CN110004099B

CN110004099B - Fermentation production method of salidroside

Info

Publication number: CN110004099B
Application number: CN201810008237.2A
Authority: CN
Inventors: 孙敬方
Original assignee: Individual
Current assignee: Sun Jing Fang
Priority date: 2018-01-04
Filing date: 2018-01-04
Publication date: 2021-06-15
Anticipated expiration: 2038-01-04
Also published as: CN110004099A

Abstract

The invention provides a method for producing and preparing salidroside by enzymatic conversion and fermentation by using apple beta-glucosidase as a novel catalyst. The efficient apple beta-glucosidase is prepared by adopting a gene recombination technology, 50 mM-1000 mM of tyrosol and 10 mM-500 mM of glucose are added, and the reaction is carried out for 20-50 h at the temperature of 37-50 ℃. Obtaining salidroside.

Description

Fermentation production method of salidroside

Technical Field

The invention relates to a method for preparing salidroside by fermenting beta-glucosidase mutant, belonging to the field of genetic engineering and microbial fermentation.

Background

Salidroside is the main effective component of plants of rhodiola of Crassulaceae. The rhodiola rosea is a perennial herbaceous plant, mainly grows in alpine, dry, anoxic, strong ultraviolet irradiation and large day and night temperature difference areas with the altitude of 1600-4000 meters, and has strong environmental adaptability and vitality. Salidroside has multiple physiological pharmacological activities of enhancing immunity, protecting cardiovascular and cerebrovascular, improving sexual function, improving memory, resisting cancer and depression, improving attention, brain and physical strength, further influencing central nervous system, especially effective for weak state of human body, remarkably improving immunity of cell or organism to foreign toxic substances, preventing heart overstrain or arrhythmia, and preventing growth and diffusion of malignant tumor in liver.

The traditional production method of salidroside is to extract and separate the plant rhodiola. Namely: pulverizing radix Rhodiolae and rhizome into coarse powder, reflux-extracting with 70% ethanol, collecting extractive solution, recovering ethanol under reduced pressure, adding equal amount of water into the concentrated solution, stirring, standing, filtering, concentrating the filtrate under reduced pressure, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butanol, and recovering solvent from ethyl acetate and n-butanol respectively to obtain crude product of salidroside.

The number of wild rhodiola rosea is rapidly reduced due to environmental deterioration and artificial over-harvest, and people also continuously explore the possibility of obtaining rhodiola rosea products by plant callus and cell culture technology.

A compact callus system is used for researching the culture of high yield of salidroside, and a plurality of callus tissues obtained from root, stem, leaf and cotyledon segregants of R. The yield of salidroside can reach 57.72mg/g of dry weight, is 5-10 times of wild plant content, and the yield of corresponding salidroside is 555.13mg/L, so that the method is very suitable for industrial mass production.

When the plant cell culture technology is used for obtaining the rhodiola sachalinensis glycoside, the economic value of the rhodiola sachalinensis is questioned because the secondary metabolite yield of the rhodiola sachalinensis is generally low in the cell culture. Therefore, how to increase the content of secondary metabolites is very important. Korean Edimen et al have studied the influence of growth regulating substances and main nutrients in rhodiola sachalinensis cell suspension culture process to find out the preferable culture conditions suitable for cell growth and salidroside accumulation, and prepare for large-scale cell culture. Under experimental conditions, an optimized culture medium which is most beneficial to rhodiola sachalinensis cell culture and salidroside accumulation is utilized, after the cells are cultured for 24 days, the biomass reaches 14.04g/L, and the content of salidroside in stem cells is only 5.66 mg/g. With the research and application of secondary metabolic pathway and metabolic engineering, the yield of salidroside can be improved through large-scale culture and biotransformation of plant cells. 3mmol/L tyrosol is added repeatedly every 24h in exponential growth phase, the output of salidroside can reach 516 mu mol/L, and the result has the possibility of industrial production.

The invention relates to a method for producing salidroside by using apple beta-glucosidase as a novel catalyst. Expressing the apple beta-glucosidase mutant in Escherichia coli (E.coli) by adopting a gene recombination technology, adding tyrosol and glucose, and carrying out enzyme conversion and fermentation to produce salidroside.

Disclosure of Invention

The invention aims to provide a method for producing salidroside by enzyme conversion and fermentation.

As one aspect of the invention, the invention provides a genetically engineered bacterium for high yield of apple beta-glucosidase, which is obtained by recombining and expressing an apple beta-glucosidase mutant in escherichia coli; wherein, the amino acid sequence of the apple beta-glucosidase mutant is shown as SEQ ID NO.1, or the sequence is replaced, deleted or added with one or more amino acids to form an amino acid sequence with the same function. The nucleotide sequence of the gene for coding the apple beta-glucosidase mutant is shown as SEQ ID NO.2, or the nucleotide sequence of the gene is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein; or a nucleotide sequence that hybridizes under specific conditions to SEQ ID NO. 2.

Further, the genetically engineered bacterium is prepared by the following method: the apple beta-glucosidase mutant MBGLM is loaded into a vector pUC 57; and (3) performing double enzyme digestion by NdeI and EcoRI, then cutting the glue, recovering the glue, connecting the glue with a pET-24d (+) vector digested by NdeI and EcoRI, and transforming escherichia coli to obtain the genetically engineered bacterium.

As one aspect of the present invention, the present invention provides a method for producing salidroside by enzymatic conversion fermentation, comprising the steps of:

step A, fermenting and producing beta-glucosidase by using the genetic engineering bacteria to obtain recombinant beta-glucosidase fermentation liquor;

step B, taking the fermentation liquor obtained in the step A as a catalyst, and taking tyrosol and glucose as substrates to synthesize salidroside;

further, the step A of producing the beta-glucosidase by fermenting the genetically engineered bacteria with high yield of the beta-glucosidase comprises the following steps: inoculating the genetically engineered bacteria to an LB liquid culture medium containing Kan, and culturing for 8-10 h at 35-40 ℃; transferring the strain to an LB-containing liquid culture medium by using an inoculation amount of 5-10%, and culturing for 1-5 h at 35-40 ℃; and (3) inducing with IPTG, cooling to 25 ℃, culturing for 40-48 h at constant temperature, inducing to produce enzyme, centrifugally collecting thalli after fermentation is finished, and ultrasonically crushing. Centrifuging to obtain supernatant after induction, namely fermentation liquor.

And further, adding the casein and the glucose in the step B, adjusting the pH value to 9.5-10.5, adding the fermentation liquor obtained in the step A, and reacting.

Further, the initial concentration of tyrosol is between 50mM and 1000 mM;

the initial concentration of the glucose is 10 mM-500 mM;

the concentration of the fermentation liquor is 0.2u/m 1-9.0 u/m 1;

the reaction temperature is 37-50 ℃, and the reaction time is 20-50 h.

According to the invention, the strain with high substrate conversion rate is selected for heterologous expression, so that the fermentation enzyme liquid with higher unit enzyme activity is obtained, the defect of low unit enzyme activity of wild bacteria is overcome, and a foundation is laid for low-cost and large-scale enzyme preparation. Meanwhile, the beta-glucosidase obtained by the invention has good activity and stability under the alkaline pH reaction condition. Meanwhile, the thermal stability is good, the half-life period at 50 ℃ is up to 150h, and the defect of poor stability of other enzyme species under the alkaline reaction condition is overcome. The enzyme reaction has the advantages of simple reaction method, no need of providing additional biological energy, short reaction time and the like. Under different substrate concentrations, high substrate conversion rate under corresponding substrate concentrations can be obtained, and a foundation is laid for industrial amplification production.

Detailed Description

EXAMPLE 1 obtaining of high-yield apple beta-glucosidase genetically engineered bacteria

(1) According to the gene sequence of apple beta-glucosidase, base optimization and artificial synthesis of a gene mutant MBGLM (the amino acid sequence of the mutant is shown as SEQ ID NO.1, the highest enzyme activity of the mutant is 198IU/ml, which is 9 times of the enzyme activity of a wild type strain), and the mutant is loaded into a vector pUC 57.

(2) The target gene was digested with NdeI and EcoRI, cut and recovered, ligated with NdeI and EcoRI digested pET-24d (+) vector, and transformed into E.coli BL21 (DE)₃)；

The transformation mixture is coated on a 50mg/L kalamycin-resistant LB plate, then transformants are picked, cultured for 6-8 h at 37 ℃, and single colony is picked to obtain MBGLM-pET-24d (+)/E.coli BL21 (DE)₃)。

And culturing in an LB/Kan liquid culture medium for 8-10 h, and then preserving the strain.

Example 2 enzyme production by fermentation

Inoculating a genetically engineered bacterium MBGLM-pET-24d (+)/E.coli BL21(DE3) into an LB liquid culture medium containing Kan, and culturing at 37 ℃ for 8-10 h; inoculating to LB liquid culture medium with 5% inoculum size, and culturing at 37 deg.C for 3 hr; inducing with 0.4mM/L IPTG, cooling to 25 ℃, culturing for 40-48 h at constant temperature, inducing enzyme production, after fermentation, centrifugally collecting thalli, and ultrasonically crushing. Centrifuging to obtain supernatant after induction, namely crude enzyme liquid.

Example 3 enzymatic Synthesis of Salidroside

250mM tyrosol, 50mM glucose and 1.25U/ml crude enzyme solution were weighed and dissolved in a buffer/n-hexane (1/1, V/V) mixed system. The reaction was carried out at 50 ℃ with shaking at 180r/min for 24 h. After the reaction is finished, extracting a certain amount of water phase reaction liquid, diluting the water phase reaction liquid by using distilled water with 2 times of volume, filtering the water phase reaction liquid by using a microporous filter membrane, and concentrating the filtrate to be dry to obtain white solid powder. mp 156-^-：268，1630，1609；¹³CNMR(CDOD)6：35.0(Ar-CH₂-)69.0(-CH₂-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-H₂O+H⁺). Pharmacodynamic study of Compound Hongjingtian tablet (Zhongjing, Zhangyao, Van primer, etc.)]The Chinese patent medicine, 1999, 21(11):588-591) data.

Taking the product salidroside, analyzing on a high performance liquid chromatograph: chromatographic conditions are as follows: a Sinochrom ODS-BP column (4.6mm multiplied by 200mm, 5um), a UV230+ type ultraviolet detector, a P230 type high-pressure constant flow pump, a column temperature of 30 ℃, a detection wavelength of 278nm, a sample injection amount of 20 mu l, and a mobile phase of 0.8mL/min methanol/water (30/70, V/V). The results are shown in Table 1.

TABLE 1 amount of salidroside formed in the reaction product and glucose conversion

Reaction time (h)	Salidroside (mg/L)	Glucose conversionPercentage (%)
			1	3.50	11.0
10	4.55	14.3
			20	5.25	16.5
30	6.07	19.1
			40	7.38	23.2
50	7.16	22.5

The result shows that the apple beta-glucosidase mutant MBGLM can completely react on tyrosol and glucose enzymatic reaction for 40 hours.

Sequence listing

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Claims

1. A genetically engineered bacterium for high yield of apple beta-glucosidase is characterized in that the genetically engineered bacterium is obtained by recombining and expressing an apple beta-glucosidase mutant in escherichia coli; wherein the amino acid sequence of the apple beta-glucosidase mutant is shown as SEQ ID NO. 1.

2. The genetically engineered bacterium of claim 1, wherein a nucleotide sequence encoding the apple β -glucosidase mutant gene is shown in SEQ ID No. 2.

3. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is prepared by the following method: the apple beta-glucosidase mutant is loaded into a vector pUC 57; and (3) performing double enzyme digestion by NdeI and EcoRI, then cutting the glue, recovering the glue, connecting the glue with a pET-24d (+) vector digested by NdeI and EcoRI, and transforming escherichia coli to obtain the genetically engineered bacterium.

4. A method for producing salidroside by enzymatic conversion and fermentation is characterized by comprising the following steps:

step A, fermenting and producing beta-glucosidase by using the genetic engineering bacteria of claim 1 to obtain recombinant beta-glucosidase fermentation liquor;

and step B, taking the fermentation liquor obtained in the step A as a catalyst, and taking tyrosol and glucose as substrates to synthesize salidroside.

5. The method of claim 4, wherein the fermentation production of the beta-glucosidase by the genetically engineered bacteria in the step A comprises the following steps: inoculating the genetically engineered bacteria to an LB liquid culture medium containing Kan, and culturing for 8-10 h at 35-40 ℃; transferring the strain to an LB-containing liquid culture medium by using an inoculation amount of 5-10%, and culturing for 1-5 h at 35-40 ℃; and (3) inducing with IPTG, cooling to 25 ℃, culturing for 40-48 h at constant temperature, inducing to produce enzyme, after fermentation is finished, centrifugally collecting thalli, ultrasonically crushing, centrifuging, and obtaining induced supernatant, namely fermentation liquor.

6. The method of claim 4, wherein the step B comprises adding tyrol and glucose, adjusting pH to 9.5-10.5, adding the fermentation liquid obtained in the step A, and reacting.

7. The method of claim 6, wherein the initial concentration of tyrosol is between 50mM and 1000 mM.

8. The method of claim 6, wherein the initial concentration of glucose is 10mM to 500 mM.

9. The method of claim 6, wherein the concentration of the fermentation broth is 0.2u/m 1-9.0 u/m 1.

10. The method of claim 6, wherein the reaction temperature is 37-50 ℃ and the reaction time is 20-50 h.