CN111733196A - Method for catalytically synthesizing L-ascorbyl oleate in high-water-activity medium - Google Patents
Method for catalytically synthesizing L-ascorbyl oleate in high-water-activity medium Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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Abstract
The invention belongs to the technical field of bioengineering, and relates to a method for catalytically synthesizing L-ascorbyl oleate in a high-water-activity medium. The method for catalytically synthesizing the L-ascorbic acid oleate in the high-water-activity medium comprises the following steps of: taking ethyl oleate and L-ascorbic acid as substrates, mixing, adding lipase, placing in a water/lipid two-phase medium with high water activity, and catalyzing and synthesizing L-ascorbic acid oleate by using recombinant lipase; the recombinant lipase is a recombinant lipase based on a lipase with an amino acid sequence shown as SEQ ID No. 1. The invention discovers for the first time that CpLIP2 lipase can catalyze ethyl oleate and L-ascorbic acid to synthesize L-ascorbic acid oleate, and the catalytic reaction can be carried out at high water activity (a)w>0.9) in a water/lipid biphasic medium. The method of the inventionThe method can produce the L-ascorbyl oleate by lipase in an aqueous medium without organic solvent, has simple reaction conditions, and provides an effective method for producing the green grease by biotransformation.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a method for catalytically synthesizing L-ascorbyl oleate in a high-water-activity medium. More specifically, the invention provides a method for synthesizing L-ascorbic acid oleate by taking ethyl oleate and L-ascorbic acid as raw materials and catalyzing ethyl oleate in a high-water-activity medium through lipase.
Background
The oil is one of important nutrient substances required by human bodies, and the oil in the food is easily oxidized and rancid in the processing, storage and transportation processes, so that the nutritional value of the food is reduced, and even the health of the human body is damaged. The addition of antioxidants to foods is often an effective way to solve this problem. The L-ascorbic acid fatty acid ester is a fat-soluble derivative of the L-ascorbic acid, and compared with the L-ascorbic acid saturated fatty acid ester, the L-ascorbic acid oleate has higher solubility in oil substances and better antioxidant effect, and is more suitable for the antioxidation of the oil.
Lipases are water-soluble enzymes with various catalytic abilities and capable of playing an active role in an oil-water interface, and can catalyze hydrolysis of esters such as fats. Under certain conditions, lipases are also capable of catalyzing the production of esters, the types of reactions being esterification, alcoholysis, acidolysis and transesterification. Since water is a product of the ester synthesis reaction, organic solvents with very low water activity are typically used and water formed during the reaction must be extracted, transesterification reactions (such as alcoholysis) do not produce water, but they should also typically be carried out at low water activity to prevent hydrolysis of the ester. Therefore, the synthesis of L-ascorbyl oleate catalyzed by the enzyme method is basically carried out in a non-aqueous phase system, such as an organic solvent. For example, an invention patent (publication No. CN 101892275B) entitled "method for synthesizing ascorbic acid fatty acid ester by catalyzing lard with lipase" discloses that isooctane: t-amyl alcohol with a volume ratio of 7: 3 is used as a reaction medium, alcoholysis reaction is carried out on lard and methanol with lipase to prepare composite fatty acid methyl ester, and then transesterification reaction is carried out on the composite fatty acid methyl ester and L-ascorbic acid to prepare ascorbic acid fatty acid ester; an invention patent (publication No. CN 102212572B) named as a method for synthesizing L-ascorbyl oleate by catalysis of yeast display lipase discloses that L-ascorbyl oleate is synthesized by catalysis of L-ascorbic acid and oleic acid by taking tetrahydrofuran as a reaction medium. There is a document (https:// doi.org/10.5650/jos.62.591) that L-ascorbic acid oleate is synthesized under catalysis of lipase using acetone as a reaction medium and L-ascorbic acid and oleic acid as substrates.
At present, L-ascorbyl oleate is not commercially produced, and the research on the synthesis of L-ascorbyl oleate is mainly carried out by using lipase in an organic solvent, but the use of the organic solvent not only causes many safety problems, but also increases the downstream processing cost of L-ascorbyl fatty acid ester serving as a green food additive. The synthesis of the organic phase causes environmental pollution, and the residual organic solvent in the sample may be harmful to human bodies. For example, the published patents: CN 102212572B (method for catalyzing and synthesizing L-ascorbyl oleate by using yeast display lipase) and CN 102212574B (method for catalyzing and synthesizing L-ascorbyl linoleate by using yeast display lipase) both use tetrahydrofuran organic solvent as a reaction medium to synthesize L-ascorbyl oleate/linoleate. Although more environmentally friendly, new reaction media such as ionic liquids, supercritical fluids, etc. have high cost and harsh operating conditions, which limit their large-scale application.
Studies have reported that high yields of fatty acid esters of glycerol or methanol are feasible in aqueous/lipid biphasic media. Candida deformans derived lipase (CdIP 1) (https:// doi.org/10.1002/yea.958) was able to esterify to methyl oleate in the presence of methanol and either oleic acid or triolein in aqueous solution. Candidapapsilosis-derived lipase (CpLIP2) catalyzes the alcoholysis of methanol and rapeseed oil in aqueous solution. Rodrigues (https:// doi.org/10.1016/j.biortech.2016.07.090) and the like immobilize the enzyme on two synthetic resins, Accurel MP 1000 and Lewatit VP OC 1600, and catalyze the transesterification of jatropha oil and methanol (2M) to synthesize biodiesel in a lipid/water system (aw-0.96). Although these studies suggest the possibility of direct bioconversion of certain specific lipases in aqueous media to synthesize methyl oleate and biodiesel, no synthesis of L-ascorbyl oleate has been reported with lipases catalyzed by ethyl oleate and L-ascorbic acid in aqueous media.
Therefore, the development of effective bioconversion in organic solvent free aqueous media is particularly challenging for green oleochemistry and also an important development direction for green oleochemistry in the future.
Disclosure of Invention
The invention aims to provide a method for catalytically synthesizing L-ascorbyl oleate in a high-water-activity medium, which can produce the L-ascorbyl oleate by lipase in an aqueous medium without an organic solvent, has simple reaction conditions and provides a new way for green and healthy oil production.
The method for catalytically synthesizing the L-ascorbic acid oleate in the high-water-activity medium comprises the following steps of: taking ethyl oleate and L-ascorbic acid as substrates, mixing, adding lipase, placing in a water/lipid two-phase medium with high water activity, and catalyzing and synthesizing L-ascorbic acid oleate by using recombinant lipase; the recombinant lipase is a recombinant lipase based on a lipase with an amino acid sequence shown in SEQ ID No.1 and derived from Candidapapesisis.
According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity, the waterWater Activity of the lipid biphasic Medium aw>0.9。
According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity according to the invention, the water/lipid biphasic medium is a 50mM phosphate buffer solution at pH6.5 and an ethyl oleate emulsion.
According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity of the present invention, the process for the catalytic synthesis of L-ascorbyl oleate by the recombinant lipase is: stirring the reaction system at 40 ℃ and 180rpm, reacting for 2.5 hours, and separating and purifying from the reaction system to obtain the L-ascorbyl oleate.
According to a further feature of the method for catalytically synthesizing L-ascorbyl oleate in a medium with high water activity of the present invention, the separation and purification method comprises: 2mL of ethyl acetate and 0.792g of sodium chloride are added into each 2.2mL of reaction system, mixed uniformly for 3min by vortex, L-ascorbyl oleate is extracted, and then centrifuged for 5min at 5000 rpm. Taking the supernatant, heating the supernatant to 77 ℃, and volatilizing ethyl acetate to obtain the L-ascorbyl oleate.
According to a further feature of the method for catalytically synthesizing L-ascorbic acid oleate in a medium with high water activity of the present invention, the concentration of the added reaction substrate L-ascorbic acid in the reaction system is 0.2-2.2mol/L, and the final concentration of ethyl oleate is 15-250 mmol/L.
According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity according to the invention, the recombinant lipase is a recombinant lipase secreted and expressed by yeast.
According to a further feature of the method for catalytically synthesizing L-ascorbyl oleate in a high water activity medium, the final concentration of the recombinant lipase in the reaction system is 0.05-2 mg/mL.
According to a further feature of the method for the catalytic synthesis of L-ascorbyl oleate in a medium with high water activity according to the invention, the recombinant lipase is a recombinant lipase displayed by yeast.
According to a further feature of the method for catalytically synthesizing L-ascorbyl oleate in a high water activity medium, the final concentration of the recombinant lipase in the reaction system is 0.01-0.5 g/mL.
The invention introduces lipase gene and cell wall α -agglutinin gene into Pichia pastoris GS115, after the Pichia pastoris cell is cultured, the lipase is expressed and secreted to the outside of cell, or the cell wall α -agglutinin is utilized to fix the lipase on the cell surface, the reaction of synthesizing L-ascorbic acid oleate by utilizing the yeast display lipase to take L-ascorbic acid and ethyl oleate as substrates is catalyzedw>0.9), the lipase used in the invention can also catalyze the synthesis of L-ascorbic acid oleate from L-ascorbic acid and ethyl oleate in the form of immobilized enzyme in a reaction medium. Therefore, the invention provides an effective biotransformation method for producing green oil.
The invention discovers for the first time that CpLIP2 lipase can catalyze ethyl oleate and L-ascorbic acid to synthesize L-ascorbic acid oleate, and the catalytic reaction can be carried out at high water activity (a)w>0.9) in a water/lipid biphasic medium, the CpLIP2 lipase displayed by the cells has high operation stability, the conversion rate (calculated by L-ascorbic acid) is more than 70.3 percent, and the yield is more than 65.8 percent.
Drawings
FIG. 1 is a figure showing the mass spectrometric identification of L-ascorbyl oleate synthesized by the lipase catalysis of the invention.
FIG. 2 is a fluorescent microscopic examination result of pPCPA-GS 115.
Detailed Description
The present invention will be further described in detail by the following specific examples in conjunction with the attached drawings, wherein the examples are implemented on the premise of the technical scheme of the present invention, and detailed implementation manners and specific operation procedures are given, but the scope of the present invention is not limited to these examples.
The first embodiment is as follows: lipase secreted and expressed by pichia in water/lipid biphasic medium (a)w>0.9) catalyzed L-ascorbic acid oilSynthesis of acid esters
Pichia pastoris GS115 used in this experiment was a commercial product available from Invitrogen corporation. The cell wall alpha-agglutinin gene sequence is derived from saccharomyces cerevisiae, and the vector pPIC3.5K is a commercial product.
The lipase is lipase (GenBank: CAC86400.1) based on an amino acid sequence of SEQ ID NO.1, is lipase derived from Candida parapsilosis, is named as CpLIP2, and has a gene sequence named as Lip 2.
According to the used expression host bacterium Pichia pastoris GS115, the sequence of Lip2 is codon optimized, alpha-factor signal peptide is used for replacing the natural signal peptide of the Lip2 gene, and a glyceraldehyde triphosphate dehydrogenase promoter (pGAP) is added to realize the constitutive secretory expression of the Lip2 in Pichia pastoris GS 115. The recombinant plasmid consists of an original vector pPIC3.5K and a glyceraldehyde triphosphate dehydrogenase promoter (pGAP) and a lipase gene downstream of an alpha-factor signal peptide, which are sequentially inserted into the original vector pPIC3.5K. The new sequence named rLip2 was synthesized by Shanghai Czeri bioengineering, Inc.
The lipase secreted and expressed by the yeast described in the embodiment is prepared by the following method: transferring the linearized recombinant plasmid into pichia pastoris GS115, inoculating the obtained transformant into a YPD culture medium, performing fermentation culture for 72-120 hours, and centrifuging to collect protein supernatant to obtain lipase fermentation enzyme liquid.
The specific experimental operations were as follows: under the action of T4 ligase, the gene of rLip2 sequence is connected with pPIC3.5K plasmid overnight to form pPIC3.5K-rLip plasmid, and the plasmid is detected and recovered through electrophoresis. In order to realize His 4 single-site replacement recombination of the target gene and Pichia pastoris GS115, the pPIC3.5K-rLip2 plasmid is subjected to enzyme digestion linearization treatment by Sal I. The purpose of the linearization treatment of the recombinant plasmid is to perform homologous recombination with an intracellular genome and improve the expression stability. Adding about 15 mu L of the target gene subjected to enzyme digestion linearization treatment into a prepared pichia pastoris GS115 competent cell, transferring the cell into an electric transfer cup, carrying out ice bath for 30min, then carrying out electric shock for 5ms under the conditions of 1500V, 400 omega and 25uF, and adding about 800 mu L of precooled sorbitol. And (3) coating about 400 mu L of the uniformly mixed electrotransformation product on an MD (MD) plate, then dotting a transformant growing on the MD plate on a tributyrin plate, carrying out inverted culture at 28 ℃ for 3-5 days, and selecting a positive recombinant strain according to the appearance of a transparent ring around a positive transformant and the size of the transparent ring, namely the recombinant pichia pastoris producing active lipase.
The tributyrin flat plate method is characterized in that by utilizing the principle that lipase or esterase can decompose tributyrin to generate fatty acid, the tributyrin emulsion is added into a culture medium and can generate a transparent ring after being decomposed, and whether the strain produces the lipase or not and the activity of the produced lipase can be judged by observing whether the transparent ring is formed or not and the size of the transparent ring.
Inoculating the recombinant Pichia pastoris into a YPD culture medium, fermenting and culturing for 96h, centrifuging at 7000rpm and 4 ℃, and collecting the supernatant containing CpLIP2 lipase.
Catalytic synthesis of L-ascorbyl oleate with CpLIP2 lipase:
(1) CpLIP2 lipase catalyzes the reaction of ethyl oleate and L-ascorbic acid, and the reaction system is shown in Table 1 below:
table 1:
| components | Volume of |
| Ethyl oleate emulsion (50g/L) | 200 μ L (final concentration 15mM) |
| L-ascorbic acid | Proper amount of |
| Enzyme solution | 1mL (final concentration 0.039mg/mL) |
| 50mM, pH6.5 phosphate buffer | Make up to 2.2mL |
(2) The reaction was carried out in a 40 ℃ incubator at 180rpm for 2.5 hours.
(3) To the reaction solution was added 2mL of ethyl acetate, followed by addition of 0.792g of sodium chloride (to eliminate an emulsion layer), vortexing and mixing for 3min, extraction of methyl oleate, followed by centrifugation at 5000rpm for 5 min.
(4) The supernatant was taken and heated to 77 ℃ to evaporate ethyl acetate, and then 500. mu.L of methanol was added to redissolve the supernatant.
(5) 200 μ L of the reaction product was subjected to LC-MS:
(a) liquid chromatography conditions: a chromatographic column: thermo Hypersil GOLD C18 column (100mm x 2.1mm, Particle sz.1.9 μm), column temperature: 40 ℃, mobile phase: phase A: 0.1% aqueous formic acid; phase B: methanol; flow rate: 300 mu L/min; the mobile phase gradients are shown in table 2 below.
Table 2:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.00 | 95 | 5 |
| 0.50 | 95 | 5 |
| 6.00 | 0 | 100 |
| 10.00 | 0 | 100 |
| 10.10 | 95 | 5 |
| 12.00 | 95 | 5 |
(b) Mass spectrum detection conditions:
an ionization mode: ESI; spraying voltage: 3.0 kV; capillary temperature: 300 ℃; source heating temperature: 350 ℃; sheath gas pressure: 45 arb; auxiliary gas pressure: 8 arb; scanning mode: full sweeping + second level; collision energy: the L-ascorbyl oleate is obtained at 30V, and the substance with molecular weight of 439.1 in the mass spectrum of figure 1 is [ C ]24H39O7]-The mass spectrum peak of the L-ascorbyl oleate is obtained.
Determination of conversion yield and conversion:
1mL of the reaction product is accurately metered to 10mL by 100 percent methanol, and then the solution is filtered by a 0.22 mu m organic filter membrane and the concentration of each component of the solution is measured by High Performance Liquid Chromatography (HPLC). HPLC detection conditions: UltiMate 3000 high performance liquid chromatography (Thermo scientific), column: thermo Hypersil GOLD C18 column (100mm x 2.1mm, Particle sz.5 μm), loading 60 μ L, mobile phase methanol/water 90/10, flow rate 1mL/min, column temperature 30 ℃, detection wavelength 250 nm.
The yield calculation formula is as follows:
the yield was the concentration of L-ascorbic acid oleate/(concentration of L-ascorbic acid + concentration of L-ascorbic acid oleate),
efficiency of the esterification reaction: the conversion was defined as the concentration of L-ascorbic acid (after reaction)/the concentration of L-ascorbic acid (before reaction) × 100%.
Through the detection and calculation of the method, the yield and the reaction efficiency of the synthesized L-ascorbyl oleate are respectively over 65.8 percent and over 70.3 percent through preliminary estimation.
Example two: yeast displayed lipases in a Water/lipid biphasic Medium (a)w>0.9) Synthesis of L-ascorbyl oleate with catalysis
A gene sequence added with a FLAG label is designed between the 5' end of the rLip sequence and the alpha-factor signal peptide, and the new sequence is named fLip and is synthesized by Shanghai Czeri bioengineering GmbH. Meanwhile, the gene sequence (AG alpha 1) of 320 amino acids at the C end of the alpha-agglutinin (AG alpha 1) is subjected to codon optimization according to the used expression host bacterium Pichia pastoris GS115 and is artificially synthesized. The recombinant plasmid consists of an original vector pPIC3.5K, a glyceraldehyde triphosphate dehydrogenase promoter (pGAP) sequentially inserted into the original vector pPIC3.5K, an alpha-factor signal peptide, a lipase gene at the downstream of a gene sequence of a FLAG label and a cell wall alpha-agglutinin gene of saccharomyces cerevisiae.
The yeast display lipase described in this example was prepared by the following method: and transferring the linearized recombinant plasmid into pichia pastoris GS115, inoculating the obtained transformant into a YPD culture medium, culturing for 72-120 hours, and centrifuging to collect thalli to obtain the yeast display lipase.
The specific experimental procedures were as follows: AG alpha 1, fLip and pPIC3.5K plasmid are connected overnight under the action of T4 ligase to form pPIC3.5K-AG alpha 1-fLip plasmid (pPCPA), and the plasmid is checked and recovered through electrophoresis. The pPCPA plasmid was linearized by digestion with Sal I. Adding about 15 mu L of the target gene subjected to enzyme digestion linearization treatment into a prepared competent cell of pichia pastoris GS115, transferring the competent cell into an electric rotating cup, carrying out ice bath for 30min, then carrying out electric shock for 5ms under the conditions of 1500V, 400 omega and 25uF, and adding about 800 mu L of precooled sorbitol. About 400. mu.L of the above-mentioned well-mixed electrotransformation product was smeared on an MD plate, then transformants grown on the MD plate were spotted on a tributyrin plate, and a positive recombinant strain was selected according to the appearance of a transparent circle around the positive transformant and the size of the transparent circle. Colonies with relatively large peripheral clearing zones were picked from tributyrin plates, inoculated into 5mL YPG liquid medium, activated at 200rpm for 24 hours. 50. mu.L of the cultured cell suspension was transferred to a new 50mL YPD culture medium and cultured at 200rpm for 72 hours. The cultured bacterial liquid is taken and placed in a 50mL centrifuge tube, 5000g is centrifuged for 5min at 4 ℃, and the supernatant is discarded. The cells were rinsed with 10mL of 1 XPBS, centrifuged at 5000g and 4 ℃ for 5min, and the supernatant was discarded. Repeat 3 times. The cells were suspended in 1 XPBS containing 1% BSA and diluted to a cell concentration of about 10 OD. mu.L of the bacterial solution was added with 0.5. mu.L of primary antibody (DYKDDDDK Tag (9A3) Mouse mAb), incubated at room temperature for two hours, and mixed once every 20min to avoid bacterial precipitation. 5000g, centrifugating for 5min at room temperature, and discarding the supernatant. The cells were washed with 800. mu.L of 1 XPBS, 5000g, centrifuged at room temperature for 5min, and the supernatant was discarded. This was repeated three times. 400 μ L of 1 XPBS containing 1% BSA suspended thallus is taken, 0.5 μ L of secondary antibody (Anti-Mouse IgG) is added, and the thallus is incubated for 45min at room temperature in a dark place and uniformly mixed every 15min to avoid thallus precipitation. 5000g, centrifugating for 5min at room temperature, and discarding the supernatant. 1mL of 1 XPBS was washed with the cells, 5000g was centrifuged at room temperature for 5min, and the supernatant was discarded. This was repeated three times. 1mL of 1 XPBS suspension was taken, and 2. mu.L of the suspension was transferred onto a slide glass, and the results were observed and recorded under a fluorescence microscope. Under 488nm light excitation, pPCPA-GS115 has green fluorescence, which shows that the lipase with the amino acid sequence of SEQ ID NO.1 connected by alpha-agglutinin is successfully displayed on the surface of the yeast (the result is shown in FIG. 2).
Yeast display CpLIP2 lipase catalyzed synthesis of L-ascorbyl oleate:
(1) yeast display CpLIP2 lipase catalyzes the reaction of ethyl oleate and L-ascorbic acid, and the reaction system is shown in Table 3 below.
Table 3:
| components | Volume of |
| Ethyl oleate emulsion (50g/L) | 200 μ L (final concentration 15mM) |
| L-ascorbic acid | Proper amount of |
| Lipase enzyme | 1 × PBS suspension (final concentration 0.08g/mL) |
| 50mM, pH6.5 phosphate buffer | Make up to 2.2mL |
(2) The reaction was carried out in a 40 ℃ incubator at 180rpm for 2.5 hours.
(3) To the reaction solution was added 2mL of ethyl acetate, followed by addition of 0.792g of sodium chloride (to eliminate an emulsion layer), vortexing and mixing for 3min, extraction of methyl oleate, followed by centrifugation at 5000rpm for 5 min.
(4) The supernatant was taken and heated to 77 ℃ to evaporate ethyl acetate, and then 500. mu.L of methanol was added to redissolve the supernatant.
(5) 200 μ L of the reaction product was assayed in a liquid chromatograph-mass spectrometer (TSQ Quantum Ultra Thermoscientific):
(a) liquid chromatography conditions: a chromatographic column: thermo Hypersil GOLD C18 column (100mm x 2.1mm, Particle sz.1.9 μm), column temperature: 40 ℃, mobile phase: phase A: 0.1% aqueous formic acid; phase B: methanol; flow rate: 300 mu L/min; the mobile phase gradients are shown in table 4 below.
Table 4:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.00 | 95 | 5 |
| 0.50 | 95 | 5 |
| 6.00 | 0 | 100 |
| 10.00 | 0 | 100 |
| 10.10 | 95 | 5 |
| 12.00 | 95 | 5 |
(b) Mass spectrum detection conditions:
an ionization mode: ESI; spraying voltage: 3.0 kV; capillary temperature: 300 ℃; source heating temperature: 350 ℃; sheath gas pressure: 45 arb; auxiliary gas pressure: 8 arb; scanning mode: full sweeping + second level; collision energy: 30V.
As a result, L-ascorbyl oleate is obtained, and the substance with molecular weight of 439.1 in the mass spectrum of figure 1 is [ C24H39O7]-I.e. L-ascorbyl oleic acidMass spectrum peak of ester.
Determination of conversion yield and conversion:
centrifuging the reaction product to remove cells, taking 1mL of supernatant, accurately metering the volume to 10mL by using 100% methanol, filtering the solution by using a 0.22 mu m organic filter membrane, and measuring the concentration of each component of the solution by using High Performance Liquid Chromatography (HPLC). HPLC detection conditions: UltiMate 3000 high performance liquid chromatography (Thermo scientific), column: thermo Hypersil gold 18 column (100mm x 2.1mm, Particle sz.5 μm), loading 60 μ L, mobile phase methanol/water 90/10, flow rate 1mL/min, column temperature 30 ℃, detection wavelength 250 nm.
The yield calculation formula is as follows:
the yield was the concentration of L-ascorbic acid oleate/(concentration of L-ascorbic acid + concentration of L-ascorbic acid oleate).
Efficiency of the esterification reaction: the conversion was defined as the concentration of L-ascorbic acid (after reaction)/the concentration of L-ascorbic acid (before reaction) × 100%.
Through the detection and calculation of the method, the yield and the reaction efficiency of the synthesized L-ascorbyl oleate are respectively over 65.8 percent and over 70.3 percent through preliminary estimation.
Example three: experimental discussion of L-ascorbyl oleate generated by catalysis of two lipases
The literature reports that Candida deformans derived lipase (CdIP 1) and Candida parapsilosis derived lipase (CpLIP2, the lipase used in the present invention) biologically convert to synthesize methyl oleate and fatty acid methyl ester under high water activity conditions, but does not report that L-ascorbic acid is reacted with oleic acid or ethyl oleate to produce L-ascorbic acid oleate.
This experiment utilizes Pichia to heterologously express Candida deformans (CdIP 1) and Candida parapsilosis lipase (CpLIP2) to investigate two different ester synthesis types of lipase at high water activity (a)w>0.9) in a water/lipid biphasic medium.
Carrying out expression and purification on a lipase gene sequence Lip1(GENBANKAJ428393.1) derived from Candida deformans (Cdlp 1) according to the method described in the first embodiment by replacing the gene sequence Lip2 with the gene sequence Lip1 in the same manner as other operation methods to obtain Cdlp 1 lipase; the reaction was then carried out as follows to examine the activity of the enzyme CdLIP2 lipase obtained in example one and CpLIP1 lipase obtained in this example for the catalytic synthesis of L-ascorbyl oleate.
Activity test of Cdlp 1 lipase and CpLIP2 lipase for catalytic synthesis of L-ascorbyl oleate
(1) The Cdlp 1 lipase catalyzes the reaction of oleic acid and L-ascorbic acid, and the reaction system is shown in Table 5 below.
Table 5:
| components | Volume of |
| Oleic acid emulsion (50g/L) | 200 μ L (final concentration 16mM) |
| L-ascorbic acid | Proper amount of |
| Enzyme solution | 1mL |
| 50mM, pH6.5 phosphate buffer | Make up to 2.2mL |
(2) CpLIP2 lipase catalyzed the reaction of ethyl oleate and L-ascorbic acid, and the reaction system is shown in Table 6 below.
Table 6:
| components | Volume of |
| Ethyl oleate emulsion (50g/l) | 200 μ L (final concentration 15mM) |
| L-ascorbic acid | Proper amount of |
| Enzyme solution | 1mL |
| 50mM, pH6.5 phosphate buffer | Make up to 2.2mL |
(3) The reaction was placed in a 40 ℃ incubator at 180rpm and reacted for 2.5 hours.
(4) To the reaction solution was added 2mL of ethyl acetate, followed by addition of 0.792g of sodium chloride (to eliminate an emulsion layer), vortexing and mixing for 3min, extraction of methyl oleate, followed by centrifugation at 5000rpm for 5 min.
(5) The supernatant was taken and heated to 77 ℃ to evaporate ethyl acetate, and then 500. mu.L of methanol was added to redissolve the supernatant.
(6) The product was taken for HPLC detection and loaded at 60. mu.L. HPLC detection conditions: the mobile phase is methanol/water 90/10, the flow rate is 1mL/min, the column temperature is 30 ℃, and the detection wavelength is 250 nm.
Mass Spectrometry (LC-MS/MS) identification of L-ascorbic acid oleate
The above 200. mu.L of the reaction product was analyzed by liquid chromatography-mass spectrometer (TSQ Quantum Ultra Thermoscientific). (1) Liquid chromatography conditions: a chromatographic column: thermo Hypersil GOLD C18 column (100mm x 2.1mm, Particle sz.1.9 μm), column temperature: 40 ℃, mobile phase: phase A: 0.1% aqueous formic acid; phase B: methanol; flow rate: 300 mu L/min; the mobile phase gradients are shown in table 7 below.
Table 7:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.00 | 95 | 5 |
| 0.50 | 95 | 5 |
| 6.00 | 0 | 100 |
| 10.00 | 0 | 100 |
| 10.10 | 95 | 5 |
| 12.00 | 95 | 5 |
(2) Mass spectrum detection conditions: an ionization mode: ESI; spraying voltage: 3.0 kV; capillary temperature: 300 ℃; source heating temperature: 350 ℃; sheath gas pressure: 45 arb; auxiliary gas pressure: 8 arb; scanning mode: full sweeping + second level; collision energy: 30V.
3. The experimental results are as follows:
(1) the successful constitutive secretion in pichia pastoris GS115 expressed CdLIP1 lipase and CpLIP2 lipase, and both CpLIP2 and CdLIP1 had lipase activity.
(2) At high water activity (a)w>0.9) in a water/lipid two-phase medium, no L-ascorbyl oleate is detected when Cdlp 1 lipase catalyzes the reaction of L-ascorbic acid and oleic acid; CpLIP2 lipase catalyzed the reaction between L-ascorbic acid and ethyl oleate, and L-ascorbyl oleate was detected in the product (as shown in FIG. 1).
(3) At high water activity (a)w>0.9) in a water/lipid two-phase medium, the CpLIP2 lipase catalyzes and synthesizes the L-ascorbyl oleate, the concentration of the ethyl oleate has larger influence on the synthesis of the L-ascorbyl oleate, the concentration of the ethyl oleate is improved, and the conversion rate of the L-ascorbic acid can be increased.
SEQUENCE LISTING
<110> river-south university, Kaiping morning glory Biochemical pharmacy Co., Ltd
<120> method for catalytically synthesizing L-ascorbyl oleate in high-water-activity medium
<130>
<160>1
<170>PatentIn version 3.5
<210>1
<211>465
<212>PRT
<213>Candida parapsilosis
<400>1
Met Arg Tyr Phe Ala Ile Ala Phe Leu Leu Ile Asn Thr Ile Ser Ala
1 5 10 15
Phe Val Leu Ala Pro Lys Lys Pro Ser Gln Asp Asp Phe Tyr Thr Pro
20 25 30
Pro Gln Gly Tyr Glu Ala Gln Pro Leu Gly Ser Ile Leu Lys Thr Arg
35 40 45
Asn Val Pro Asn Pro Leu Thr Asn Val Phe Thr Pro Val Lys Val Gln
50 55 60
Asn Ala Trp Gln Leu Leu Val Arg Ser Glu Asp Thr Phe Gly Asn Pro
65 70 75 80
Asn Ala Ile Val Thr Thr Ile Ile Gln Pro Phe Asn Ala Lys Lys Asp
85 90 95
Lys Leu Val Ser Tyr Gln Thr Phe Glu Asp Ser Gly Lys Leu Asp Cys
100 105 110
Ala Pro Ser Tyr Ala Ile Gln Tyr Gly Ser Asp Ile Ser Thr Leu Thr
115 120 125
Thr Gln Gly Glu Met Tyr Tyr Ile Ser Ala Leu Leu Asp Gln Gly Tyr
130 135 140
Tyr Val Val Thr Pro Asp Tyr Glu Gly Pro Lys Ser Thr Phe Thr Val
145 150 155 160
Gly Leu Gln Ser Gly Arg Ala Thr Leu Asn Ser Leu Arg Ala Thr Leu
165 170 175
Lys Ser Gly Asn Leu Thr Gly Val Ser Ser Asp Ala Glu Thr Leu Leu
180 185 190
Trp Gly Tyr Ser Gly Gly Ser Leu Ala Ser Gly Trp Ala Ala Ala Ile
195 200 205
Gln Lys Glu Tyr Ala Pro Glu Leu Ser Lys Asn Leu Leu Gly Ala Ala
210 215 220
Leu Gly Gly Phe Val Thr Asn Ile Thr Ala Thr Ala Glu Ala Val Asp
225 230 235 240
Ser Gly Pro Phe Ala Gly Ile Ile Ser Asn Ala Leu Ala Gly Ile Gly
245 250 255
Asn Glu Tyr Pro Asp Phe Lys Asn Tyr Leu Leu Lys Lys Val Ser Pro
260 265 270
Leu Leu Ser Ile Thr Tyr Arg Leu Gly Asn Thr His Cys Leu Leu Asp
275 280 285
Gly Gly Ile Ala Tyr Phe Gly Lys Ser Phe Phe Ser Arg Ile Ile Arg
290 295 300
Tyr Phe Pro Asp Gly Trp Asp Leu Val Asn Gln Glu Pro Ile Lys Thr
305 310 315 320
Ile Leu Gln Asp Asn Gly Leu Val Tyr Gln Pro Lys Asp Leu Thr Pro
325 330 335
Gln Ile Pro Leu Phe Ile Tyr His Gly Thr Leu Asp Ala Ile Val Pro
340 345 350
Ile Val Asn Ser Arg Lys Thr Phe Gln Gln Trp Cys Asp Trp Gly Leu
355 360 365
Lys Ser Gly Glu Tyr Asn Glu Asp Leu Thr Asn Gly His Ile Thr Glu
370 375 380
Ser Ile Val Gly Ala Pro Ala Ala Leu Thr Trp Ile Ile Asn Arg Phe
385 390 395 400
Asn Gly Gln Pro Pro Val Asp Gly Cys Gln His Asn Val Arg Ala Ser
405 410 415
Asn Leu Glu Tyr Pro Gly Thr Pro Gln Ser Ile Lys Asn Tyr Phe Glu
420 425 430
Ala Ala Leu His Ala Ile Leu Gly Phe Asp Leu Gly Pro Asp Val Lys
435 440 445
Arg Asp Lys Val Thr Leu Gly Gly Leu Leu Lys Leu Glu Arg Phe Ala
450 455 460
Phe
465
Claims (10)
1. A method for catalytically synthesizing L-ascorbyl oleate in a medium with high water activity is characterized by comprising the following steps: taking ethyl oleate and L-ascorbic acid as substrates, mixing, adding lipase, placing in a water/lipid two-phase medium with high water activity, and catalyzing and synthesizing L-ascorbic acid oleate by using recombinant lipase; the recombinant lipase is a recombinant lipase based on a lipase with an amino acid sequence shown in SEQ ID No.1 and derived from Candidapapesisis.
2. The process for the catalytic synthesis of L-ascorbyl oleate in a high water activity medium according to claim 1, characterized in that: water activity a of the water/lipid biphasic mediumw>0.9。
3. The method of claim 1, wherein: the water/lipid biphasic medium is 50mM phosphate buffer solution with pH6.5 and ethyl oleate emulsion.
4. The method of claim 1, wherein: the process for synthesizing the L-ascorbyl oleate by the catalysis of the recombinant lipase comprises the following steps: stirring the reaction system at 40 ℃ and 180rpm, reacting for 2.5 hours, and separating and purifying from the reaction system to obtain the L-ascorbyl oleate.
5. The method according to claim 4, wherein the separation and purification method comprises the following steps: 2mL of ethyl acetate and 0.792g of sodium chloride are added into each 2.2mL of reaction system, mixed uniformly for 3min by vortex, L-ascorbyl oleate is extracted, and then centrifuged for 5min at 5000 rpm. Taking the supernatant, heating the supernatant to 77 ℃, and volatilizing ethyl acetate to obtain the L-ascorbyl oleate.
6. The method of claim 1, wherein: the concentration of the added reaction substrate L-ascorbic acid in the reaction system is 0.2-2.2mol/L, and the final concentration of the ethyl oleate is 15-250 mmol/L.
7. The method of claim 1, wherein: the recombinant lipase is a recombinant lipase secreted and expressed by yeast.
8. The method of claim 7, wherein: the final concentration of the recombinant lipase in the reaction system is 0.05-2 mg/mL.
9. The method of claim 1, wherein: the recombinant lipase is a recombinant lipase displayed by yeast.
10. The method of claim 9, wherein: the final concentration of the recombinant lipase in the reaction system is 0.01-0.5 g/mL.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070270507A1 (en) * | 2003-08-14 | 2007-11-22 | Albrecht Weiss | Use of Pit Emulsions in Enzymatic Reactions |
| EP2298727A1 (en) * | 2009-09-05 | 2011-03-23 | Cognis IP Management GmbH | Method for producing esters of short-chains alcohols from triglyceride-rich oils |
| CN102212572A (en) * | 2011-04-28 | 2011-10-12 | 浙江大学 | Method for catalytically synthesizing L-ascorbyl oleate with yeast display lipase |
| CN104480156A (en) * | 2014-12-19 | 2015-04-01 | 暨南大学 | L-ascorbyloleate as well as preparation method and application thereof |
-
2020
- 2020-07-17 CN CN202010691269.4A patent/CN111733196A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070270507A1 (en) * | 2003-08-14 | 2007-11-22 | Albrecht Weiss | Use of Pit Emulsions in Enzymatic Reactions |
| EP2298727A1 (en) * | 2009-09-05 | 2011-03-23 | Cognis IP Management GmbH | Method for producing esters of short-chains alcohols from triglyceride-rich oils |
| CN102212572A (en) * | 2011-04-28 | 2011-10-12 | 浙江大学 | Method for catalytically synthesizing L-ascorbyl oleate with yeast display lipase |
| CN104480156A (en) * | 2014-12-19 | 2015-04-01 | 暨南大学 | L-ascorbyloleate as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JOANA RODRIGUES ET AL.: "Biodiesel production from crude jatropha oil catalyzed by immobilized lipase/acyltransferase from Candida parapsilosis in aqueous medium", 《BIORESOURCE TECHNOLOGY》 * |
| VIRGINIE NEUGNOT ET AL.: "The lipase/acyltransferase from Candida parapsilosis: Molecular cloning and characterization of purified recombinant enzymes", 《EUR. J. BIOCHEM.》 * |
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