A method for synthesizing phenylpropenyl esters by ferulic acid esterase catalysis

Abstract

The present invention provides a method for synthesizing phenylpropenyl esters using ferulic acid esterase. The method comprises reacting methyl phenylpropenyl ester and ethyl phenylpropenyl ester with an alcohol or a linear fatty alcohol containing a phenyl or naphthyl group. The method is mild and environmentally friendly, has simple steps, uses a cost-effective substrate, produces few by-products, is conducive to industrial production, and is suitable for widespread application.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis, and particularly relates to a method for synthesizing phenylpropenyl ester by catalyzing ferulic acid esterase. Background Art

[0002] Phenylacetate is an ester derivative formed by using phenylacrylic acid as an acyl donor and alcohols, thiols, and amines as acyl acceptors. Phenylacetate combines the antioxidant and anti-inflammatory physiological activities of phenylacrylic acid with the biological activities of alcohols, thiols, and amines. It also has better water or fat solubility than phenylacrylic acid, making it easier to exert its physiological functions in specific biological environments. For example, ferulic acid in phenylacrylic acid has been shown to have stronger pharmacological activity and lower toxicity than ferulic acid. . Compared with ferulic acid, ferulic acid aromatic esters have better fat solubility. Ferulic acid aromatic esters have anti-hyperglycemic, antioxidant, anti-inflammatory and cancer prevention effects. Ferulic acid sugar esters are better water-soluble than ferulic acid, and have antibacterial, antioxidant, anti-glycation, intestinal microecology regulation and blood vessel protection, and effectively control the occurrence and development of diabetes. For example, the antioxidant activity of 6-O-feruloyl glucose ester is higher than that of ferulic acid. Ferulic acid amide can lower blood pressure, inhibit acetylcholinesterase and promote insulin secretion. Its inhibitory activity on acetylcholinesterase is higher than that of ferulic acid.

[0003] Phenylacetate can be extracted from natural plants, but natural plants have complex components and the resulting products require complex purification steps. For example, ferulic acid oligosaccharide esters are obtained by adding ferulic acid esterase to hydrolyze hemicellulose, and the enzymatic hydrolysis product is further purified.

[0004] Besides natural product extraction, phenylpropenyl esters can also be obtained through chemical synthesis and enzymatic synthesis, most of which are non-aqueous phase syntheses. Taking benzyl ferulate as an example, its chemical synthesis requires two steps: ferulic acid is catalyzed into ferulic acid chloride, which is then reacted with benzyl alcohol to synthesize benzyl ferulate. The entire reaction process is carried out in an organic solvent, but the large-scale use of organic solvents is not economical or environmentally friendly. Enzymatic synthesis methods can utilize enzymes such as lipase, ferulic acid esterase, ferulic acid acyltransferase, and carboxylic acid reductase. Lipase must catalyze the synthesis of ferulic acid esters in a solvent-free system or an organic solvent system. Ferulic acid esterase catalyzes the enzymatic synthesis of alkyl ferulates and sugar ferulate esters in a microemulsion system with a water content of 1-5%. Currently, only two examples of aqueous phase synthesis of phenylpropenyl esters have been reported: the first is enzymatic synthesis in aqueous phase using a complex and expensive coenzyme A substrate using ferulic acid acyltransferase, and the second is the synthesis of phenylpropenyl esters in aqueous phase using a carboxylic acid reductase using the expensive cofactor ATP.

[0005] Therefore, it is necessary to develop a new green, environmentally friendly and economical method for synthesizing phenylpropenyl esters to address the problem of dependence on organic solvents in non-aqueous enzyme catalysis and the problem of using expensive and complex cofactors in existing aqueous catalysis. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for synthesizing phenylpropenyl ester catalyzed by ferulic acid esterase.

[0007] The technical solution of the present invention is to provide a feruloyl esterase AmCE1, whose amino acid sequence is shown as SEQ ID NO 2, and the feruloyl esterase is derived from Anaeromyces mucronatus.

[0008] A ferulic acid esterase AmCE1, characterized in that its amino acid sequence is as shown in SEQ ID NO 2, by mutating the serine S at position 67 to isoleucine I.

[0009] A method for synthesizing phenylpropenyl esters in an aqueous phase using feruloyl esterase as catalytic agent is disclosed, characterized in that: methyl ferulate, methyl cinnamate, methyl caffeate, methyl p-coumarate, or trans-methyl ferulate is used as an acyl donor; benzyl alcohol, phenylethanol, phenylpropanol, phenylbutanol, p-hydroxyphenylethanol, 4-hydroxy-3-methoxybenzyl alcohol, aribonucleic acid, naphthaleneethanol, phenyl-β-D-glucoside, arbutin, phenyl-β-D-galactopyranoside, propanol, butanol, or octanol is used as an acyl acceptor; and phenylpropenyl ester is reacted to produce phenylpropenyl ester under the catalysis of feruloyl esterase; the catalytic reaction temperature is 20-55° C.; the substrate and feruloyl esterase are removed from the catalytic reaction system, and the remainder is water, phosphate, or Tris-HCl buffer; a cosolvent for dissolving the substrate may or may not be added to the reaction system; the pH value of the catalytic reaction system is 3-11; and the rotation speed of the catalytic reaction system is 0-220 rpm.

[0010] The reaction steps are:

[0011] (1) Heterologous expression and acquisition of ferulic acid esterase AmCE1

[0012] Heterologous expression and acquisition of feruloyl esterase AmCE1

[0013] The gene sequence of ferulic acid esterase AmCE1 used for fermentation preparation of the catalytic enzyme is shown in SEQ ID NO. 1, and the amino acid sequence encoded by it is shown in SEQ ID NO. 2. The entire gene was synthesized by GenScript Biotechnology Co., Ltd. After gene synthesis, it was ligated with the expression vector pET-30a(+) and transformed into E. coli BL21(DE3) for protein expression. Successfully transformed expression strains were screened using kanamycin-resistant culture medium, expanded, and then transferred to shake flask culture. The recombinant strain was added to LB liquid culture medium containing kanamycin at a 5% addition rate and cultured at 220 rpm and 37°C. When the OD600 is 0.5 to 0.6, add IPTG at a concentration of 1 mM and induce expression at 20°C and 220 rpm for 20 hours. Collect the cells by centrifugation at 6000 rpm for 10 minutes and resuspend them in 20 mL of 50 mM pH 7.4 PBS. Disrupt the cells by ultrasonication at 360 kW for 40 minutes and then centrifuge at 12000 rpm for 30 minutes to remove cell debris. Take the supernatant and filter it with a 0.22 μm filter membrane. Store it as crude enzyme at -20°C for subsequent use.

[0014] Recombinant AmCE1 is tagged with a 6His tag at its amino terminus, so purification is simple using Ni-NTA affinity column chromatography as follows. The supernatant was directly loaded onto a Ni-NTA affinity column, and the column was washed with two volumes of wash buffer [3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 500 mM NaCl, and 10 mM imidazole (pH 7.4)]. Recombinant AmCE1 bound to the column was eluted with elution buffer [3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 500 mM NaCl, and 88.4 mM imidazole (pH 7.4)], and the protein from the eluted fraction was dialyzed to remove excess imidazole. The purity of the purified recombinant AmCE1 was assessed by SDS-PAGE (containing 10% (w / v) acrylamide) and the pure enzyme was stored at -20°C until further use.

[0015] (2) Enzymatic catalysis

[0016] Acyl donor esters and acyl acceptor alcohols are added to a buffer solution, followed by crude or purified AmCE1 enzyme. The substrate and feruloyl esterase are removed from the catalytic reaction system, leaving water, phosphate, or Tris-HCl buffer as the remainder. A cosolvent for dissolving the substrate may or may not be added to the reaction system. The catalytic reaction temperature is 35° C., the pH value of the catalytic reaction system is 8.5, and the rotation speed of the catalytic reaction system is 200 rpm. After the reaction, an ester exchange reaction occurs, wherein the acyl donor removes methanol or ethanol, and the acyl acceptor removes hydrogen from the alcohol, reacting with the remaining phenylpropenoyl group of the acyl donor. Thus, alcohols with benzene rings or naphthyl groups and straight-chain fatty alcohols replace the methyl or ethyl ester in the original substrate to generate new phenylpropenoyl esters.

[0017] The selected substrates are methyl phenylpropene and ethyl phenylpropene, and the alcohols are alcohols with a benzene ring, a naphthyl group and straight-chain fatty alcohols.

[0018] The cosolvent is 1% to 10% v / v DMSO.

[0019] The method for synthesizing phenylpropenyl esters by using ferulic acid esterase in aqueous phase catalysis uses methyl ferulate as an acyl donor and phenyl alcohols such as benzyl alcohol, phenylethyl alcohol, phenylpropanol and phenylbutanol as acyl acceptors to catalyze the synthesis of benzyl ferulate, phenylethyl ferulate, phenylpropyl ferulate and phenylbutyl ferulate.

[0020] The method for synthesizing phenylpropenyl esters by using feruloyl esterase in aqueous phase catalysis uses methyl cinnamate, methyl caffeate and methyl paracoumarate as acyl donors and phenylethanol as acyl acceptor to synthesize phenylethyl cinnamate, phenylethyl caffeate and phenylethyl paracoumarate.

[0021] The method for synthesizing phenylpropenyl ester by using ferulic acid esterase in water phase catalysis uses trans-methyl ferulate as an acyl donor and p-hydroxyphenylethanol as an acyl acceptor to synthesize trans-p-hydroxyphenylethyl ferulate.

[0022] The method for synthesizing phenylpropenyl esters by aqueous phase catalysis of ferulic acid esterase uses methyl ferulate as an acyl donor and alcohols having a phenyl group and a methoxy group or a hydroxyl group modified on the phenyl group with a side chain as an acyl acceptor, i.e., p-hydroxyphenylethanol and 4-hydroxy-3-methoxybenzyl alcohol as acyl acceptors to synthesize p-hydroxyphenylethyl ferulate and 4-hydroxy-3-methoxybenzyl ferulate.

[0023] The method for synthesizing phenylpropenyl ester by using ferulic acid esterase in water phase catalysis uses methyl ferulate as an acyl donor and aribendol as an acyl acceptor to synthesize aribendol ferulate.

[0024] The method for synthesizing phenylpropenyl ester by using ferulic acid esterase in water phase catalysis uses methyl ferulate as an acyl donor and naphthaleneethanol as an acyl acceptor to synthesize naphthyl ethyl ferulate.

[0025] The method for synthesizing phenylpropenyl esters by aqueous phase catalysis of ferulic acid esterase uses methyl ferulate as an acyl donor and glycosides with phenyl groups, namely phenyl-β-D-glucoside, arbutin, and phenyl-β-D-pyranoside, as acyl acceptors to synthesize phenyl-β-D-glucoside ferulate, arbutin ferulate, and phenyl-β-D-pyranoside ferulate.

[0026] The method for synthesizing phenylpropenyl esters by using ferulic acid esterase in aqueous phase catalysis uses methyl ferulate as an acyl donor and straight-chain alcohols such as propanol, butanol and octanol as acyl acceptors to catalyze the synthesis of propyl ferulate, butyl ferulate and octyl ferulate.

[0027] Compared with the prior art, the present invention has the following outstanding substantive features and significant progress:

[0028] 1. Feruloyl esterase was used to catalyze the synthesis of phenylpropenoic acid esters in aqueous phase. This is the first time that feruloyl esterase has been used to enzymatically synthesize various phenylpropenoic acid esters in an aqueous system. This is efficient and green, and reduces the use of organic reagents.

[0029] 2. The steps are simple, avoiding tedious group protection and deprotection steps, with few by-products and avoiding complicated impurity removal steps.

[0030] The above two points are the results obtained through a large number of experiments. They are not obvious and cannot be inferred by those skilled in the art based on existing technology and knowledge. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the SDS-PAGE image of the purified AmCE1 protein.

[0032] Figure 2 Diagram of the enzymatic reaction process.

[0033] Figure 3 This is the HPLC chromatogram of the products of the enzymatic reaction of methyl ferulate and phenylethanol.

[0034] Figure 4 This is the mass spectrum of the enzymatic product phenethyl ferulate. The relative molecular mass of phenethyl ferulate is 298, and the positive ion mode corresponds to a molecular weight of 299 in the figure. DETAILED DESCRIPTION

[0035] Below by embodiment, technical scheme of the present invention is described in further detail, but following embodiment is for illustrating example of the present invention, does not constitute any limitation to claims of the present invention.Should be understood that these embodiments are only for illustrating the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.Embodiment is with methyl ferulate and phenylethyl alcohol synthesis as example.Reagents involved in the present invention, unless otherwise specified, are analytically pure.

[0036] Example 1

[0037] Expression and preparation of feruloyl esterase AmCE1

[0038] 1.1 Bacterial strains and culture conditions

[0039] E. coli DH5α was used as a cloning strain, E. coli BL21(DE3) was used as an expression strain (kananamycin resistance), and the recombinant E. coli strains were grown in LB medium containing kanamycin (50 μg / mL) at 37°C.

[0040] 1.2 Construction of expression vector

[0041] The gene sequence of ferulic acid esterase AmCE1 used for fermentation preparation of the catalytic enzyme is shown in SEQ ID NO. 1, and the amino acid sequence encoded by it is shown in SEQ ID NO. 2. The entire gene was synthesized by GenScript Biotechnology Co., Ltd. After gene synthesis, it was ligated with the expression vector pET-30a(+) and transformed into E. coli BL21(DE3) for protein expression. Successfully transformed expression strains were screened using kanamycin-resistant culture medium, expanded, and then transferred to shake flask culture. The recombinant strain was added to LB liquid culture medium containing kanamycin at a 5% addition rate and cultured at 220 rpm and 37°C. When the OD600 is 0.5 to 0.6, add IPTG at a concentration of 1 mM and induce expression at 20°C and 220 rpm for 20 hours. Collect the cells by centrifugation at 6000 rpm for 10 minutes and resuspend them in 20 mL of 50 mM pH 7.4 PBS. Disrupt the cells by ultrasonication at 360 kW for 40 minutes and then centrifuge at 12000 rpm for 30 minutes to remove cell debris. Take the supernatant and filter it with a 0.22 μm filter membrane. Store it as crude enzyme at -20°C for subsequent use.

[0042] Recombinant AmCE1 is tagged with a 6His tag at its amino terminus, making purification simple using Ni-NTA affinity chromatography as follows. The supernatant was directly loaded onto a Ni-NTA affinity column, and the column was washed with two volumes of wash buffer [3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 500 mM NaCl, and 10 mM imidazole (pH 7.4)]. Recombinant AmCE1 bound to the column was eluted with elution buffer [3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 500 mM NaCl, and 88.4 mM imidazole (pH 7.4)]. Protein from the eluted fraction was dialyzed to remove excess imidazole. Purified recombinant AmCE1 was assessed for purity by SDS-PAGE (containing 10% (w / v) acrylamide). The concentration of pure enzyme protein was determined using a BioSharp BCA assay kit. The size of recombinant AmCE1 is 31.7 kDa. Figure 1 The SDS-PAGE images shown show that the apparent molecular weight of the enzyme is consistent with its theoretical molecular weight.

[0043] Example 2

[0044] Enzyme-catalyzed synthesis of benzyl ferulate

[0045] In this example, benzyl ferulate was synthesized using the pure enzyme prepared in Example 1 as a catalyst, methyl ferulate as an acyl donor, and benzyl alcohol as an acyl acceptor.

[0046] (1) Dissolve methyl ferulate in benzyl alcohol and add it to 100 mM Tris-HCl buffer at pH 8.5. The final concentration of methyl ferulate is 250 mM and that of benzyl alcohol is 810 mM. Add the purified enzyme solution until the enzyme content reaches 0.4 mg / mL. Shake the reaction in a constant temperature shaking incubator at 35°C and 200 rpm for 3 h.

[0047] (2) After the reaction, 75 μL of acetic acid was added to terminate the reaction. An equal volume of methanol was added to dissolve the product. 50 μL of the methanol solution was diluted 50 times with methanol / water = 8 / 2. The product was filtered through a 0.22 μm membrane and then detected by high-performance liquid chromatography. The high-performance liquid chromatography conditions were a 250 mm × 4.6 mm Waters Symmetry Shield RP18 4.6 mm X 250 mm C18 column, with a mobile phase of methanol: water containing 0.1% TFA (V:V) = 80:20, a flow rate of 1 mL / min, a column temperature of 25°C, a detection wavelength of 310 nm, and an injection volume of 3 μL. The product yield was determined by normalization, and the conversion rate was 25.05%.

[0048] Example 3

[0049] Enzyme-catalyzed synthesis of phenethyl ferulate

[0050] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and phenylethanol was used as an acyl acceptor to synthesize phenylethyl ferulate.

[0051] (1) Methyl ferulate was dissolved in phenylethanol and added to 100 mM Tris-HCl buffer at pH 8.5 (the final concentration of methyl ferulate was 250 mM and the final concentration of phenylethanol was 810 mM). Purified enzyme solution was added to an enzyme content of 0.4 mg / mL. The reaction was stirred in a constant temperature shaking incubator at 35°C and 200 rpm for 3 h.

[0052] After the reaction, 75 μL of acetic acid was added to terminate the reaction, and 3 volumes of acetonitrile were added to dissolve the product. 50 μL of the acetonitrile solution was diluted 20 times with acetonitrile / water = 8 / 2, and filtered through a 0.22 μm membrane before HPLC detection. The HPLC conditions were a 250 mm × 4.6 mm Waters Symmetry Shield RP18 4.6 mm X 250 mm C18 column, with a mobile phase of acetonitrile: 0.1% TFA water (V:V) = 80:20, a flow rate of 1 mL / min, a column temperature of 25°C, a detection wavelength of 310 nm, and an injection volume of 3 μL. The product yield was detected using the normalization method, and the conversion rate was 55.26%. Figure 3 As shown in FIG, the HPLC chromatogram of the enzymatic reaction product shows the formation of the product phenylethyl ferulate. Figure 4 The following is the mass spectrum of the enzymatic product, phenethyl ferulate. The relative molecular mass of phenethyl ferulate is 298, and the molecular weight in the positive ion mode corresponds to 299. This confirms that the method is feasible and that the product is indeed generated.

[0053] Example 4

[0054] Enzyme-catalyzed synthesis of phenylpropyl ferulate

[0055] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and phenylpropanol was used as an acyl acceptor to synthesize phenylpropyl ferulate.

[0056] Methyl ferulate was dissolved in phenylpropanol and added to a 100 mM Tris-HCl buffer at pH 8.5 (final concentration of methyl ferulate was 250 mM, final concentration of phenylpropanol was 810 mM). Purified enzyme solution was added to an enzyme content of 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 3 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization method. The conversion rate was 23.92%.

[0057] Example 5

[0058] Enzyme-catalyzed synthesis of phenylbutyl ferulate

[0059] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and phenylbutanol was used as an acyl acceptor to synthesize phenylbutanol ferulate.

[0060] Methyl ferulate was dissolved in phenylbutanol and added to 100 mM Tris-HCl buffer, pH 8.5 (final concentration of methyl ferulate was 250 mM, final concentration of phenylbutanol was 810 mM). Purified enzyme solution was added to an enzyme content of 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was determined by normalization method. The conversion rate was 14.96%.

[0061] Example 6

[0062] Enzyme-catalyzed synthesis of phenylethyl cinnamate

[0063] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl cinnamate was used as an acyl donor, and phenylethanol was used as an acyl acceptor to synthesize phenylethyl cinnamate.

[0064] Methyl cinnamate was dissolved in phenylethanol and added to 100 mM Tris-HCl buffer, pH 8.5 (final concentration of methyl cinnamate was 250 mM, final concentration of phenylethanol was 810 mM). Purified enzyme solution was added to an enzyme content of 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was determined by normalization, resulting in a conversion rate of 0.89%.

[0065] Example 7

[0066] Enzyme-catalyzed synthesis of caffeic acid phenethyl ester

[0067] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, caffeic acid methyl ester was used as an acyl donor, and phenylethanol was used as an acyl acceptor to synthesize caffeic acid phenethyl ester.

[0068] Caffeic acid methyl ester and phenylethanol were added to 100 mM Tris-HCl buffer at pH 8.5 (final concentration of caffeic acid methyl ester was 250 mM, and final concentration of phenylethanol was 810 mM). Purified enzyme solution was added to an enzyme concentration of 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was determined by normalization, yielding a conversion rate of 6.42%.

[0069] Example 8

[0070] Enzyme-catalyzed synthesis of trans-p-coumaric acid methyl ester

[0071] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, trans-p-coumaric acid methyl ester was used as an acyl donor, and phenylethanol was used as an acyl acceptor to synthesize trans-p-coumaric acid phenylethanol ester.

[0072] To methyl trans-p-coumarate and phenylethanol (50 mM trans-p-coumarate, 810 mM phenylethanol final concentration) in 100 mM Tris-HCl buffer at pH 8.5, purified enzyme solution was added to an enzyme concentration of 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was determined by normalization, yielding a conversion rate of 19.615%.

[0073] Example 9

[0074] Enzymatic Synthesis of Trans-4-Hydroxyphenylethyl Ferulate

[0075] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, trans-methyl ferulate was used as an acyl donor, and p-hydroxyphenylethanol was used as an acyl acceptor to synthesize trans-p-hydroxyphenylethyl ferulate.

[0076] Trans-methyl ferulate and p-hydroxyphenylethanol were added to 100 mM Tris-HCl buffer at pH 8.5 (final concentration of methyl ferulate was 250 mM, and final concentration of p-hydroxyphenylethanol was 810 mM). Purified enzyme solution was added until the enzyme content reached 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization method, resulting in a conversion rate of 8.62%.

[0077] Example 10

[0078] Enzyme-catalyzed synthesis of p-hydroxyphenylethyl ferulate

[0079] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and p-hydroxyphenylethanol was used as an acyl acceptor to synthesize p-hydroxyphenylethyl ferulate.

[0080] Methyl ferulate and p-hydroxyphenylethanol (50 mM methyl ferulate, final concentration of p-hydroxyphenylethanol: 810 mM) were added to a purified enzyme solution to a final enzyme concentration of 0.4 mg / mL in 100 mM Tris-HCl buffer at pH 8.5. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was determined by normalization, yielding a conversion rate of 8.62%.

[0081] Example 11

[0082] Enzyme-catalyzed synthesis of 4-hydroxy-3-methoxybenzyl ferulate

[0083] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and 4-hydroxy 3-methoxybenzyl alcohol was used as an acyl acceptor to synthesize 4-hydroxy 3-methoxybenzyl ferulate.

[0084] Methyl ferulate and 4-hydroxy-3-methoxybenzyl alcohol were added to 100 mM Tris-HCl buffer at pH 8.5 (final concentration of methyl ferulate was 250 mM, and final concentration of 4-hydroxy-3-methoxybenzyl alcohol was 810 mM). Purified enzyme solution was added until the enzyme content reached 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was determined by normalization, resulting in a conversion rate of 5.20%.

[0085] Example 12

[0086] Enzyme-catalyzed synthesis of aribendol ferulate

[0087] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and aribendol was used as an acyl acceptor to synthesize aribendol ferulate.

[0088] Methyl ferulate and aribendol were added to 100 mM Tris-HCl buffer at pH 8.5 (final concentration of methyl ferulate was 250 mM, final concentration of aribendol was 810 mM). Purified enzyme solution was added until the enzyme content reached 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization method. The conversion rate was 0.99%.

[0089] Example 13

[0090] Enzymatic Synthesis of Naphthyl Ethyl Ferulate

[0091] In this example, the crude enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and naphthaleneethanol was used as an acyl acceptor to synthesize naphthyl ferulate.

[0092] Methyl ferulate and naphthaleneethanol were added to 100 mM Tris-HCl buffer at pH 8.5 (12 mg of methyl ferulate and 25 μL of naphthaleneethanol). 30 μL of the crude enzyme solution was added to a reaction volume of 250 μL. The reaction was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 3 h. The reaction solution was normalized to determine the product yield as in Example 2, and the conversion rate was 19.93%.

[0093] Example 14

[0094] Enzyme-catalyzed synthesis of phenyl β-D-glucoside ferulate

[0095] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and phenyl-β-D-glucoside was used as an acyl acceptor to synthesize phenyl β-D-glucoside ferulate.

[0096] Methyl ferulate and phenyl-β-D-glucoside were added to 100 mM Tris-HCl buffer at pH 8.5 (final concentration of methyl ferulate was 250 mM, and final concentration of phenyl-β-D-glucoside was 810 mM). Purified enzyme solution was added until the enzyme content reached 0.4 mg / mL. The reaction was incubated in a constant temperature shaking incubator at 35°C and 200 rpm for 4 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization method. The conversion rate was 1.59%.

[0097] Example 15

[0098] Enzymatic Synthesis of Arbutin Ferulate

[0099] In this example, the crude enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and arbutin was used as an acyl acceptor to synthesize arbutin ferulate.

[0100] Methyl ferulate and arbutin (methyl ferulate added in an amount of 12 mg and arbutin added in an amount of 25 mg) were added to a 100 mM Tris-HCl buffer at pH 8.5. 30 μL of the crude enzyme solution was added to a reaction system of 250 μL. The reaction was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 1.3 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization, with a conversion rate of 2.99%.

[0101] Example 16

[0102] Enzymatic Synthesis of Phenyl-β-D-Galactopyranoside Ferulate

[0103] In this example, the crude enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and phenyl-β-D-galactopyranoside was used as an acyl acceptor to synthesize phenyl-β-D-galactopyranoside ferulate.

[0104] Methyl ferulate and phenyl-β-D-galactopyranoside were added to 100 mM Tris-HCl buffer at pH 8.5 (methyl ferulate added in an amount of 12 mg, phenyl-β-D-galactopyranoside added in an amount of 25 mg). 30 μL of the crude enzyme solution was added to a reaction volume of 250 μL. The reaction was incubated in a constant temperature shaking incubator at 37° C. and 200 rpm for 3 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization, with a conversion rate of 3.37%.

[0105] Example 17

[0106] Enzymatic Synthesis of Propyl Ferulate

[0107] In this example, the crude enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and propanol was used as an acyl acceptor to synthesize propyl ferulate.

[0108] Methyl ferulate and propanol (methyl ferulate added in an amount of 12 mg and propanol added in an amount of 25 μL) were added to a 100 mM Tris-HCl buffer at pH 8.5. 30 μL of the crude enzyme solution was added to a reaction system of 250 μL. The reaction was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 3 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization, with a conversion rate of 29.86%.

[0109] Example 18

[0110] Enzymatic Synthesis of Butyl Ferulate

[0111] In this example, the crude enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and butanol was used as an acyl acceptor to synthesize butyl ferulate.

[0112] Methyl ferulate and butanol were added to 100 mM Tris-HCl buffer at pH 8.5 (methyl ferulate added in an amount of 12 mg, butanol added in an amount of 25 μL). 30 μL of the crude enzyme solution was added to a reaction system of 250 μL. The reaction was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 3 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization method. The conversion rate was 48.75%.

[0113] Example 19

[0114] Enzymatic Synthesis of Octyl Ferulate

[0115] In this example, the crude enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and octanol was used as an acyl acceptor to synthesize octyl ferulate.

[0116] Methyl ferulate and octanol were added to 100 mM Tris-HCl buffer at pH 8.5 (methyl ferulate added in an amount of 12 mg, octanol added in an amount of 25 μL). 30 μL of the crude enzyme solution was added to a reaction system of 250 μL. The reaction was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 3 h. The reaction solution was treated as in Example 2, and the product yield was detected by normalization, with a conversion rate of 13.18%.

[0117] Example 20

[0118] Enzymatic synthesis of trans-4-hydroxyphenylethyl ferulate using DMSO as a cosolvent.

[0119] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, trans-methyl ferulate was used as an acyl donor, and p-hydroxyphenylethanol was used as an acyl acceptor to synthesize trans-p-hydroxyphenylethyl ferulate.

[0120] In a 100 mM Tris-HCl buffer at pH 8.5, trans-ferulic acid methyl ester and p-hydroxyphenylethanol (trans-ferulic acid methyl ester 50 mM, p-hydroxyphenylethanol final concentration 810 mM) were added with 10% v / v DMSO, and finally, the purified enzyme solution was added to an enzyme content of 0.4 mg / mL. The reaction was shaken in a constant temperature shaking incubator at 35° C. and 200 rpm for 4 h. The reaction solution was treated as in Example 2 and the product yield was detected by normalization. The conversion rate was 22.69%, which was 2.6 times the yield when no cosolvent was added (Example 9).

[0121] Example 21

[0122] Enzymatic synthesis of phenyl β-D-glucoside ferulate using DMSO as a cosolvent.

[0123] In this example, the pure enzyme prepared in Example 1 was used as a catalyst, methyl ferulate was used as an acyl donor, and phenyl-β-D-glucoside was used as an acyl acceptor to synthesize phenyl β-D-glucoside ferulate.

[0124] Methyl ferulate and phenyl-β-D-glucoside were added to 100 mM Tris-HCl buffer at pH 8.5 (final concentration of methyl ferulate was 250 mM, final concentration of phenyl-β-D-glucoside was 810 mM), 10% v / v DMSO was added, and purified enzyme solution was added to an enzyme content of 0.4 mg / mL. The reaction was shaken in a constant temperature shaking incubator at 35° C. and 200 rpm for 4 h. The reaction solution was treated as in Example 2 and the product yield was detected by normalization. The conversion rate was 5.8%, and the yield was 3.6 times that when no cosolvent was added (Example 14).

[0125] Example 22

[0126] Preparation of Feruloyl Esterase Mutant S67I

[0127] AmCE1 was site-directed mutagenesis performed by inverse PCR to prepare single and double mutant libraries.

[0128] The feruloyl esterase AmCE1 from Anaeromyces mucronatus (gene sequence shown in SEQ ID NO: 1, encoded protein sequence shown in SEQ ID NO: 2) was amplified by PCR using primers A (SEQ ID NO: 3) and B (SEQ ID NO: 4), respectively. The PCR products were then digested and inserted into the Nde I and Xho I sites of the expression vector pET30a(+) to obtain the recombinant plasmid pET-30a(+)-AmCE1.

[0129] According to the described mutation position S67 of embodiment 1, reverse primer (mutation primer sequence is as shown in Table 1) is designed at mutation position S67, utilize upstream and downstream mutation primer amplification target fragment, and on primer, introduce corresponding sudden change, carry out inverse PCR with recombinant plasmid pET-30a (+)-AmCE1 as template, PCR product is after the Dpn I enzymatic digestion template is handled, the plasmid DNA of use is extracted from the Escherichia coli host, because endogenous dam methylase, the adenine of this sequence has been methylated, therefore can be cut off by DMT.And with synthetic DNA such as PCR owing to not methylated, therefore can not be cut off, eliminate false positive and be transformed into Escherichia coli BL21 (DE3), send order-checking through picking bacterium colony after the screening of Kan. After measuring correctly, obtain the recombinant bacterium that suddenlys change successfully, the recombinant bacterium that suddenlys change successfully is carried out induction expression, can obtain the feruloyl esterase mutant. The system of above-mentioned inverse PCR is:

[0130] PrimeSTAR® GXL DNA Polymerase 0.25μL

[0131] 5XPS Buffer 5μL

[0132] Template plasmid pET-30a(+)-AmCE1 1 μL

[0133] dNTP 2 μL

[0134] Upstream of mutation primer (Table 2) 0.5 μL

[0135] Downstream of mutation primer (Table 2) 0.5 μL

[0136] Make up to 25 μL with sterile water.

[0137] The PCR reaction program is:

[0138] 95℃, 2 min; 98℃, 10 s, 50-65℃, 30 s, 34 cycles; 72℃, 7 min; 72℃, 10 min.

[0139] Table 1 Mutation primers for mutation sites

[0140] Primer name Serial number Primer sequence (5'-3') S67I Upstream NO:3 TctgatgatttatgaagattccatgctggaggacGACT S67I downstream NO:4 ATCTTCATAaaTCATCAGaccatgcaggtAGTAGACAAC

[0141] Example 23

[0142] Determination of transacylation / hydrolysis rates of feruloyl esterase mutants

[0143] In this example, the S67I mutant prepared by the method of Example 22 was used. After expansion, the culture was transferred to a shake flask. The recombinant strain was added to LB liquid medium containing kanamycin at a 5% addition rate and cultured at 220 rpm and 37°C. When the OD600 reached 0.5 to 0.6, the strain was induced with IPTG (1 mM) at 20°C for 20 hours, and the crude enzyme was collected by centrifugation.

[0144] Methyl ferulate and phenylethanol (200 mg of methyl ferulate and 100 μL of phenylethanol) were added to 800 μL of 50 mM PBS buffer at pH 7.4. Finally, 100 μL of crude enzyme solution was added and the reaction mixture was allowed to stand at room temperature for 4 hours. Acetonitrile was added to completely dissolve the reaction mixture. The reaction mixture was treated as in Example 3, and the product yields of phenylethyl ferulate and ferulic acid were determined using the normalization method. S67I exhibited a higher acyl transfer and hydrolysis rate, 2.19 times that of the wild-type, demonstrating that S67I is more efficient in utilizing the substrate to synthesize phenylethyl ferulate.

Claims

1. A ferulic acid esterase AmCE1 mutant, characterized in that The amino acid sequence is obtained by mutating the serine S at position 67 to isoleucine I as shown in SEQ ID NO 2.

2. A method for synthesizing phenylpropenyl esters by aqueous phase catalysis using ferulic acid esterase, characterized in that: Methyl ferulate, methyl cinnamate, methyl caffeate, methyl p-coumarate, or trans-methyl ferulate is used as an acyl donor, and benzyl alcohol, phenylethanol, phenylpropanol, phenylbutanol, p-hydroxyphenylethanol, 4-hydroxy-3-methoxybenzyl alcohol, aribonucleic acid, naphthylethanol, phenyl-β-D-glucoside, arbutin, phenyl-β-D-galactopyranoside, propanol, butanol, or octanol is used as an acyl acceptor, catalyzed by wild-type feruloyl esterase AmCE1, to produce phenylpropenyl ester. The reaction temperature is 35° C. The substrate and feruloyl esterase are removed from the catalytic reaction system, and the remainder is Tris-HCl buffer. A cosolvent for dissolving the substrate may or may not be added to the reaction system. The pH value of the catalytic reaction system is 8.

5. The rotation speed of the catalytic reaction system is 200 rpm. The reaction steps are: (1) Heterologous expression and acquisition of ferulic acid esterase AmCE1 The gene sequence of ferulic acid esterase AmCE1 used for fermentation preparation of catalytic enzyme is shown in SEQ ID NO. 1, and the amino acid sequence encoded by it is shown in SEQ ID NO.

2. The whole gene was synthesized by GenScript Biotechnology Co., Ltd., and after gene synthesis, it was ligated with the expression vector pET-30a(+) and transformed into E. coli BL21(DE3) for protein expression. The successfully transformed expression strain was screened using a kanamycin-resistant culture medium. After expansion culture, it was transferred to a shake flask culture. The recombinant strain was added to LB liquid culture medium containing kanamycin at a dosage of 5%, and cultured at 220 rpm and 37°C. When the OD600 was 0.5 to 0.6, IPTG was added to a final concentration of 1 mM, and expression was induced at 20°C and 220 rpm for 20 hours. The bacteria were collected by centrifugation at 6000 rpm for 10 minutes, and 20 mL of 50 mM pH 7.4 was used. Resuspend the cells in PBS, disrupt the cells by ultrasonication at 360 kW for 40 min, and then centrifuge at 12,000 rpm for 30 min to remove cell debris. The supernatant was filtered through a 0.22 μm filter membrane and stored at -20°C as crude enzyme for later use. Recombinant AmCE1 is tagged with a 6His tag at its amino terminus, so purification is simple using Ni-NTA affinity column chromatography. The crude enzyme solution is directly loaded onto the Ni-NTA affinity column, and the column is washed with a wash buffer containing 3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 500 mM NaCl, and 10 mM imidazole at pH 7.4; the recombinant AmCE1 bound to the column is eluted with an elution buffer containing 3.8 mM NaH2PO4, 16.2 mM Na2HPO4, 500 mM NaCl, and 88.4 mM imidazole at pH 7.4, and the protein from the eluted fraction is dialyzed to remove excess imidazole. The purity of the purified recombinant AmCE1 is checked by SDS-PAGE electrophoresis, and the pure enzyme is stored at -20°C for subsequent use. (2) Enzymatic catalysis An acyl donor (methyl phenylpropenyl ester or ethyl phenylpropenyl ester) and an acyl acceptor alcohol are added to a buffer solution, followed by the addition of crude or purified AmCE1 enzyme. The substrate and ferulic acid esterase are removed from the catalytic reaction system, leaving a Tris-HCl buffer solution as the remainder. A cosolvent for dissolving the substrate may or may not be added to the reaction system. The catalytic reaction temperature is 35° C., the pH value of the catalytic reaction system is 8.5, and the rotation speed of the catalytic reaction system is 200 rpm. After the reaction, the acyl donor removes methanol or ethanol, the acyl acceptor removes hydrogen from the alcohol, and reacts with the remaining phenylpropenyl group of the acyl donor, thereby replacing the methyl or ethyl ester in the original substrate with alcohols containing a benzene ring or a naphthyl group and a straight-chain fatty alcohol to generate a new phenylpropenyl ester.

3. The method for synthesizing phenylpropenyl ester by aqueous phase catalysis of ferulic acid esterase according to claim 2, characterized in that: The selected substrates used methyl ferulate, methyl cinnamate, methyl caffeate, methyl p-coumarate or trans-methyl ferulate as acyl donors, and benzyl alcohol, phenylethanol, phenylpropanol, phenylbutanol, p-hydroxyphenylethanol, 4-hydroxy-3-methoxybenzyl alcohol, aribonucleic acid, naphthylethanol, phenyl-β-D-glucoside, arbutin, phenyl-β-D-galactopyranoside, propanol, butanol or octanol as acyl acceptors.

4. The method for synthesizing phenylpropenyl ester by aqueous phase catalysis of ferulic acid esterase according to claim 2, characterized in that: The cosolvent is 1% to 10% v / v DMSO.

5. The method for synthesizing phenylpropenyl ester by aqueous phase catalysis of ferulic acid esterase according to claim 2, characterized in that: Benzyl ferulate, phenethyl ferulate, phenpropyl ferulate and phenbutyl ferulate were synthesized catalytically using methyl ferulate as the acyl donor and phenyl alcohols such as benzyl alcohol, phenylethanol, phenylpropanol and phenylbutanol as the acyl acceptors.

6. The method for synthesizing phenylpropenyl ester by aqueous phase catalysis of ferulic acid esterase according to claim 2, characterized in that: Phenethyl cinnamate, phenylethyl caffeate and phenylethyl p-coumarate were synthesized using methyl cinnamate, methyl caffeate and methyl p-coumarate as acyl donors and phenylethanol as acyl acceptor.

7. The method for synthesizing phenylpropenyl ester by aqueous phase catalysis of ferulic acid esterase according to claim 2, characterized in that: Trans-ferulic acid p-hydroxyphenylethyl ester was synthesized using trans-ferulic acid methyl ester as acyl donor and p-hydroxyphenylethanol as acyl acceptor.

8. The method for synthesizing phenylpropenyl ester by aqueous phase catalysis of ferulic acid esterase according to claim 2, characterized in that: Ferulate p-hydroxyphenylethyl ester and 4-hydroxy-3-methoxybenzyl ferulate were synthesized with methyl ferulate as acyl donor and hydroxyphenylethanol and 4-hydroxy-3-methoxybenzyl alcohol as acyl acceptors.

9. The method for synthesizing phenylpropenyl esters by aqueous phase catalysis of ferulic acid esterase according to claim 2, characterized in that: Alibendol ferulate was synthesized using methyl ferulate as the acyl donor and alibendol as the acyl acceptor.

10. The method for synthesizing phenylpropenyl esters by aqueous phase catalysis using ferulic acid esterase according to claim 2, characterized in that: Naphthyl ferulate was synthesized using methyl ferulate as the acyl donor and naphthaleneethanol as the acyl acceptor.

11. The method for synthesizing phenylpropenyl esters by aqueous phase catalysis using ferulic acid esterase according to claim 2, characterized in that: Phenyl-β-D-glucoside ferulate, arbutin ferulate and phenyl-β-D-galactopyranoside ferulate were synthesized using methyl ferulate as an acyl donor and glycosides with a phenyl group, namely phenyl-β-D-glucoside, arbutin and phenyl-β-D-galactopyranoside, as acyl acceptors.

12. The method for synthesizing phenylpropenyl esters by aqueous phase catalysis using ferulic acid esterase according to claim 2, characterized in that: Propyl ferulate, butyl ferulate and octyl ferulate were synthesized catalyzed by using methyl ferulate as the acyl donor and straight-chain alcohols such as propanol, butanol and octanol as the acyl acceptors.

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