis of salidroside, which aims to solve the problem that the prior art lacks a method for efficiently synthesizing the salidroside.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for biosynthesis of salidroside using a nucleotide sequence having a nucleotide sequence of SEQ ID NO:1 to carry out catalytic reaction on a substrate 4-hydroxy phenethyl alcohol to obtain the salidroside.
Further, the glycosyltransferase is selected from a crude enzyme solution or a purified enzyme solution comprising the glycosyltransferase.
Further, the method comprises the steps of: and (3) inoculating the recombinant cells which are cultured overnight and can express the glycosyltransferase into a fermentation culture medium for expansion culture until the OD600 is between 0.6 and 0.8, adding an inducer for induction fermentation for 18 to 48 hours, and collecting a supernatant product to obtain the salidroside, wherein the 4-hydroxyphenylethanol is added together with the inducer.
Further, the conditions for the above-mentioned expansion culture are 30℃to 37℃and 200rpm to 220rpm.
Further, the conditions for the above induced fermentation are 28℃to 30℃and 200rpm to 220rpm.
Further, the inducer is IPTG.
Further, the final concentration of the inducer is 0.1mM-1mM.
Further, the fermentation medium formula comprises: naCl, tryptone, yeast extract, glucose, 4-hydroxyphenylethanol, antibiotics and MOPS.
Further, the fermentation medium formula comprises: 10 g/L of the NaCl,10 g/L of the tryptone, 5 g/L of the yeast extract, 20 g/L to 30 g/L of the glucose, 40 g/L to 50 g/L of MOPS and 50 mg/L to 100 mg/L of kanamycin.
Further, the glycosyl donor is UDPG.
By applying the technical scheme of the invention, the glycosyltransferase (with the amino acid sequence shown in SEQ ID NO: 1) has high catalytic activity, so that 4-hydroxy phenethyl alcohol (tyrosol) can be used as a substrate to catalyze and synthesize the salidroside with high efficiency, and the yield of the salidroside is higher than that of the salidroside synthesized by other glycosyltransferases in the prior art. The invention solves the problem of lack of a method for efficiently synthesizing salidroside in the prior art, and has wide application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 shows SDS-PAGE patterns of glycosyltransferase OfT GT1 protein expression according to example 2 of the present invention, wherein lane 1 represents a protein Marker; lane 2 shows total protein of whole bacteria after bacterial disruption; lane 3 shows total protein in the supernatant after bacterial disruption; lane 4 shows the purified protein.
FIG. 2 shows an HPLC detection chart of the content of salidroside in the strain fermentation broth according to example 4 of the present invention, wherein FIG. 2A shows an HPLC chart of a salidroside standard; FIG. 2B shows an HPLC plot of BL21 (DE 3)/pET 28a-OfT GT1 fermentation 48 h for producing salidroside.
FIG. 3 shows the LC-MS mass spectrum of example 4 according to the invention, wherein A shows the mass spectrum of the salidroside standard and B shows the mass spectrum of BL21 (DE 3)/pET 28 a-OfT.sup.8GT 1 fermentation 48: 48 h for the production of salidroside.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
As mentioned in the background art, the salidroside has the beneficial effects of resisting oxidation, resisting depression, resisting fatigue, relieving altitude stress and the like, however, the yield of the natural salidroside limited by the quantity of plant rhodiola rosea is very limited, and the problems of complicated steps, poor regioselectivity and the like of the chemical synthesis of the salidroside exist. Therefore, the biological method with mild conditions becomes the first choice method for synthesizing the salidroside. Currently, glycosyltransferases used in the art to synthesize salidroside have the disadvantage of low activity (see example 3 for specific alignment data). Therefore, the invention discovers that the existing glycosyltransferase (with the amino acid sequence shown as SEQ ID NO: 1) derived from the sweet osmanthus (Osmanthus fragrans) can synthesize the salidroside for the first time, and the yield of the salidroside synthesized by the enzyme is higher than that of other glycosyltransferases, thereby being beneficial to the industrialized production of the salidroside.
In a typical embodiment of the present invention, a method for biosynthesis of salidroside is provided, using a polypeptide having the sequence of SEQ ID NO:1 to carry out catalytic reaction on a substrate 4-hydroxy phenethyl alcohol to obtain salidroside. The glycosyltransferase is selected from a crude enzyme solution or a purified enzyme solution comprising the glycosyltransferase.
The amino acid sequence of the glycosyltransferase SEQ ID NO:1 is as follows:
MGSIAGNDKPHAVCIPYPAQGHINPMFNLAKLLHHQGFYITFVNTEFNHKRLLKSRGPAALDGLPDFRFETIPDGLPSSDGDATQDIPSLCESTTKTCLAPFCSLLTKLNNAAPVMPPVTCIVSDGAMSFTLKAAEQFGLPEVLLWTTSACGFLGYTQYANLIERGYTPLKEMSQVTNGYLETTIDWIPGMKDIRLRDLPTFLRTTDSNDIMLNFVLREIEAVPKAKAIILNTFDALEHDVLDALSSMNPVIYSVGPLQLMMNHIQNEKHTLTSSSLWKEELECIKWLDKKEPNSVVYVNFGSIAVVTAQQLTEFAWGLANSKKSFLWIIRPDLVAGDLAMLPPEFVTETEDRSMLISWCPQEEVLKHPAIGGFLTHSGWNSTIESIVAGVPLICWPFFAEQQTNCRYSCVEWGMGMEIDNNVKRDEVEELVKELMDGEKGMKMKEKAMEWKKKAEEATAPGGSSFMNFEELVKLLQ.
the nucleotide sequence of the glycosyltransferase SEQ ID NO:2 is as follows:
ATGGGTAGTATCGCCGGTAATGATAAACCGCATGCCGTGTGTATTCCGTATCCGGCCCAGGGTCATATTAATCCGATGTTTAATCTGGCCAAGCTGCTGCATCATCAGGGCTTTTATATTACCTTTGTGAACACCGAATTCAACCATAAACGTCTGCTGAAAAGTCGCGGCCCGGCCGCATTAGATGGTTTACCTGATTTTCGTTTCGAGACCATTCCGGATGGCCTGCCGAGCAGTGATGGTGACGCTACACAGGATATTCCGAGCCTGTGCGAAAGTACCACCAAAACCTGCCTGGCCCCGTTTTGTAGTCTGCTGACCAAACTGAATAACGCAGCACCGGTTATGCCGCCGGTGACATGTATTGTGAGTGATGGTGCCATGAGTTTTACCCTGAAAGCAGCCGAACAGTTTGGTCTGCCGGAAGTTCTGCTGTGGACCACCAGTGCCTGTGGTTTTCTGGGCTATACCCAGTATGCCAATCTGATTGAACGCGGCTATACCCCGCTGAAAGAAATGAGTCAGGTTACCAATGGTTACCTGGAAACCACCATTGATTGGATTCCGGGCATGAAAGATATTCGTCTGCGCGATCTGCCGACCTTTCTGCGTACCACCGATAGCAATGATATTATGCTGAATTTCGTGCTGCGTGAAATTGAAGCAGTTCCGAAAGCAAAAGCCATTATTCTGAATACCTTCGATGCCCTGGAACATGATGTGCTGGATGCCCTGAGCAGTATGAATCCGGTTATCTATAGCGTGGGTCCGCTGCAGCTGATGATGAATCATATTCAGAATGAGAAGCACACCCTGACCAGCAGTAGTCTGTGGAAAGAAGAACTGGAATGTATTAAGTGGCTGGATAAAAAGGAGCCGAATAGTGTGGTGTATGTGAATTTTGGTAGCATTGCCGTTGTTACCGCCCAGCAGCTGACCGAATTTGCCTGGGGTCTGGCCAATAGCAAAAAATCATTTCTGTGGATCATCCGCCCGGATCTGGTTGCAGGTGACCTGGCAATGCTGCCGCCTGAATTTGTTACCGAAACCGAAGATCGCAGCATGCTGATTAGCTGGTGTCCGCAGGAAGAAGTGCTGAAACATCCGGCAATTGGTGGCTTTCTGACCCATAGTGGCTGGAATAGTACCATTGAAAGTATTGTTGCCGGTGTGCCGCTGATTTGCTGGCCTTTCTTTGCCGAACAGCAGACCAATTGTCGTTATAGTTGTGTGGAATGGGGTATGGGTATGGAAATTGATAATAACGTGAAGCGTGACGAAGTGGAAGAACTGGTTAAAGAACTGATGGATGGCGAAAAAGGTATGAAAATGAAGGAAAAGGCGATGGAATGGAAAAAGAAAGCCGAAGAAGCCACCGCCCCGGGTGGTAGCAGCTTCATGAATTTTGAAGAACTGGTGAAGCTGCTGCAGTAA.
The method for biosynthesis of salidroside can be used for in vitro synthesis by using crude enzyme liquid or purified enzyme liquid, or in vivo synthesis by fermenting recombinant cells containing the enzyme gene. In order to facilitate the biosynthesis process of salidroside, in a preferred embodiment of the present invention, an in vivo synthesis method is used, which comprises: and (3) inoculating the recombinant cells which are cultured overnight and can express the glycosyltransferase into a fermentation culture medium for expansion culture until the OD600 is between 0.6 and 0.8, adding an inducer for induced fermentation for 18 to 48 hours, and collecting a supernatant product to obtain the salidroside, wherein the 4-hydroxyphenylethanol (filtered and sterilized by adopting a sterile membrane) is added together with the inducer. The method is simple and rapid, and the produced salidroside has high yield, and can be applied to industrial production of salidroside.
After the recombinant cells are inoculated into the fermentation medium, an expansion culture is required to increase the number of the recombinant cells, and the expansion culture conditions are closely related to the number and growth conditions of the recombinant cells. When the number of recombinant cells reaches a certain concentration, the cells need to be subjected to induction fermentation culture. In order to make the recombinant cells produce salidroside as much as possible, it is preferable to culture under optimal induced fermentation culture conditions for the recombinant cells to produce salidroside. In a preferred embodiment of the present invention, the conditions for the above-mentioned expansion culture are 30℃to 37℃and 200rpm to 220rpm; preferably, the conditions for the above induced fermentation are 28℃to 30℃and 200rpm to 220rpm.
Depending on the expression vector, a suitable inducer is selected. The appropriate concentration of the inducer is selected according to the expression of the target protein in the different recombinant cells. In a preferred embodiment of the present application, the inducer is IPTG; more preferably, the final concentration of the inducer is 0.1mM-1mM; further preferably, the final concentration of the inducer is 0.1mM. Under the preferable condition, the method can maximally produce the salidroside, improve the yield of the salidroside and accelerate the production efficiency of the salidroside.
The fermentation medium provides proper conditions for the growth and metabolism of recombinant cells, different recombinant cells have different requirements for nutrient components in the fermentation medium, and different fermentation media can be selected according to the types of the recombinant cells. In a preferred embodiment of the present application, the fermentation medium formulation described above comprises: naCl, tryptone, yeast extract, glucose, 4-hydroxyphenylethanol, antibiotics and MOPS (3- (N-methylpyrazine) -propanesulfonic acid); preferably, 10 g/L of the NaCl,10 g/L of the tryptone, 5 g/L of the yeast extract, 20 g/L to 30 g/L of the glucose and 0.5 g/L of the 4-hydroxyphenylethanol; preferably, the antibiotic is kanamycin; more preferably, the kanamycin concentration is 50 mg/L to 100 mg/L. The addition of antibiotics is an indispensable step in the conventional heterologous expression process, plays a role in killing infectious microbes, and the type and concentration of antibiotics can be determined according to the type of the expression vector. Under the preferable condition, the method provides the optimal nutrition condition for the recombinant cells, and is an important guarantee for high production of salidroside by the recombinant cells.
In order to ensure that the reaction for producing salidroside proceeds smoothly, a glycosyl donor is indispensable. In a preferred embodiment of the invention, the glycosyl donor is UDPG. In an in vitro catalytic experiment, the glycosyl donor can be provided by directly adding UDPG into a catalytic reaction system, and in an in vivo catalytic experiment, the glycosyl donor can be provided by adding glucose into a fermentation medium, and the glucose is metabolized by recombinant cells to generate UDPG. Under the action of glycosyl donor, glycosyl transferase acts to produce salidroside. Based on the preparation method, the glycosyltransferase is utilized to react with a substrate 4-hydroxyphenylethanol (tyrosol), so that the 4-hydroxyphenylethanol (tyrosol) can be catalyzed to obtain salidroside. The glycosyltransferase has high catalytic activity, high yield of the produced salidroside, wide application prospect and high economic value.
The invention is described in further detail below in connection with specific examples which are not to be construed as limiting the scope of the invention as claimed. Unless otherwise indicated, the experimental methods used in the following examples are all conventional.
EXAMPLE 1 construction of recombinant E.coli BL21 (DE 3)/pET 28a-OfT GT1
The OfT GT1 gene sequence from Osmanthus fragrans is searched through NCBI, and the codon optimization of the escherichia coli and the synthesis of plasmid pET28a-OfT GT1 are carried out through nine-day gene technology (Tianjin) limited company, wherein the nucleotide sequence is shown as SEQ ID NO: 2. The OfT GT1 gene fragment was ligated to plasmid pET28a-OfT8GT1 to construct a recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21 (DE 3) to obtain recombinant E.coli BL21 (DE 3)/pET 28a-OfT8GT1. The specific transformation method comprises the following steps: BL21 (DE 3) competent cells (1.5 ml EP tube containing 50. Mu.L competent cells) were thawed on ice, 50 ng plasmid was added to the competent cells, mixed well, ice-bath 30. 30 min, heat-shocked 90 s in a 42℃water bath, 600. Mu.L resuscitation fluid (LB medium) was added, incubated at 37℃50. 50 min, and after incubation, the whole bacterial solution was spread on a plate (LB medium) containing 50 mg/L kanamycin, and placed in a 37℃incubator for overnight culture.
Example 2 protein expression validation of glycosyltransferase OfT GT1
After overnight culture, the cloned strain is selected and inoculated into LB liquid medium containing 50 mg/L kanamycin, the bacteria are shaken at 37 ℃ until the OD600 reaches 0.8-1, IPTG with the final concentration of 0.15 mM is added, after shaking table induction culture is performed for 16-20 hours at 30 ℃, the bacteria are collected by centrifugation at 4 ℃ under 5000 rpm, the bacteria are resuspended in PBS buffer solution, the ultrasonic waves are performed by an ultrasonic breaker, and the obtained product is placed into an ice-water mixture for 180W breaking for 3-5 min.
SDS-PAGE of the supernatant and pellet of OfT GT1 crude enzyme lysate showed that there was a distinct protein band between 40-55KD as shown in FIG. 1, with a molecular weight close to that of his-OfT GT1, 55.4 KD, and OfT GT1 protein expression clearly seen.
EXAMPLE 3 in vitro catalysis of OfT8GT1 to Synthesis of salidroside from 4-hydroxyphenylethanol
OfT8GT1 purified enzyme was obtained by the method of examples 1 and 2, the specific procedure being as follows:
(1) Bacterial heavy suspension: the cells of 8 g were resuspended in 80: 80 mL equilibration buffer and thoroughly stirred with a glass rod until no pellet was present.
(2) And (3) thallus crushing: crushing the heavy suspension in a high-pressure homogenizer for 1 time of 500 bar times of crushing and 2 times of 800 bar times of crushing, and opening cooling circulating water in advance for cooling.
(3) Purifying: 2mL Thermo HisPur ™ Nickel-NTA resin (model: 88221) and 80 mL crude enzyme solution were thoroughly mixed at 4℃for one hour, and then the mixed crude enzyme solution was subjected to filtration and purification on a chromatographic column.
(4) Washing: the impurity protein was washed with 20 mM imidazole buffer, 5-10 column volumes of Ni packing were washed until the protein concentration of the effluent was measured to be below 0.1 mg/ml. (determination of protein concentration by Bradford method)
(5) Eluting: eluting target protein with 250 mM imidazole buffer, and washing 5-10 times column volume of Ni packing until the protein concentration of effluent liquid is below 0.1 mg/ml. (determination of protein concentration by Bradford method)
(6) Concentrating and changing liquid: concentrating and changing the purified enzyme OfT GT1 collected by nickel column purification by using a30 KD ultrafiltration tube, adding 8-10 ml balance buffer solution for desalting, and centrifuging 3750 rpm for 20min. This step was repeated 3-5 times until the concentration was 0.5-1 ml volumes of protein, and the protein concentration was finally measured.
(7) Protein concentration measured by Bradford method: coomassie Brilliant Blue (CBB) determination of protein content belongs to one of the dye binding methods. Coomassie brilliant blue is red in the free state with maximum light absorption at 488nm, and when it becomes cyan upon binding to protein, the protein-pigment conjugate has maximum light absorption at 595 nm. The light absorption value is proportional to the protein content, so that the method can be used for quantitative determination of protein.
The method comprises the following specific steps:
(1) 1.00g of Bovine Serum Albumin (BSA) was weighed by a balance and dissolved in deionized water to prepare 100ml of solution having a concentration of 10mg/ml. 100ul,80ul,60ul,40ul,20ul of BSA solution was placed in a 1.5ml EP tube with a pipette, 900ul,920ul,940ul,960ul, 480 ul of Phosphate Buffered Saline (PBS) was added to prepare 1ml of solution, and the solution was mixed uniformly by shaking. A set of BSA solutions was obtained at concentrations of 1.0mg/ml, 0.8mg/ml,0.6mg/ml, 0.4mg/ml, 0.2mg/ml, respectively.
(2) The pipette was used to remove 100ul of the freshly prepared set of BSA solutions, and the solutions were placed in a 1.5ml EP tube, each with 900ul of PBS solution added, and mixed well by shaking. A set of BSA solutions was obtained at concentrations of 0.10 mg/ml, 0.08mg/ml,0.06mg/ml, 0.04mg/ml, 0.02mg/ml, respectively. A further 1ml of PBS solution (BSA solution concentration 0 mg/ml) was taken for the control.
(3) A50 ul set of BSA solutions were removed with a pipette, and added dropwise to the well plate, each with 200ul of Coomassie Brilliant Blue (CBB). After 10min of standing, the absorbance of the set of BSA solutions was measured with an enzyme-labeled instrument, and a plot of absorbance versus BSA concentration was made with orgin to measure a standard curve: y=0.5421x+0.5047, y=od 595nm, x=protein concentration (mg/mL).
Sample detection: and adding a 30 mu L sample and a 150 mu L Brandford solution into the ELISA plate, reacting for 5min, measuring OD595nm, and calculating the protein concentration of the sample according to a BSA standard curve.
The above purified and concentrated OfT GT1 was taken for the following reaction:
System 1: 1mM 4-hydroxyphenylethanol, 2 mM UDP-glucose (UDPG), 10. Mu. g OfT8GT1 purified enzyme, 0.05M Tris-HCl 7.4,2 mM ascorbic acid, and 100. Mu.L of deionized water were added, and the reaction system was the same as that of CN 114317480A;
system 2: 2mM 4-hydroxyphenylethanol, 2.5 mM UDP-glucose, 20. Mu. g OfT8GT1 purified enzyme, 0.1M PBS 7.0, 100. Mu.L of deionized water was added, and the reaction system was identical to CN 116716270A;
System 3:5 mM 4-hydroxyphenylethanol, 2.5 mM UDP-glucose, 50. Mu. g OfT8GT1 purified enzyme, 0.1M PBS 7.0, 100. Mu.L of deionized water was used, and the reaction system was identical to CN 116716270A.
After the above systems are all placed in a 37 ℃ water bath kettle to react for 0.5-1 hour, 100 mu L of glacial methanol is added into the reaction system to terminate the reaction, the reaction is cooled in ice for 2 min, the reaction is centrifuged at 12000 rpm for 5min, a 0.22 mu m organic phase filter membrane is used for filtering the reaction solution to prepare a sample, and the synthetic amount of the salidroside in the reaction system is analyzed by HPLC. Detecting the liquid phase peak area of 4-hydroxy phenethyl alcohol (tyrosol) and the liquid phase peak area of salidroside, wherein the ratio of the liquid phase peak area of the salidroside to the liquid phase peak area of 4-hydroxy phenethyl alcohol (tyrosol) is the conversion rate. The catalytic activity of the glycosyltransferase was evaluated based on the conversion.
As can be seen from the data in Table 1 below, after the in vitro catalytic reaction is completed, the synthesis of salidroside is detected obviously, and the conversion rate of the substrate 4-hydroxyphenylethanol reaches 90% or more. Compared with glycosyltransferase reported in the patent published in the prior art, ofT GT1 is more prone to synthesizing salidroside by taking 4-hydroxyphenylethanol as a substrate when taking 1mM 4-hydroxyphenylethanol and 1mM 3, 4-dihydroxyphenylethanol (hydroxytyrosol) as substrates respectively, and the highest conversion rate can reach 90%. In addition, compared with different addition amounts of 4-hydroxyphenylethanol with different concentrations and different glycosyltransferases, ofT GT1 of the invention has higher catalytic efficiency and lower enzyme dosage in the aspect of catalyzing and synthesizing salidroside, and is beneficial to realizing higher economic value.
TABLE 1
Note that: + represents a conversion of 20-40%, ++ represents a conversion of 40-60%, +++ stands for transformation the rate is 60-80%, ++++ represents transformation the rate is 80-90%, rate of 80 90%.
EXAMPLE 4 fermentation culture of recombinant E.coli BL21 (DE 3)/pET 28a-OfT GT1
Preparing seed liquid: colony PCR was picked to verify correct monoclonal into LB medium containing 50 mg/L kanamycin, cultured at 37℃overnight at 200: 200 rpm for 18: 18 h.
Fermentation culture: detecting the bacterial liquid concentration of the seed liquid, calculating the inoculation volume (mL) according to the initial bacterial liquid concentration OD600 of 0.1, transferring the bacterial liquid to a 50mL fermentation medium (LB+20 g/L glucose+MOPS) containing 80-120 mg/L kanamycin, adding 0.5 g/L4-hydroxy phenethyl alcohol (tyrosol) and 0.1 mM IPTG for induction when the bacterial liquid concentration OD600 is 0.8-1, reducing the temperature to 28-30 ℃, and continuing 200 rpm induction culture for 18-48 h.
EXAMPLE 5 Synthesis of salidroside by recombinant E.coli BL21 (DE 3)/pET 28a-OfT GT1
Fermenting 48 h, taking 1mL sample, centrifuging, and taking supernatant for HPLC detection under the following conditions: the chromatographic column ATLANTIS T is 3.6 mm 3 μm, the mobile phase is H 2 O+0.1% TFA and ACN+0.1% TFA, the flow rate is 1.0 mL/min, the column temperature is 40 ℃, the detection wavelength is 220 nm, and the detection duration is 12.0 min. Simultaneously detecting organic acid and glucose: the chromatographic column Aminex HPX-87H column 300*7.8 mm, mobile phase H 2O+5 mM H2SO4, flow rate 0.6 mL/min, column temperature 50 ℃, RID detector, detection duration 25.0 min.
The results show that when the fermentation is carried out for 48 h, BL21 (DE 3)/pET 28a-OfT8GT1 recombinant strain can produce 653.6 mg/L salidroside by taking 4-hydroxy phenethyl alcohol (tyrosol) as a precursor, the molar conversion rate reaches 83.1 percent, the data is shown in the following table 2, the HPLC diagram is shown in figure 2B, the retention time Rt of the salidroside is 5.1 min, and the retention time is consistent with that of a standard product of the salidroside (figure 2A). LC-MS spectra are shown in figure 3B (relevant experimental parameters and results are shown in Table 4), wherein [ M-H ] - = 299.3 is consistent with the salidroside standard spectra are shown in figure 3A (relevant experimental parameters and results are shown in Table 3), and the molecular weight [ M-H ] - = 299.2 is consistent, so that the glycosyltransferase OfT GT1 can be determined to catalyze 4-hydroxyphenylethanol (tyrosol) to synthesize the salidroside.
TABLE 2
Table 3 peak results
And/indicates that the data is not provided.
Table 4 peak results
And/indicates that the data is not provided.
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the invention discovers for the first time that the existing glycosyltransferase derived from sweet osmanthus (Osmanthus fragrans) can utilize microorganism to efficiently catalyze the reaction of glycosyl donor UDP-glucose and substrate 4-hydroxy phenethyl alcohol to generate single glycosylation product rhodiola glycoside, wherein 0.5g/L of 4-hydroxy phenethyl alcohol is added as substrate, and the concentration of rhodiola glycoside in supernatant can reach 653.6mg/L through fermentation culture. Compared with other glycosyltransferases in the prior art, the glycosyltransferase has high activity, high yield of the generated salidroside, unique advantages, wide prospect and high value, and is expected to be applied to the industrial production of the salidroside.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for biosynthesis of salidroside, which is characterized by using SEQ ID NO:1, carrying out catalytic reaction on a substrate 4-hydroxy phenethyl alcohol by glycosyltransferase with an amino acid sequence shown in the formula 1 to obtain the salidroside;
wherein the glycosyl donor is UDPG.
2. The method of claim 1, wherein the glycosyltransferase is selected from a crude enzyme solution or a purified enzyme solution comprising the glycosyltransferase.
3. The method according to claim 1, characterized in that the method comprises:
And inoculating the recombinant cells which are cultured overnight and can express the glycosyltransferase into a fermentation culture medium for expansion culture until the OD600 is between 0.6 and 0.8, adding an inducer for induction fermentation for 18 to 48 hours, and collecting a supernatant product to obtain the salidroside, wherein the 4-hydroxyphenylethanol is added together with the inducer.
4. A method according to claim 3, wherein the conditions of the expansion culture are 30-37 ℃, 200-220 rpm.
5. A method according to claim 3, wherein the conditions for inducing fermentation are 28-30 ℃, 200-220 rpm.
6. A method according to claim 3, wherein the inducer is IPTG.
7. A method according to claim 3, wherein the final concentration of the inducer is between 0.1mM and 1mM.
8. A method according to claim 3, wherein the formulation of the fermentation medium comprises: naCl, tryptone, yeast extract, glucose, 4-hydroxyphenylethanol, antibiotics and MOPS.
9. The method of claim 8, wherein the formulation of the fermentation medium comprises: 10 g/L of NaCl,10 g/L of tryptone, 5 g/L of yeast extract, 20 g/L-30 g/L of glucose, 0.5 g/L of 4-hydroxyphenylethanol, 40 g/L-50 g/L of MOPS and 50 mg/L-100 mg/L of kanamycin.
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