阅读说明：本技术 一种基于酶法合成阿魏酸及阿魏酸甲酯方法的优化与改进 (optimization and improvement of method for synthesizing ferulic acid and methyl ferulate based on enzyme method ) 是由 廖海黎 周嘉裕 陈安琪 李秋娥 张田 黄新河 于 2019-11-07 设计创作，主要内容包括：本发明公开了一种基于酶法合成阿魏酸及阿魏酸甲酯方法的优化与改进,包括以下步骤：步骤1：将含有质粒<I>LCCOMT</I>-pET28a的大肠杆菌BL21菌株进行活化；步骤2：对步骤1得到的菌液进行扩大培养后,同时加入IPTG、咖啡酸/咖啡酸甲酯进行共发酵；步骤3：将共发酵后的发酵液离心,用乙酸乙酯萃取后即可得到阿魏酸/阿魏酸甲酯；本发明采用基因工程酶(LCCOMT)基因工程菌的共发酵制备阿魏酸或阿魏酸甲酯,工艺简单、成本低、过程可控且环保。(The invention discloses optimization and improvement of methods for synthesizing ferulic acid and methyl ferulate based on an enzyme method, which comprises the following steps of step 1, plasmid-containing LCCOMT -escherichia coli BL21 strain of pET28a for activation; step 2: after the bacterial liquid obtained in the step 1 is subjected to amplification culture, IPTG and caffeic acid/methyl caffeate are added for co-fermentation; and step 3: centrifuging the fermented liquid after co-fermentation, and extracting with ethyl acetate to obtain ferulic acid/methyl ferulate; the method adopts the co-fermentation of genetically engineered enzyme (LCCOMT) genetically engineered bacteria to prepare the ferulic acid or the ferulic acid methyl ester, and has the advantages of simple process, low cost, controllable process and environmental protection.)

1, optimization and improvement of a method for synthesizing ferulic acid and methyl ferulate based on an enzyme method, which is characterized by comprising the following steps:

step 1: activating the Escherichia coli BL21 strain containing the plasmid LCCOMT-pET28 a;

step 2: after the bacterial liquid obtained in the step 1 is subjected to amplification culture, IPTG (isopropyl-beta-D-thioglycollate) and caffeic acid or methyl caffeate are added for co-fermentation;

and step 3: centrifuging the fermented liquid, and extracting with ethyl acetate to obtain ferulic acid or methyl ferulate.

2. The optimization and improvement of the method for synthesizing ferulic acid and methyl ferulate based on enzyme method according to claim 1, wherein the activation process in step 1 is as follows:

s11: streaking and inoculating the strain on an LB solid culture medium plate containing 10 mu g/mL kanamycin, and inversely culturing the strain in a constant-temperature incubator at 37 ℃ for overnight;

s12: selecting a single colony, inoculating the single colony to an LB liquid culture medium containing 10 mu g/mL kanamycin, and carrying out primary culture for 14-16 h;

s13: and (4) sucking the bacterial liquid obtained in the step S12 into a centrifugal tube, and adding sterilized glycerol to the centrifugal tube, so that the bacterial liquid can be stored for a long time at the temperature of minus 80 ℃.

3. The optimization and improvement of the enzymatic synthesis-based ferulic acid and methyl ferulate method according to claim 1, wherein the scale-up culture in step 2 is as follows:

culturing the strain at 37 ℃ and 200rpm until the OD600 is between 0.6 and 0.8.

4. The optimization and improvement of the methods for synthesizing ferulic acid and methyl ferulate based on the enzyme method according to claim 1, wherein the co-fermentation conditions in the step 2 when the additive is caffeic acid are that the fermentation time is 24-48 h, the temperature is 20 ℃, the amount of IPTG is 95.32mg/L, and the co-fermentation conditions when the additive is methyl caffeate are that the fermentation time is 24-48 h, the temperature is 30 ℃, and the amount of IPTG is 95.32 mg/L.

5. The optimization and improvement of the enzymatic synthesis-based ferulic acid and methyl ferulate method according to claim 1, wherein the step 3 comprises centrifuging the fermentation broth at 4 ℃ for 10min, collecting the supernatant, and extracting with ethyl acetate several times to obtain ferulic acid or methyl ferulate.

Technical Field

The invention relates to the technical field of bioengineering, in particular to optimization and improvement of methods for synthesizing ferulic acid and methyl ferulate based on an enzyme method.

Background

Ferulic acid is kinds of phenolic acid commonly existing in the plant world, and has a chemical name of 4-hydroxy-3-methoxycinnamic acid, and has a plurality of health care functions, such as free radical removal, thrombosis resistance, bacterial resistance, inflammation resistance, tumor inhibition, hypertension prevention and treatment, heart disease prevention, sperm motility enhancement, human epidermal melanocyte proliferation inhibition, melanin synthesis, tyrosinase activity and the like, wherein the main application of the ferulic acid comprises 1 whitening components in health care cosmetics, 2, the ferulic acid can be used as a synthetic precursor of vanillin in the food industry, the annual sales of the ferulic acid in the whole world exceeds 100 billion yuan RMB, 3, the ferulic acid is used as an antioxidant and a preservative of food, 4, more than 20 finished medicines taking the ferulic acid as a main component have a market prospect of .

For example, the methyl ferulate can be added into cosmetics, not only can the whitening effect be achieved, but also the skin can have a silk texture after long-term use, and the methyl ferulate can also be used as a synthetic intermediate of a powerful whitening agent isooctyl ferulate.

The method for synthesizing ferulic acid by using an environment-friendly biological method is a necessary trend, so that the method for synthesizing ferulic acid by using modern genetic engineering is a necessary trend, at present, a method for synthesizing ferulic acid by using enzyme methods with the patent number of 2015100756780 and a method for synthesizing ferulic acid methyl ester by using enzyme methods with the patent number of 2019102612912 solve the heavy pollution defect of chemical synthesis and chemical extraction methods.

Disclosure of Invention

The invention provides optimization and improvement of methods for synthesizing ferulic acid and methyl ferulate by adopting genetic engineering enzyme in vivo transformation and based on an enzyme method.

The invention adopts the technical scheme that the optimization and improvement of methods for synthesizing ferulic acid and methyl ferulate based on an enzyme method comprise the following steps:

step 1: activating the Escherichia coli BL21 strain containing the plasmid LCCOMT-pET28 a;

step 2: after the bacterial liquid obtained in the step 1 is subjected to amplification culture, IPTG (isopropyl-beta-D-thioglycollate) and caffeic acid or methyl caffeate are added for co-fermentation;

and step 3: centrifuging the fermented liquid, and extracting with ethyl acetate to obtain ferulic acid or methyl ferulate.

2. The optimization and improvement of the method for synthesizing ferulic acid and methyl ferulate based on enzyme method according to claim 1, wherein the activation process in step 1 is as follows:

s11: streaking and inoculating the strain on an LB solid culture medium plate containing 10 mu g/mL kanamycin, and inversely culturing the strain in a constant-temperature incubator at 37 ℃ for overnight;

s12: selecting a single colony, inoculating the single colony to an LB liquid culture medium containing 10 mu g/mL kanamycin, and carrying out primary culture for 14-16 h;

s13: and (4) sucking the bacterial liquid obtained in the step S12 into a centrifugal tube, and adding sterilized glycerol to the centrifugal tube, so that the bacterial liquid can be stored for a long time at the temperature of minus 80 ℃.

In step , the scale-up in step 2 is as follows:

culturing the strain at 37 ℃ and 200rpm until the OD600 is between 0.6 and 0.8.

, co-fermenting the additive caffeic acid in the step 2 under the conditions of 24-48 h of fermentation time, 20 ℃ and 95.32mg/L of IPTG, and co-fermenting the additive caffeic acid methyl ester under the conditions of 24-48 h of fermentation time, 30 ℃ and 95.32mg/L of IPTG.

, the step 3 comprises centrifuging the fermentation broth at 4 deg.C for 10min, collecting supernatant, and extracting with ethyl acetate for several times to obtain ferulic acid or methyl ferulate.

The invention has the beneficial effects that:

(1) the method does not need to carry out ultrasonic disruption on the genetic engineering bacteria, does not need expensive nickel affinity columns to carry out affinity purification on the LCCOMT recombinant protein, does not need to add S-adenosylmethionine with higher price, does not need dialysis bags and the like, and therefore, the cost is lower;

(2) the invention adopts a genetic engineering enzymatic internal transformation method, omits the processes of affinity chromatography, dialysis and the like for extracting LCCOMT recombinase, has shorter flow, simpler operation and shortened process time;

(3) the method does not need LCCOMT separation and purification, so that the treatment without using hydrochloric acid and a nickel affinity column needs an alkaline solution, and the process is more environment-friendly;

(4) the process has fewer operation steps and stronger operability, and can meet the requirements of operators at different levels.

Drawings

FIG. 1 is a schematic process flow diagram of the present invention.

FIG. 2 is a schematic diagram of the prior art of synthesizing ferulic acid by genetic engineering.

FIG. 3 is an HPLC chart of ferulic acid obtained in the example of the invention.

FIG. 4 is an HPLC chart of methyl ferulate obtained in the example of the present invention.

Detailed Description

The invention is further illustrated in the following description of specific embodiments and in the accompanying drawings.

As shown in figure 1, optimization and improvement of the method for synthesizing ferulic acid and methyl ferulate based on enzyme method comprises the following steps:

step 1: activating the Escherichia coli BL21 strain containing the plasmid LCCOMT-pET28 a;

the strain source of the genetic engineering bacteria is as follows: the genetically engineered bacterium used in the experiment is Escherichia coli BL21 strain which contains plasmid LCCOMT-pET28a and is preserved in the laboratory.

The preparation method of the strain is as follows (2015100756780, the acquisition method of the strain is disclosed in detail in methods for synthesizing ferulic acid by enzyme method):

1. PCR amplifying the full-length reading frame of the COMT gene of the ligusticum wallichii from the cDNA by using primers (A1: CGCGGATCCATGAATACGGAGCTGATCCCACC; A2: CGGAATTCACATTAAGCAGATGCCAGACACCC) with two enzyme cutting sites of EcoR1 and BamH1 respectively;

2. carrying out double enzyme digestion on the amplified rhizoma ligustici wallichii COMT gene fragment and pET28a by using EcoR1 and BamH1 respectively to obtain fragments with sticky ends at two ends; connecting the two fragments by using T4DNA ligase, wherein the system comprises 10 x T4DNA ligasebuffer 2.5ul, rhizoma ligustici wallichii COMT DNA fragment 6ul after double digestion, pET28a vector 2ul after double digestion and T4 DNAllagase 1ul, and supplementing 25ul by using sterilized double distilled water; ligation was carried out overnight at 16 ℃; obtaining a connected recombinant plasmid;

3. 10ul of the recombinant plasmid was used to transform BL21 competent cells. Coating the successfully transformed recombinant BL21 genetic engineering bacteria on a plate containing 50mg/L kanamycin to screen positive clones;

4. and (3) identifying the preliminarily screened recombinant strain by colony PCR and plasmid double digestion, and sequencing to determine the correctness of a reading frame to obtain the expected recombinant strain.

The activation process is as follows:

s11: streaking the strain on an LB solid culture medium plate containing 10 mu g/mL kanamycin (the final concentration of the kanamycin is 10 mu g/mL), and inversely culturing the strain in a constant-temperature incubator at 37 ℃ for overnight;

s12: selecting a single colony, inoculating the single colony into 50mL LB liquid culture medium containing 10 mu g/mL kanamycin to perform primary culture for 14-16 h;

s13: and (4) sucking 800 mu L of the bacterial liquid obtained in the step S12 into a 1.5mL centrifuge tube, and adding 150 mu L of sterilized glycerol to the centrifugal tube, so that the bacterial liquid can be stored for a long time at the temperature of minus 80 ℃.

Step 2: after the bacterial liquid obtained in the step 1 is subjected to amplification culture, IPTG and caffeic acid are added for co-fermentation;

1mL of the overnight-cultured recombinant strain was inoculated into 100mL of LB liquid medium containing 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl, and 50mg/L of kanamycin, and cultured at 37 ℃ and 200rpm until OD600 became 0.6-0.8.

IPTG was added as an inducer for efficient expression of LCCOMT at a final concentration of 1 mol/L. Simultaneously adding a substrate caffeic acid or methyl caffeate; wherein the final concentration of caffeic acid is 0.2g/L, and the final concentration of methyl caffeate is 0.16 g/L.

The conditions for carrying out the co-fermentation in the invention are 20-25 ℃, 150rpm, and 10-12 h of co-fermentation culture. On the basis, orthogonal tests are carried out, and then the optimal fermentation conditions are obtained. The optimal fermentation conditions for preparing ferulic acid obtained by orthogonal test are as follows: the fermentation time is 24-48 h, the temperature is 20 ℃, and the dosage of IPTG is 95.32 mg/L. The optimal fermentation conditions for preparing the ferulic acid methyl ester obtained by the orthogonal test are as follows: the fermentation time is 24-48 h, the temperature is 30 ℃, and the dosage of IPTG is 95.32 mg/L.

And step 3: centrifuging the fermented liquid, and extracting with ethyl acetate to obtain ferulic acid or methyl ferulate.

The obtained fermentation liquor was centrifuged at 13000g for 10min at 4 ℃ and the supernatant was collected. Extracting with ethyl acetate for 4 times to obtain ferulic acid or methyl ferulate crude product.

Then carrying out high-phase liquid chromatography detection

Finally, according to the previous single-factor experiment result, three main parameters of co-fermentation time, co-fermentation temperature, IPTG dosage and the like are selected to carry out L9 (4) by using an orthogonal design assistant³) And (4) performing orthogonal experiments. So as to obtain the most suitable co-fermentation process and co-fermentation effect.

steps are carried out for the optimal process conditions obtained by orthogonal experiments to find that the co-fermentation time has larger influence on the yield and has better economical efficiency, therefore, the subsequent experiments are prolonged to 24 and 48 hours through the co-fermentation time to determine the final process conditions.

Filtering the obtained ferulic acid and the ferulic acid methyl ester crude product with 0.22 μm organic phase ultrafiltration membrane, and collecting the filtrate supernatant for high performance liquid chromatography detection. High performance liquid chromatography conditions: the column was an Elite Hypersil ODS2(4.6 mm. times.200 mm, 5 μm) and the loading was 10. mu.L.

Wherein the sample source is: caffeic acid, S-adenosine-L-ammonium methosulfate (SAM) from Biotechnology engineering (Shanghai) Ltd, caffeic acid methyl ester standard from Shanghai leaf Biotech Ltd, ferulic acid standard from Chengdu City drug laboratory, and ferulic acid methyl ester standard from Chengdu Klomao Biotech Ltd.

Ferulic acid detection system: mobile phase 0.1% phosphoric acid-methanol (66:34), flow rate: 1mL/min, column temperature 30 ℃, and detection wavelength 323 nm.

Detection system of ferulic acid methyl ester: mobile phase 0.1% methyl phosphate (55:45), flow rate: 1mL/min, column temperature 30 ℃, detection wavelength 330 nm.

The final yield was calculated from the integrated peak areas of substrate and product.

Orthogonal experiment optimization:

and selecting three parameters such as co-fermentation temperature, co-fermentation time, IPTG dosage and the like according to the previous single-factor experiment result, and performing an orthogonal experiment by using an orthogonal design assistant. Using L9 (4)³) Orthogonal tables the experiments were arranged and the results are shown in tables 1, 2 and 3.

TABLE 1 orthogonal experiment factors and horizon

TABLE 2 Ferulic acid Quadrature test results

TABLE 3 results of orthogonal experiments with methyl ferulate

As can be seen from the above orthogonal experimental results, the influence on the yield of ferulic acid is in order of magnitude: co-fermentation time, IPTG dosage and co-fermentation temperature. The result of the most suitable genetic engineering strain co-fermentation synthesis of ferulic acid obtained by orthogonal experiments is as follows: the co-fermentation time is 18h, the IPTG dosage is 95.32mg/L, and the co-fermentation temperature is 20 ℃. The experimental result shows that the yield of ferulic acid synthesized by co-fermentation of the genetic engineering bacteria under the optimal process condition can reach 39.19% and 78 mg/L.

The influence on the yield of methyl ferulate was in order: IPTG dosage, co-fermentation temperature and co-fermentation time. The result of the most suitable genetic engineering strain co-fermentation synthesis of ferulic acid obtained by orthogonal experiments is as follows: the co-fermentation time is 16h, the IPTG dosage is 95.32mg/L, and the co-fermentation temperature is 30 ℃. Experimental results show that the yield of the ferulic acid methyl ester synthesized by the co-fermentation of the genetic engineering strain under the optimal process conditions can reach 24.14 percent and 78 mg/L.

The optimum process conditions obtained by the orthogonal experiment are further explored by to find that the co-fermentation time has larger influence on the yield and has better economical efficiency, so in the subsequent experiments, the fermentation temperature and the IPTG dosage are fixed, and the co-fermentation time is respectively prolonged to 24 hours and 48 hours.

Final process conditions of ferulic acid: the co-fermentation temperature is 20 ℃, the IPTG dosage is 95.32mg/L, and the fermentation is respectively carried out for 24h and 48 h. The experimental result shows that the yield of the ferulic acid can reach 48.43% (96.86mg/L) after 24 hours of co-fermentation. The yield of the ferulic acid can reach 89.85% (178mg/L) after 48 hours of co-fermentation.

Final process conditions for methyl ferulate: the co-fermentation temperature is 30 ℃, the IPTG dosage is 95.32mg/L, and the fermentation is respectively carried out for 24h and 48 h. The experimental result shows that the yield of the ferulic acid methyl ester can reach 99.21% (158.4mg/L) after 24 hours of co-fermentation. The yield of the ferulic acid can reach 100% (160mg/L) after 48 hours of co-fermentation. The results are shown in Table 4.

TABLE 4 yield of ferulic acid and methyl ferulate after Co-fermentation

The invention adopts a genetic engineering in-vivo transformation method, only needs to add an Inducer (IPTG) and substrates caffeic acid and caffeic acid methyl ester while fermenting genetic engineering bacteria, does not need to add expensive S-adenosylmethionine, and can obtain ferulic acid or ferulic acid methyl ester crude products by centrifugation and extraction after fermentation. Compared with the prior art, 1, the method does not need to carry out ultrasonic disruption on the genetically engineered bacteria, so that an ultrasonic disruption instrument does not need to be purchased, an expensive nickel affinity column (380 yuan/g) is not needed to carry out affinity purification on the LCCOMT recombinant protein, S-adenosylmethionine (24 yuan/g) with higher price does not need to be added, a dialysis bag (1cm inner diameter, 180 yuan/m) is not needed, and the like, and the cost is lower. 2. Because the co-fermentation method is adopted, the biosynthesis is directly carried out in the bacteria, and the processes of affinity chromatography, dialysis (24h) and the like for extracting the LCCOMT recombinase are omitted. Shorter flow, simpler operation and shorter process time. 3. The separation and purification process of the LCCOMT recombinase requires hydrochloric acid and treatment of a nickel affinity column, so that an alkaline solution is required, and the process is more environment-friendly. 4. The process has fewer operation steps and stronger operability, and can meet the requirements of operators at different levels. Therefore, the method has the advantages of simple process, low cost, controllable process, environmental protection, important application value and good economy.