CN110195084B - Production method of p-hydroxyphenylethanol and application of p-hydroxyphenylethanol to producing penicillium chrysogenum - Google Patents

Production method of p-hydroxyphenylethanol and application of p-hydroxyphenylethanol to producing penicillium chrysogenum Download PDF

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CN110195084B
CN110195084B CN201910398145.4A CN201910398145A CN110195084B CN 110195084 B CN110195084 B CN 110195084B CN 201910398145 A CN201910398145 A CN 201910398145A CN 110195084 B CN110195084 B CN 110195084B
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朱虎
常爱平
王颖璐
陈雯丹
何侨妹
李丽
鱼晓丹
戴晓芸
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Abstract

The invention discloses application of Penicillium chrysogenum DXY-1 (Penicillium chrysogenum DXY-1) in production of p-hydroxyphenylethanol. The invention also discloses a production method of the p-hydroxyphenylethanol, which comprises the step of using Penicillium chrysogenum DXY-1 (Penicillium chrysogenum DXY-1). The invention adopts penicillium chrysogenum to produce the p-hydroxyphenylethanol for the first time, has simple process and has important significance for expanding the industrial production of the p-hydroxyphenylethanol.

Description

Production method of p-hydroxyphenylethanol and application of p-hydroxyphenylethanol to producing penicillium chrysogenum
Technical Field
The invention relates to application of penicillium chrysogenum DXY-1 and a production method of p-hydroxyphenylethanol.
Background
P-hydroxyphenylethanol is also known as 4-hydroxyphenylethanol and is commonly known as Tyrosol (Tyrosol), and is mainly used for synthesizing cardiovascular drugs metoprolol. The p-hydroxyphenylethanol can be synthesized by a chemical method. Firstly, hydroxyacetophenone is used as a raw material to synthesize p-hydroxyphenyl glyoxal, and p-hydroxyphenyl ethanol is obtained through reduction. The p-hydroxyphenylethanol is obtained by oxidizing beta-aminophenylethanol serving as a raw material.
In addition, the p-hydroxyphenylethanol can also be obtained by adopting a microbial fermentation method. For example, CN109370967a discloses the use of an engineered bacterium in tyrosol production, wherein an amino acid transaminase, a glutamate dehydrogenase, a keto acid decarboxylase, and an alcohol dehydrogenase are introduced into the engineered bacterium for the synthesis of tyrosol. For another example, CN108753636a discloses a method for constructing yeast for producing tyrosol and hydroxytyrosol, wherein a biosynthetic pathway of tyrosol or hydroxytyrosol is constructed in yeast BY4741 to improve the yield of tyrosol or hydroxytyrosol. Introducing PcAAS and ADH sequences into a yeast BY4741 to obtain a PcAAS-ADH recombinant yeast for producing tyrosol; introducing a pdc1 gene knockout box and a tyrA expression box into the PcAAS-ADH recombinant yeast to obtain the PcAAS-ADH-delta pdc1-tyrA recombinant yeast for producing the tyrosol; the DNA sequence of HpaBC was introduced into PcAAS-ADH-. DELTA.pdc 1-tyrA recombinant yeast to obtain a hydroxytyrosol-producing PcAAS-ADH-HpaBC-. DELTA.pdc 1-tyrA recombinant yeast. For another example, CN106754607a discloses a method for constructing a recombinant strain for producing tyrosol, which comprises constructing a recombinant vector pRSFDuet-ARO10-ARO8 by using a genetic engineering technology, transforming the recombinant vector pRSFDuet-ARO10-ARO8 into genetically engineered escherichia coli BL21 (DE 3) with a gene pheA and a gene feaB knocked out, obtaining a strain BE0, performing a whole-cell catalytic reaction, using 10mM tyrosine as a substrate, fermenting for 20h to produce tyrosol with a yield of 6.73mM, wherein the tyrosine conversion rate can reach 67.3%, performing chemical mutagenesis on the basis of the recombinant strain to obtain a recombinant strain BE2 with optimized performance and high tyrosol yield, performing a whole-cell catalytic reaction, using 10mM tyrosine as a substrate, fermenting for 20h to produce tyrosol with a yield of 8.71mM, and the tyrosine conversion rate can reach 87.1%. The method obtains the p-hydroxyphenylethanol through a gene process. Therefore, there is still a need to expand the production process of p-hydroxyphenylethanol.
Penicillium chrysogenum is a fungus belonging to the order of the Filosporium (from the order of the Cordyporales) of the subdivision Deuteromycotina, belonging to the genus Penicillium of the family of the Cordysoraceae, distributed in the soil, air and in septic organic materials. At present, no report is available on the production of p-hydroxyphenylethanol by penicillium chrysogenum.
Disclosure of Invention
An object of the present invention is to provide a use of penicillium chrysogenum for the production of p-hydroxyphenylethanol. The invention also aims to provide a production method of the p-hydroxyphenylethanol, which has a simple process.
In one aspect, the invention provides a use of Penicillium chrysogenum DXY-1 (Penicillium chrysogenum DXY-1) in the production of p-hydroxyphenylethanol, wherein the preservation number of the Penicillium chrysogenum DXY-1 is CCTCC NO: M2018731. The classification name of the biological material is Penicillium chrysogenum DXY-1, the Latin literature name is Penicillium chrysogenum DXY-1, the full name of the collection unit is China center for type culture collection for short, the address of the collection unit is Wuhan university in Wuhan city, hubei province, china, the collection date is 11 months and 15 days in 2018, and the collection number is CCTCC NO: M2018731.
In another aspect, the invention provides a method for producing p-hydroxyphenylethanol, which comprises the step of using Penicillium chrysogenum DXY-1 (Penicillium chrysogenum DXY-1), wherein the preservation number of the Penicillium chrysogenum DXY-1 is CCTCC NO: M2018731.
The production method according to the present invention preferably comprises the following specific steps:
(1) Fermenting and culturing the penicillium chrysogenum DXY-1 to obtain fermentation liquor;
(2) Extracting the fermentation liquor to obtain an extract; and
(3) Separating and purifying the extract to obtain the p-hydroxyphenylethanol.
According to the production method of the present invention, preferably, in the step (1), the activated penicillium chrysogenum DXY-1 is inoculated into a culture solution, and shake culture is carried out for 50-100 h at 25-30 ℃ and 100-300 rmp to obtain a seed culture solution; then transferring the seed culture solution to a culture medium, and performing shake culture at 25-30 ℃ and 100-300 rmp for 5-9 days to obtain a fermentation solution.
According to the production method of the present invention, preferably, in the step (1), the inoculum amount of the seed culture solution is 2 to 9wt% based on the weight of the medium.
According to the production method of the present invention, preferably, in the step (1), the culture solution comprises the following components based on 100ml of seawater: 3 to 7 weight percent of glucose, 0.5 to 3.5 weight percent of peptone, 0.3 to 0.6 weight percent of NaCl and 100ml of seawater; based on 100ml of seawater, the culture medium comprises the following components: 3 to 7 weight percent of glucose, 0.5 to 3.5 weight percent of peptone, 0.3 to 0.6 weight percent of NaCl and 100ml of seawater.
According to the production method of the present invention, preferably, in the step (2), the mycelium pellet in the fermentation liquid is crushed, then ethyl acetate is added for extraction for 1 to 5 times, and the supernatant is concentrated to obtain the extract; wherein the amount of ethyl acetate used in each extraction is 0.8-1.5 times of the volume of the fermentation liquor.
According to the production method of the present invention, preferably, in the step (3), the extract is dissolved in methanol to form a methanol solution; an ultraviolet detector is connected with a gel column chromatography in series, and methanol is used as an eluent for separation to obtain an eluent; separating the eluate into multiple eluate components, performing activity detection with purple bacillus CV026 screening model, and separating the active eluate components by liquid chromatography to obtain p-hydroxy phenyl ethanol.
According to the production method of the present invention, preferably, in the step (3), the eluate is developed with a thin layer chromatography plate, and similar components are combined and concentrated according to a vanillin coloration result of the thin layer scanning method to obtain a plurality of eluted components.
According to the production method of the present invention, preferably, the gel column chromatography employs a sephadex column; the conditions of the liquid chromatography were as follows: 20-35 vol% methanol water solution is used as a mobile phase, and the flow rate is 0.5-1.0 mL/min.
The invention adopts penicillium chrysogenum to produce the p-hydroxyphenylethanol for the first time, has simple process and has important significance for expanding the industrial production of the p-hydroxyphenylethanol.
Deposit description
The classification name of the biological material is Penicillium chrysogenum DXY-1, the Latin Wen Xueming is Penicillium chrysogenum DXY-1, the full name of the preservation unit and the preservation center are abbreviated as China typical culture preservation center, the address of the preservation unit is Wuhan university in Wuhan city, hubei China, the preservation date is 10 months 31 days in 2018, and the preservation number is CCTCC NO: M2018731.
Detailed Description
The invention provides application of Penicillium chrysogenum DXY-1 (Penicillium chrysogenum DXY-1) in production of p-hydroxyphenylethanol. The preservation number of the penicillium chrysogenum DXY-1 is CCTCC NO: M2018731. The invention adopts penicillium chrysogenum DXY-1 to obtain the p-hydroxyphenylethanol for the first time.
The ocean contains abundant microbial resources, and the relatively unique ecological environment of the ocean, such as high pressure, high salt, low temperature, oxygen deficiency, oligotrophism and the like, endows the ocean with a unique metabolic mechanism of microbes, so that the probability of finding novel lead compounds is greatly increased. From secondary metabolites of marine microorganisms, many chemical drugs having excellent therapeutic effects have been developed, which indicates that marine microorganisms are an important repository of natural drugs. The Penicillium chrysogenum DXY-1 of the invention is preferably Penicillium chrysogenum of marine origin, more preferably Penicillium chrysogenum of Taiwan strait silt origin. The invention adopts marine penicillium chrysogenum DXY-1 to obtain the p-hydroxyphenylethanol for the first time.
The invention also provides a production method of the p-hydroxyphenylethanol, which comprises the step of using the Penicillium chrysogenum DXY-1. The collection number of the penicillium chrysogenum DXY-1 is CCTCC NO: M2018731.
The use and production method of the present invention may have the same steps, which are described in the following in a unified manner. The invention comprises the following specific steps: (1) Fermenting and culturing the penicillium chrysogenum DXY-1 to obtain fermentation liquor; (2) extracting the fermentation broth to obtain an extract; and (3) separating and purifying the extract to obtain the p-hydroxyphenylethanol.
In the step (1), the activated penicillium chrysogenum DXY-1 is cultured in a culture solution to obtain a seed culture solution; then culturing the seed culture solution on a culture medium to obtain a fermentation solution. The activation method of the present invention is not particularly limited, and for example, plate activation is employed. After the plate activation, a proper amount of hyphae is scraped and inoculated in the culture solution. The ratio of the two is not particularly limited.
The culture solution of the invention can be prepared by the following formula: based on 100ml of seawater, the culture solution comprises the following components: 3 to 7 weight percent of glucose, 0.5 to 3.5 weight percent of peptone, 0.3 to 0.6 weight percent of NaCl and 100ml of seawater. Preferably, the culture solution of the present invention may be formulated as follows: 3 to 6 weight percent of glucose, 1 to 3 weight percent of peptone, 0.3 to 0.6 weight percent of NaCl, and 100ml of seawater. According to one embodiment of the invention, the formula of the culture solution is as follows: 3wt% glucose, 1wt% peptone, 0.3wt% NaCl, and 100ml seawater.
The culture medium comprises the following components: based on 100ml of seawater, the culture medium comprises the following components: 3 to 7 weight percent of glucose, 0.5 to 3.5 weight percent of peptone, 0.3 to 0.6 weight percent of NaCl and 100ml of seawater. (ii) a Preferably, the culture medium of the present invention may be formulated as follows: 4 to 6 weight percent of glucose, 1 to 3 weight percent of peptone, 0.4 to 0.6 weight percent of NaCl, and 100ml of seawater. According to one embodiment of the invention, the formulation of the culture medium of the invention is: 5wt% glucose, 2wt% peptone, 0.5wt% NaCl, and 100ml seawater.
In some embodiments, activated Penicillium chrysogenum DXY-1 is inoculated into a culture medium and shake-cultured at 25-30 ℃ and 100-300 rmp for 50-100 h to obtain a seed culture medium. The culture temperature may be 25 to 30 ℃, preferably 26 to 29 ℃, and more preferably 27 to 28 ℃. The oscillation rate may be 100 to 300rmp, preferably 120 to 200rmp, more preferably 150 to 180rmp. The culture time may be 50 to 100 hours, preferably 60 to 90 hours, and more preferably 72 to 85 hours.
In some embodiments, the seed culture broth is transferred to a culture medium and shake-cultured at 25-30 ℃ and 100-300 rmp for 5-9 days to obtain a fermentation broth. The culture temperature may be 25 to 30 ℃, preferably 26 to 29 ℃, and more preferably 27 to 28 ℃. The oscillation rate may be from 100 to 300rmp, preferably from 120 to 200rmp, more preferably from 150 to 180rmp. The culture time may be 5 to 9 days, preferably 6 to 8 days, and more preferably 6 to 7 days. Thus, the full fermentation can be ensured, and the fermentation efficiency is improved. The amount of the seed culture solution to be inoculated may be2 to 9wt%, preferably 3 to 8wt%, more preferably 5 to 6wt% based on the weight of the culture medium.
In the step (2), the mycelium pellets in the fermentation liquid are crushed to obtain a treatment liquid. For example, a homogenizer is used to break the mycelium pellets in the fermentation broth. Extracting with ethyl acetate, mixing the supernatants, and concentrating to obtain extract. The number of extractions may be 1 to 5, preferably 2 to 3. Concentration can be carried out using a rotary evaporator. The amount of ethyl acetate used per extraction may be 0.8 to 1.5 times, preferably 0.9 to 1.3 times, more preferably 1 to 1.2 times the volume of the fermentation broth. This is advantageous for the sufficient recovery of p-hydroxyphenylethanol.
In step (3), the extract is dissolved in methanol to form a methanol solution. The amount and ratio of the two is not particularly limited. Optionally, the methanol solution is filtered through a polymeric membrane filter to remove impurities. The pore size of the filter membrane may be in the range of 0.1 to 1 μm, preferably 0.1 to 0.5 μm, more preferably 0.1 to 0.22. Mu.m. An ultraviolet detector is connected with a gel column chromatography in series, and methanol is used as an eluent for separation to obtain an eluent. For example, a methanol solution is uniformly added to the gel column drop by drop along the inner wall of the gel column by a dropper, a washing solution formed by washing a sample bottle with a small amount of methanol is also added to the gel column, and then a valve below the gel column is opened. And when the liquid level of the sample is lowered to be close to the surface layer of the gel column, adding methanol along the inner wall of the gel column, repeatedly adding methanol for several times to ensure that the methanol solution containing the extract completely enters the gel column, and then adding a large amount of methanol for elution. The gel column of the present invention may employ a Sephadex column, such as Sephadex LH-20.
In step (3), the eluate is separated into a plurality of eluted components. According to one embodiment of the present invention, the eluate is developed using a thin layer chromatography plate, and similar components are combined and concentrated according to the vanillin coloration result of thin layer scanning (TLC) to obtain a plurality of eluted components.
In the step (3), the eluted components are subjected to activity detection by using a screening model of purple bacillus CV026, and the active eluted components are separated by liquid chromatography (such as HPLC) to obtain the p-hydroxyphenylethanol. The conditions of the liquid chromatography were as follows: 20-35 vol% methanol water solution is used as a mobile phase, and the flow rate is 0.5-1.0 mL/min. Preferred conditions for liquid chromatography are as follows: 22-30 vol% methanol water solution is used as a mobile phase, and the flow rate is 0.7-0.9 mL/min. More preferred conditions for liquid chromatography are as follows: 25-28 vol% methanol water solution is used as a mobile phase, and the flow rate is 0.8-0.85 mL/min. The adoption of such conditions is favorable for obtaining high-purity p-hydroxyphenylethanol. In liquid chromatography, detection can be performed using ultraviolet light having a wavelength of 210 nm. The liquid chromatography may employ a C18 column, such as ZORBAX SB-Aq.
According to one embodiment of the present invention, the extract is dissolved in methanol to form a methanol solution, and the methanol solution is filtered through a polymeric membrane having a pore size of 0.1 to 0.22 μm to remove impurities; adopting a UV-900 ultraviolet detector and a Sephadex LH20 gel column for chromatographic series connection, and separating by using methanol as an eluent to obtain eluent; separating the eluent into a plurality of elution components, carrying out activity detection by using a screening model of the violobacter violaceus CV026, carrying out liquid chromatography separation on the active elution components, taking 25vol% methanol water solution as a mobile phase, detecting by adopting ultraviolet light with the wavelength of 210nm at the flow rate of 0.8mL/min, and carrying out separation and purification to obtain the p-hydroxyphenylethanol.
< test method >
Ultraviolet spectrum: the product was dissolved in methanol and the sample was scanned with an ultraviolet spectrophotometer (Shimadzu, japan, UV 2450/2550) at a wavelength of 200-800 nm.
Nuclear magnetic resonance: dissolving the product in deuterated dimethyl sulfoxide, and performing nuclear magnetic analysis, wherein the analysis type is hydrogen spectrum (A) 1 H-NMR), carbon spectrum ( 13 C-NMR). The NMR spectrometer was Bruker MSL 500 Brooks.
The following illustrates the starting materials used in the examples:
culture solution: 3wt% of glucose, 1wt% of peptone, 0.3wt% of NaCl and 100ml of seawater.
Culture medium: 5wt% of glucose, 2wt% of peptone, 0.5wt% of NaCl and 100ml of seawater.
Example 1
The production method of the p-hydroxyphenylethanol comprises the following steps:
(1) Fermenting and culturing Penicillium chrysogenum DXY-1 to obtain fermentation liquor
Inoculating activated penicillium chrysogenum DXY-1 (from Taiwan strait silt) in a culture solution, and shake-culturing at 28 deg.C and 150rmp for 72h to obtain a seed culture solution; then, the seed culture solution was transferred to a culture medium and shake-cultured at 28 ℃ and 150rmp for 7 days to obtain a fermentation broth. The inoculum size of the seed culture solution was 5wt% of the medium weight.
(2) Extracting the fermentation broth to obtain an extract
Crushing mycelium pellets in the fermentation liquor, adding ethyl acetate for extraction for 3 times, combining supernatant, and concentrating by adopting a rotary evaporator to obtain an extract; the amount of ethyl acetate used per extraction was 1 time the volume of the fermentation broth.
(3) Separating and purifying the extract to obtain p-hydroxyphenylethanol
Dissolving the extract in methanol to form methanol solution, and filtering with filter membrane with pore diameter of 0.22 μm. An ultraviolet detector (UV-900) is connected with a gel column chromatography (Sephadex LH 20) in series, and methanol is used as an eluent to separate the filtered methanol solution to obtain eluent. Spreading the eluate with thin layer chromatography plate, and combining and concentrating similar components according to vanillin color development result of thin layer scanning method to obtain 13 eluate components. And (3) carrying out activity detection on the eluted components by using a screening model of bacillus violaceus CV026, and separating the active eluted components by liquid chromatography (25 vol% methanol water solution is used as a mobile phase, the flow rate is 0.8mL/min, ultraviolet light with the wavelength of 210nm is adopted, and a C18 chromatographic column ZORBAX SB-Aq is adopted) to obtain a p-hydroxyphenylethanol product.
Examples of the experiments
The structure of the p-hydroxyphenylethanol product obtained in example 1 was identified.
The ultraviolet spectrum shows that the characteristic absorption peak of benzene appears at the wavelength of 275nm, and the compound is supposed to contain a benzene ring. The K absorption band formed by a conjugated double bond system appears at the wavelength of 220nm, which indicates that the compound contains more double bonds.
Hydrogen spectrum of nuclear magnetic resonance ( 1 H NMR) gave 10 hydrogen signals. An active hydrogen signal (delta) appears in a low field region H 9.11,s, 4-OH), 4 benzenyl hydrogen signals (delta) appear in the aromatic region H 6.99，d，H-4；δ H 6.99，d，H-8；δ H 6.63，d，H-5；δ H 6.63,d,H-7; ) Two by two overlap and splitThe d peak indicates that the compound possibly contains a 1,4-disubstituted benzene ring structure, and a methylene signal (delta) appears in a high field region H 2.60,t, H-2), a methylene signal (. Delta.) to oxygen H 3.50, q, H-1) and an active hydrogen signal (. Delta.) attached to the fatty chain H 4.55, t, 1-OH). This suggests that the compound is a benzene derivative.
Carbon spectrum of nuclear magnetic resonance ( 13 C NMR) gave 8 carbon signals, respectively the carbon signals (. Delta.) on the 6 benzene rings C 129.8 of the total weight of the mixture; 115.3;130.0 of the total weight of the alloy; 155.8 of the total weight of the mixture; 130.0 of the total weight of the alloy; 115.3 And 2 high field regions SP 3 Hybridized carbon Signal (. Delta.) C 62.9 38.6) with a carbon signal to oxygen at 62.9 and a carbon signal to the benzene ring at 38.6. This suggests that the compound is p-hydroxyphenylethanol. The compound is determined to be the p-hydroxy phenethyl alcohol by comparing the nuclear magnetic data of the p-hydroxy phenethyl alcohol in the literature. The chemical structural formula of the compound is as follows:
Figure BDA0002058847070000091

Claims (10)

1. the application of Penicillium chrysogenum DXY-1 (Penicillium chrysogenum DXY-1) in the production of p-hydroxyphenylethanol is disclosed, wherein the preservation number of the Penicillium chrysogenum DXY-1 is CCTCC NO: M2018731.
2. A production method of p-hydroxyphenylethanol is characterized by comprising the step of using Penicillium chrysogenum DXY-1 (Penicillium chrysogenum DXY-1), wherein the preservation number of the Penicillium chrysogenum DXY-1 is M2018731.
3. The production method according to claim 2, characterized by comprising the following specific steps:
(1) Fermenting and culturing the penicillium chrysogenum DXY-1 to obtain fermentation liquor;
(2) Extracting the fermentation liquor to obtain an extract; and
(3) Separating and purifying the extract to obtain the p-hydroxyphenylethanol.
4. The production method according to claim 3, wherein in the step (1), the activated penicillium chrysogenum DXY-1 is inoculated into a culture solution and shake-cultured at 25-30 ℃ and 100-300 rmp for 50-100 h to obtain a seed culture solution; then transferring the seed culture solution to a culture medium, and performing shake culture at 25-30 ℃ and 100-300 rmp for 5-9 days to obtain a fermentation solution.
5. The production method according to claim 4, wherein in the step (1), the amount of the inoculated seed culture solution is 2 to 9wt% based on the weight of the medium.
6. The production method according to claim 5, wherein, in the step (1), the culture solution comprises the following components based on 100ml of seawater: 3 to 7 weight percent of glucose, 0.5 to 3.5 weight percent of peptone, 0.3 to 0.6 weight percent of NaCl and 100ml of seawater; based on 100ml of seawater, the culture medium comprises the following components: 3 to 7 weight percent of glucose, 0.5 to 3.5 weight percent of peptone, 0.3 to 0.6 weight percent of NaCl and 100ml of seawater.
7. The production method according to any one of claims 3 to 6, wherein in the step (2), the mycelium pellets in the fermentation liquid are crushed, then ethyl acetate is added for extraction for 1 to 5 times, and the supernatant is concentrated to obtain an extract; wherein, the amount of ethyl acetate used in each extraction is 0.8 to 1.5 times of the volume of the fermentation liquor.
8. The production method according to any one of claims 3 to 6, wherein in the step (3), the extract is dissolved in methanol to form a methanol solution; an ultraviolet detector is connected with a gel column chromatography in series, and methanol is used as an eluent for separation to obtain an eluent; separating the eluate into multiple eluate components, performing activity detection with purple bacillus CV026 screening model, and separating the active eluate components by liquid chromatography to obtain p-hydroxy phenyl ethanol.
9. The production method according to claim 8, wherein in the step (3), the eluate is developed using a thin layer chromatography plate, and similar components are combined and concentrated according to a vanillin development result of the thin layer scanning method to obtain a plurality of eluted components.
10. The production method according to claim 9, wherein the gel column chromatography employs a sephadex column; the conditions of the liquid chromatography were as follows: 20-35 vol% methanol water solution is used as a mobile phase, and the flow rate is 0.5-1.0 mL/min.
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