CN114657220A - Method for preparing sulforaphane - Google Patents

Method for preparing sulforaphane Download PDF

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CN114657220A
CN114657220A CN202210490467.3A CN202210490467A CN114657220A CN 114657220 A CN114657220 A CN 114657220A CN 202210490467 A CN202210490467 A CN 202210490467A CN 114657220 A CN114657220 A CN 114657220A
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myrosinase
enzymolysis
sulforaphane
attgg4
preparing
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姜宏
毛相朝
刘岩
王丽丽
韩达
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Ocean University of China
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    • C12Y302/01147—Thioglucosidase (3.2.1.147), i.e. myrosinase

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Abstract

The invention discloses a method for preparing sulforaphane, which belongs to the technical field of functional enzymes, and specifically comprises the following steps: carrying out enzymolysis on glucoraphanin or a raw material containing glucoraphanin by using myrosinase AtTGG4 or an enzyme preparation containing the myrosinase AtTGG4 to prepare the glucoraphanin, wherein the enzymolysis conditions are as follows: performing enzymolysis for 10-20 minutes at 20-30 ℃; the amino acid sequence of the myrosinase AtTGG4 is shown in SEQ ID NO. 1. The method for preparing the sulforaphane by utilizing the myrosinase AtTGG4 to carry out enzymolysis on the sulforaphane, has the advantages of high enzymolysis speed and high enzymolysis efficiency, the enzymolysis efficiency can reach 99.86 percent when the enzymolysis is carried out for 15 minutes, the sulforaphane can be recycled for more than 10 times, and the method is very suitable for industrial application and has important significance on the industrial production of the sulforaphane.

Description

Method for preparing sulforaphane
Technical Field
The invention relates to a method for quickly and efficiently preparing sulforaphane by using myrosinase AtTGG4, belonging to the technical field of functional enzymes.
Background
Sulforaphane is one of natural active ingredients with the strongest anticancer effect found in vegetables at present, and has excellent effects on preventing and treating cancers, cardiovascular diseases, mental diseases, diabetes, inflammation and the like. Sulforaphane is obtained by hydrolyzing sulforaphane with myrosinase, which is the only enzyme found so far that can hydrolyze glucosinolates and is widely present in cruciferous plants.
At present, the sulforaphane is prepared by taking broccoli seeds as raw materials, releasing myrosinase by crushing and other modes, and hydrolyzing self-sulforaphane by utilizing plant endogenous myrosinase. In addition, the brassicaceae plant can be used as a raw material, exogenous myrosinase is added to prepare the sulforaphane, the yield of the method is improved compared with the former method, the myrosinase needs to be separated and purified from the plant in advance, and the price is high. Therefore, the development of a high-efficiency and low-cost preparation method of the sulforaphane is of great significance.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for quickly and efficiently preparing the sulforaphane by using the myrosinase AtTGG 4.
The invention is realized by the following technical scheme:
a method for preparing sulforaphane comprises the following steps: adopting myrosinase AtTGG4 or an enzyme preparation containing myrosinase AtTGG4 to carry out enzymolysis on glucoraphanin or a raw material containing the glucoraphanin to prepare the sulforaphane, wherein the enzymolysis conditions are as follows: carrying out enzymolysis for 10-20 minutes at 20-30 ℃.
The myrosinase AtTGG4 is myrosinase disclosed in the prior art (namely, myrosinase TGG4 disclosed in another invention patent application CN 112899177A of the applicant), the amino acid sequence of the myrosinase is shown in SEQ ID NO.1, and a person skilled in the art can construct a recombinant expression vector by using a gene recombination technology, obtain recombinant engineering bacteria by transfection, and perform fermentation culture on the recombinant engineering bacteria to obtain the myrosinase AtTGG 4. Specifically, an enzyme preparation containing myrosinase AtTGG4 can be prepared by the following method: the gene recombination technology is utilized to construct a recombinant expression vector containing the coding gene of the myrosinase AtTGG4, yarrowia lipolytica is transfected to obtain recombinant engineering bacteria, fermentation culture is carried out, fermentation liquor is centrifuged, thalli are collected, and freeze-drying is carried out to obtain enzyme powder, namely the enzyme preparation containing the myrosinase AtTGG 4.
The amino acid sequence of myrosinase AtTGG4 is shown below (SEQ ID NO. 1):
SQKVCNPECKAKEPFHCDNTHAFNRTGFPRNFTFGAATSAYQIEGAAHRALNGWDYFTHRYPEKVPDRSSGDLACDSYDLYKDDVKLLKRMNVQAYRLSIAWSRVLPKGRLTGGVDENGITYYNNLINELKANGIEPYVTIFHWDVPQTLEDEYGGFLSTRIVEDYTNYAELLFQRFGDRVKFWITLNQPFSLATKGYGDGSYPPGRCTGCELGGDSGVEPYTVAHNQLLAHAKTVSLYRKRYQKFQGGKIGTTLIGRWFAPLNEFSELDKAAAKRAFDFFVGWFLDPLVYGKYPTIMREMVGDRLPEFTPEQSALVKGSLDFLGLNYYVTQYATDAPPPTQLNAITDARVTLGFYRNGVPIGVVAPSFVYYPPGFRQILNYIKDNYKNPLTYITENGVADLDLGNVTLATALADNGRIQNHCSHLSCLKCAMKDGCNVAGYFAWSLMDNYEFGNGYTLRFGMNWVNFTNPADRKEKASGKWFSKFLAK。
further, the specific mode of the fermentation culture is as follows: the recombinant engineering strain is picked up, inoculated into 10ml YPD liquid culture medium, cultured for 16 hours at 30 ℃ and 200rpm, then inoculated into 250 ml PPB culture medium according to the inoculum size of 1 percent, and fermented for 5 days at 30 ℃ and 200 rpm.
Further, the sulforaphane-containing material is selected from broccoli seeds.
Further, the enzymolysis conditions are as follows: enzymatic hydrolysis was carried out at 25 ℃ for 15 minutes.
Further, the specific mode of the enzymolysis is as follows: adding myrosinase AtTGG4 or an enzyme preparation containing myrosinase AtTGG4 into a substrate solution with the pH value of 4.0-6.0, and carrying out enzymolysis for 10-20 minutes at the temperature of 20-30 ℃; the substrate in the substrate solution is glucoraphanin or crushed broccoli seeds.
Further, the specific mode of the enzymolysis is as follows: 40U of the enzyme preparation containing myrosinase AtTGG4 was added to 10ml of a substrate solution with pH 5.0 and subjected to enzymatic hydrolysis at 25 ℃ for 15 minutes; recovering enzyme preparation containing myrosinase AtTGG4, and repeatedly performing enzymolysis for more than 10 times; the substrate solution is prepared by the following method: heating broccoli seeds for 1 h at 110 ℃ to inactivate endogenous enzymes, crushing to obtain broccoli seed powder, and adding water to prepare a substrate solution with the concentration of 0.1 g/ml.
The recombinant engineering bacteria for expressing the myrosinase AtTGG4 are constructed by yarrowia lipolytica, methanol is not required to be added for induction during fermentation, and the myrosinase AtTGG4 can be efficiently expressed. Because the expressed myrosinase AtTGG4 is attached to or embedded on the cell surface, the collected thallus can be directly used as an enzyme preparation for the preparation of the sulforaphane, the recycling of the myrosinase can be realized by recovering cells, the high-efficiency and multi-batch repeated preparation is carried out, and the preparation cost is greatly reduced.
The invention utilizes myrosinase AtTGG4 to carry out enzymolysis on the sulforaphane to prepare the sulforaphane, the enzymolysis speed is high, the enzymolysis efficiency is high, when the enzymolysis is carried out for 15 minutes at 25 ℃, the yield of the sulforaphane is 10 mg/g, the enzymolysis efficiency can reach 99.86%, and the sulforaphane still has better enzymolysis efficiency (98.16%) at the low temperature of 20 ℃. The enzymolysis efficiency, the enzymolysis speed and the enzymolysis temperature are obviously superior to other myrosinase. The time required by myrosinase reported in the prior art is more than 30 minutes, and the yield of the sulforaphane is low, for example, when the myrosinase mentioned in the research on the extraction process optimization and the activity function of the sulforaphane in the broccoli carries out enzymolysis for 7.1 hours at 36.8 ℃, the yield of the sulforaphane is 0.408 mg/g. The myrosinase mentioned in the research on the process of enzymolysis and extraction of sulforaphane in broccoli seeds is assisted by ultrasonic extraction, the enzymolysis time at 35 ℃ is 8 hours, and the yield of the sulforaphane is 6.682 mg/g. The exogenous enzyme extracted from Chinese cabbage mentioned in the section of Enhanced production of sulforaphane by exogenous enzymes obtained from Chinese cabbage flowing and harvesting of hydrolyzed and digested by yeast in Chinese cabbage, is subjected to enzymolysis at 30 ℃ for 30 minutes, the yield of sulforaphane is 240 mu mol/1139 mu mol, and the enzymolysis efficiency is 21.07%. In addition, the myrosinase AtTGG4 has different enzymolysis speeds when different substrates are enzymolyzed, the optimal enzymolysis time for enzymolysis of the sulforaphane is 1.5 hours (recorded in CN 112899177 a), and the optimal enzymolysis time for enzymolysis of the sulforaphane is 15 minutes, so that the myrosinase AtTGG4 has strong substrate specificity, and the enzymolysis speed and the enzymolysis efficiency are greatly different when substrates with extremely close enzymolysis structures (the structural difference between the sulforaphane and the sulforaphane is very small, and the difference is only one carbon-carbon double bond). Furthermore, after the enzyme preparation prepared by the invention is recycled for 10 times, the yield of the sulforaphane can still reach more than 87 percent, so the enzyme preparation is very suitable for industrial application and has important significance for industrial production of the sulforaphane.
In addition, in the prior art, the optimum enzymolysis conditions for researching myrosinase are judged by taking product glucose as a standard, but the yield cannot be directly equal to the sulforaphane, so that the optimum pH, enzyme adding amount and reaction time for producing the sulforaphane are determined through experimental optimization, and the small test for preparing the sulforaphane is realized.
The various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art.
Drawings
FIG. 1: and (3) high performance liquid detection images of the product and the sulforaphane standard substance.
FIG. 2: and (5) identifying a product by mass spectrometry.
FIG. 3: graph showing the effect of pH change on product yield.
FIG. 4: the product yield changes under different enzyme adding amounts and different reaction times are shown schematically.
FIG. 5: schematic representation of relative yield of sulforaphane after repeated use of the enzyme powder formulation.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents and materials used in the following examples are conventional instruments, reagents and materials known in the art and are commercially available. Unless otherwise specified, the experimental methods and the detection methods in the following examples are conventional experimental methods and detection methods in the prior art.
EXAMPLE 1 preparation of myrosinase AtTGG4 Using Yeast engineered bacteria
Constructing a recombinant expression vector containing the coding gene of the myrosinase AtTGG4 by using a conventional gene recombination technology, and transfecting yarrowia lipolytica to obtain recombinant engineering bacteria; selecting recombinant strains, inoculating the recombinant strains in 10ml of YPD liquid culture medium, culturing at 30 ℃ and 200rpm for 16 hours, inoculating the recombinant strains into 250 ml of PPB culture medium according to the inoculum size of 1%, and fermenting at 30 ℃ and 200rpm for 5 days; and (3) centrifuging the fermentation liquor at 6000rpm for 5 minutes, collecting thalli, and freeze-drying to obtain an enzyme powder preparation of the myrosinase AtTGG 4.
Example 2 determination of enzyme powder preparation Activity
The glucose master solution of 1 mg/ml was diluted to different concentration gradients (100%, 80%, 60%, 40%, 20%), 100. mu.l of each concentration was extracted, 300. mu.l of DNS solution was added, boiling water bath was carried out for 10 minutes, after cooling, 800. mu.l of distilled water was added for dilution, 200. mu.l was taken out in a 96-well plate, and then absorbance at 540 nm was measured with a microplate reader to draw a glucose standard curve.
The enzyme powder preparation prepared in example 1 was added with water to prepare an enzyme solution having a concentration of 30 mg/ml, 80. mu.l of the enzyme solution was taken, 120. mu.l of a glucoraphanin standard solution (chromatographic grade, purity ≥ 95%) was added, 300. mu.l of a citrate buffer solution having pH 5 was added, reaction was carried out at 75 ℃ for 20 minutes, centrifugation was carried out at 12000 rpm after cooling for 2 minutes, 100. mu.l of the supernatant was taken and 150. mu.l of a DNS solution was added, well mixed, subjected to boiling water bath for 10 minutes, cooled and centrifuged again (centrifugation at 12000 rpm for 2 minutes), 200. mu.l of the supernatant was taken and 200. mu.l of distilled water was added, 200. mu.l of the solution was sucked into a 96-well plate after mixing, and the absorbance at 540 nm was measured using a microplate reader. One unit of myrosinase activity (U) is defined as the amount of glucose (μmol) produced per unit mass of enzyme powder preparation (g) in a unit time (min). The enzyme activity of the enzyme powder preparation is measured to be 48.48U/g.
Example 3 determination of optimum pH for the preparation of sulforaphane
The optimum pH in the sulforaphane preparation process is researched by taking the sulforaphane glycoside in the purified broccoli seeds as a substrate:
heating broccoli seeds for 1 h at 110 ℃ to inactivate endogenous enzymes, crushing to obtain broccoli seed powder, and dissolving the seed powder in a distilled water ratio of 10% (w/v, g/ml) to obtain a substrate solution; the substrate solution was taken in 4 portions (10 ml portions), pH was adjusted to 3, 4, 5 and 6 with a citrate buffer solution, 40U of the enzyme powder preparation (prepared in example 1) was added, and the enzymatic reaction was carried out at 25 ℃ for 20 minutes.
Taking 1 ml of enzymolysis liquid sample, extracting with 2 ml of ethyl acetate, then carrying out nitrogen blow concentration, and then dissolving with 1 ml of acetonitrile. Detecting the enzymolysis product by High Performance Liquid Chromatography (HPLC), wherein the chromatographic conditions are as follows: the flow rate is 0.8ml/min, the detection wavelength is 245nm, and the column temperature is 30 ℃. The sulforaphane standard is used as a reference. As shown in FIG. 1, it was found that the enzymatic hydrolysate contained sulforaphane.
And (3) detecting and analyzing the enzymolysis product by using a mass spectrum, wherein the result is shown in figure 2, comparing the molecular formula and the relative molecular mass of the sulforaphane, determining that M/z 178.0 is a sulforaphane molecular ion peak which is expressed as [ M + H ] +, and further confirming that the product contains the sulforaphane.
Detecting the concentration of the sulforaphane in the acetonitrile solution by using a High Performance Liquid Chromatography (HPLC), wherein the chromatographic conditions are as follows: the flow rate is 0.8ml/min, the detection wavelength is 245nm, and the column temperature is 30 ℃. As a result, as shown in FIG. 3, the optimum pH for sulforaphane preparation was 5.
Example 4 determination of optimum enzyme addition amount and reaction time for the preparation of sulforaphane
Since sulforaphane is sensitive to temperature, the product is not easily obtained at higher temperature and lower temperature, and 25 ℃ is selected as the reaction temperature by referring to data of Kim et al. The enzyme addition and the reaction time were optimized at 25 ℃ and pH 5:
taking 3 parts of substrate solution (10 ml each, pH = 5), respectively adding 20U, 30U and 40U of enzyme powder preparation, carrying out enzymolysis reaction for 20 min at 25 ℃ and 150 rpm, sampling at different time intervals, sampling 1 ml each time, extracting with 2 ml of ethyl acetate, then blowing nitrogen for concentration, finally dissolving with 1 ml of acetonitrile, detecting the concentration of the sulforaphane in the acetonitrile solution by using High Performance Liquid Chromatography (HPLC), converting into the content of the sulforaphane after enzymolysis of each 1 g of broccoli seeds, and obtaining the result shown in figure 4, wherein when the enzyme adding amount is 40U and the reaction time is 15 minutes, the content of the sulforaphane is the highest and is 10 mg/g.
Example 5 extraction and quantification of sulforaphane from broccoli seeds
The broccoli seeds used in example 3 were pulverized and dissolved in 80% (v/v) methanol solution pre-cooled at-20 ℃, left to stand for 30 minutes and extracted with shaking for 30 minutes, and the sulforaphane content was measured by HPLC under the following chromatographic conditions: the flow rate was 0.8ml/min, the detection wavelength was 229nm, and the column oven was 30 ℃. The amount of sulforaphane in the substrate is 58.30 mu mol/g broccoli seeds by calculation according to a liquid phase detection diagram, the yield of sulforaphane in example 4 is 10 mg/g, the yield is 58.22 mu mol/g broccoli seeds after conversion into sulforaphane, the substrate conversion rate is the ratio of the amount of sulforaphane participating in conversion to the amount of sulforaphane in the seeds, and the substrate conversion rate can reach 99.86 percent by calculation.
Taking substrate solution (10 ml, pH = 5), adding 40U enzyme powder preparation, performing enzymolysis reaction at 20 deg.C and 150 rpm for 15 min, and detecting sulforaphane concentration by High Performance Liquid Chromatography (HPLC) to 9.83 mg/g, with substrate conversion rate of 98.16%.
EXAMPLE 6 Multi-batch, high efficiency preparation of sulforaphane
Taking a substrate solution (10 ml, pH = 5), adding 40U of enzyme powder preparation, carrying out enzymolysis reaction for 15 minutes at 25 ℃ and 150 rpm, centrifuging for 5 minutes at 6000rpm, collecting precipitates (namely the recovered enzyme powder preparation), adding the precipitates into the substrate solution for enzymolysis, recycling for 10 times, detecting the concentration of the sulforaphane in an acetonitrile solution by using High Performance Liquid Chromatography (HPLC), and researching the recycling effect of the myrosinase by taking the concentration of the sulforaphane at the first enzymolysis as 100%, wherein the result is shown in figure 5. The result shows that the relative yield of the sulforaphane can still reach more than 87 percent after the sulforaphane is repeatedly used for 10 times, which indicates that the myrosinase AtTGG4 is stable and can be recycled.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Sequence listing
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<120> a method for preparing sulforaphane
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Arg Ile Val Glu Asp Tyr Thr Asn Tyr Ala Glu Leu Leu Phe Gln Arg
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Lys Arg Tyr Gln Lys Phe Gln Gly Gly Lys Ile Gly Thr Thr Leu Ile
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Gly Arg Trp Phe Ala Pro Leu Asn Glu Phe Ser Glu Leu Asp Lys Ala
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Ala Ala Lys Arg Ala Phe Asp Phe Phe Val Gly Trp Phe Leu Asp Pro
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Leu Val Tyr Gly Lys Tyr Pro Thr Ile Met Arg Glu Met Val Gly Asp
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Arg Leu Pro Glu Phe Thr Pro Glu Gln Ser Ala Leu Val Lys Gly Ser
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Claims (9)

1. A method for preparing sulforaphane is characterized by comprising the following steps: carrying out enzymolysis on glucoraphanin or a raw material containing glucoraphanin by using myrosinase AtTGG4 or an enzyme preparation containing the myrosinase AtTGG4 to prepare the glucoraphanin, wherein the enzymolysis conditions are as follows: performing enzymolysis for 10-20 minutes at 20-30 ℃; the amino acid sequence of the myrosinase AtTGG4 is shown in SEQ ID NO. 1.
2. The method for preparing sulforaphane according to claim 1, wherein: the enzyme preparation containing myrosinase AtTGG4 is prepared by the following method: the gene recombination technology is utilized to construct a recombinant expression vector containing the coding gene of the myrosinase AtTGG4, yarrowia lipolytica is transfected to obtain recombinant engineering bacteria, fermentation culture is carried out, fermentation liquor is centrifuged, thallus is collected and freeze-dried, and enzyme powder is obtained, namely the enzyme preparation containing the myrosinase AtTGG 4.
3. The method for preparing sulforaphane according to claim 2, wherein: the specific mode of the fermentation culture is as follows: the recombinant engineered strain was inoculated into 10ml YPD liquid medium, cultured at 30 ℃ and 200rpm for 16 hours, and then inoculated into 250 ml PPB medium at an inoculum size of 1%, and fermented at 30 ℃ and 200rpm for 5 days.
4. The method for preparing sulforaphane according to claim 1, wherein: the sulforaphane-containing raw material is selected from broccoli seeds.
5. The method for preparing sulforaphane according to claim 1, wherein: the enzymolysis conditions are as follows: enzymatic hydrolysis was carried out at 25 ℃ for 15 minutes.
6. The method for preparing sulforaphane according to any one of claims 1 to 5, wherein: the specific mode of enzymolysis is as follows: adding myrosinase AtTGG4 or an enzyme preparation containing myrosinase AtTGG4 into a substrate solution with the pH value of 4.0-6.0, and carrying out enzymolysis for 10-20 minutes at the temperature of 20-30 ℃; the substrate in the substrate solution is glucoraphanin or crushed broccoli seeds.
7. The method of preparing sulforaphane according to claim 6, wherein: the substrate solution is prepared by the following method: heating broccoli seeds for 1 h at 110 ℃ to inactivate endogenous enzymes, crushing to obtain broccoli seed powder, and adding water to prepare a substrate solution with the concentration of 0.1 g/ml.
8. The method of preparing sulforaphane according to claim 6, wherein: after enzymolysis, the enzyme preparation containing myrosinase AtTGG4 is recovered, and is continuously added into the substrate solution, and the enzymolysis is repeated for more than 10 times.
9. The method for preparing sulforaphane according to claim 7 or 8, wherein: the specific mode of enzymolysis is as follows: 40U of the enzyme preparation containing myrosinase AtTGG4 was added to 10ml of a substrate solution with pH 5.0 and subjected to enzymatic hydrolysis at 25 ℃ for 15 minutes; recovering enzyme preparation containing myrosinase AtTGG4 after enzymolysis, adding into substrate solution, and repeating enzymolysis for more than 10 times
The application of myrosinase AtTGG4 in degrading glucoraphanin or preparing glucoraphanin is disclosed, wherein the amino acid sequence of the myrosinase AtTGG4 is shown in SEQ ID NO. 1.
CN202210490467.3A 2022-05-07 2022-05-07 Method for preparing sulforaphane Pending CN114657220A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120213890A1 (en) * 2011-02-22 2012-08-23 Caudill Seed Company, Inc. Spray dried myrosinase and use to produce isothiocynates
CN112899177A (en) * 2021-02-02 2021-06-04 中国海洋大学 Recombinant yarrowia lipolytica expressing myrosinase TGG4 and application thereof
CN113736763A (en) * 2021-10-13 2021-12-03 中国海洋大学 Myrosinase Rmryr and application thereof in preparation of sulforaphane and sulforaphane
CN114164129A (en) * 2021-11-18 2022-03-11 江南大学 Recombinant pichia pastoris for heterologous expression of myrosinase and application of recombinant pichia pastoris in preparation of sulforaphane

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120213890A1 (en) * 2011-02-22 2012-08-23 Caudill Seed Company, Inc. Spray dried myrosinase and use to produce isothiocynates
CN112899177A (en) * 2021-02-02 2021-06-04 中国海洋大学 Recombinant yarrowia lipolytica expressing myrosinase TGG4 and application thereof
CN113736763A (en) * 2021-10-13 2021-12-03 中国海洋大学 Myrosinase Rmryr and application thereof in preparation of sulforaphane and sulforaphane
CN114164129A (en) * 2021-11-18 2022-03-11 江南大学 Recombinant pichia pastoris for heterologous expression of myrosinase and application of recombinant pichia pastoris in preparation of sulforaphane

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