Method for catalyzing grease hydrolysis decarboxylation by cell-like multiphase emulsion optical enzyme system
阅读说明：本技术 一种类细胞多相乳液光酶体系催化油脂水解脱羧的方法 (Method for catalyzing grease hydrolysis decarboxylation by cell-like multiphase emulsion optical enzyme system ) 是由 李志刚 梁倩 杨博 陈华勇 马佩瑶 蔡璐遥 邓佩柔 于 2021-07-27 设计创作，主要内容包括：本发明属化学生产和化学合成领域,公开了一种类细胞多相乳液光酶体系催化油脂水解脱羧的方法。向脂肪酶液中加入可溶性盐、聚合物和/或亲水醇、脂肪酸光脱羧酶和油脂,配成类细胞多相乳液体系；或者将可溶性盐、聚合物和/或亲水醇、脂肪酸光脱羧酶和脂肪酸混合均匀配成类细胞多相乳液体系；类细胞多相乳液体系于搅拌条件下,蓝光照射下进行酶催化反应,反应结束后,加入乙酸乙酯萃取,静置或离心至分为三层,收集上层液产物为水解及脱羧后产物。脂肪酶可催化甘油酯的深度水解,脂肪酸光脱羧酶催化游离脂肪酸的脱羧,可实现反应快速、智能控制的同时,解除底物抑制,建立一种高效、可控、酶易回收且产物可同步分离的新型多酶联用催化体系。(The invention belongs to the field of chemical production and chemical synthesis, and discloses a method for catalyzing hydrolysis decarboxylation of oil by a cell-like multiphase emulsion optical enzyme system. Adding soluble salt, polymer and/or hydrophilic alcohol, fatty acid light decarboxylase and grease into lipase liquid to prepare a cell-like multiphase emulsion system; or mixing soluble salt, polymer and/or hydrophilic alcohol, fatty acid light decarboxylase and fatty acid uniformly to prepare a cell-like multiphase emulsion system; and (3) carrying out enzyme catalysis reaction under blue light irradiation of the cell-like multiphase emulsion system under the stirring condition, adding ethyl acetate for extraction after the reaction is finished, standing or centrifuging until the reaction is divided into three layers, and collecting the upper layer liquid product as a product after hydrolysis and decarboxylation. The lipase can catalyze deep hydrolysis of glyceride, the decarboxylation of free fatty acid is catalyzed by the fatty acid light decarboxylase, the substrate inhibition is removed while the reaction is fast and intelligently controlled, and a novel multi-enzyme combined catalytic system which is efficient and controllable, easy to recover enzyme and synchronous in product separation is established.)
1. A method for catalyzing grease hydrolysis decarboxylation by a cell-like heterogeneous emulsion photo-enzyme system is characterized by comprising the following steps:
(1) preparation of a cell-like multiphase emulsion System
Adding soluble salt, polymer and/or hydrophilic alcohol, fatty acid light decarboxylase and grease into lipase liquid to prepare a cell-like multiphase emulsion system;
or mixing soluble salt, polymer and/or hydrophilic alcohol, fatty acid light decarboxylase and fatty acid uniformly to prepare a cell-like multiphase emulsion system;
(2) carrying out enzyme catalysis reaction on a cell-like multiphase emulsion system under the irradiation of blue light under the stirring condition, adding ethyl acetate for extraction after the reaction is finished, standing or centrifuging until the reaction is divided into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer from top to bottom, and collecting an upper liquid product which is a product after hydrolysis and decarboxylation.
2. The method according to claim 1, wherein the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the oil and fat, the fatty acid light decarboxylase and the lipase in the step (1) is (0.1-20): (0.1-20): (0.1-10): (0.001-20): 1;
or the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the fatty acid light decarboxylase and the fatty acid in the step (1) is (0.1-20): (0.1-20): (0.002-20): 1.
3. the method according to claim 2, wherein the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the oil and fat, the fatty acid light decarboxylase and the lipase in the step (1) is (0.2-10): (0.2-10): (0.2-4): (0.005-15): 1;
or the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the fatty acid light decarboxylase and the fatty acid in the step (1) is (0.2-10): (0.2-10): (0.005-12): 1.
4. the method according to claim 1 or 2 or 3, wherein the polymer and/or hydrophilic alcohol of step (1) is one or more of polyethylene glycol, polypropylene glycol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, or ethylene glycol.
5. The method of claim 4, wherein the polymer and/or hydrophilic alcohol of step (1) is polyethylene glycol 400 or polyethylene glycol 600.
6. The method according to claim 5, wherein the soluble salt in the step (1) is one or more of sodium citrate, sodium chloride, sodium sulfate, ammonium sulfate, sodium carbonate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
7. The method according to claim 1, wherein the oil or fat in step (1) is one or more of edible oil, non-edible oil and waste oil or fat.
8. The method according to claim 1, wherein the lipase liquid in step (1) is further added with phospholipid, and the mass ratio of phospholipid to lipase liquid is (0.1-10): 1.
9. the method of claim 1, wherein the pH of the multiple emulsion system of step (1) is from 6.5 to 10.5; the reaction conditions of the step (2) are as follows: the temperature is 30-37 ℃, and the reaction time is 2-48 h.
10. The method according to claim 1, wherein the lipase in step (1) is lipase AYS with a concentration of 0.005g/mL-0.25 g/mL; the fatty acid light decarboxylase is artificially fermented whole cells or simply purified crude enzyme or purified pure enzyme, and the concentration is 0.0002g/mL-1.0 g/mL.
Technical Field
The invention belongs to the field of chemical production and chemical synthesis, relates to a separation and application technology of enzyme, and particularly relates to a method for catalyzing hydrolysis decarboxylation of oil by a cell-like multiphase emulsion optical enzyme system.
Background
Alk (en) enes are compounds that play an important role in chemical production and chemical synthesis, belong to non-renewable resources, and are only available in short term by means of oil and gas production. Petroleum is a non-renewable resource and its reserves are very limited. The conventional alkane (alkene) preparation method generally adopts metal ion catalyst, high pressure and H2Under the conditions of (1). Compared with the traditional chemical catalysis, the enzyme catalysis has the advantages of low energy consumption, low pollution, mild reaction conditions, high selectivity and the like, but needs to be powered by expensive NADPH. In recent years, Beisson and a team thereof find a light-powered fatty acid light decarboxylase (CvFAP) (Beisson, F.science.2017,357,903-907.) from a unicellular chlorella viridis variant strain NC64A, the enzyme catalysis is dominant, a coupled lipase catalysis technology catalyzes hydrolysis decarboxylation of grease to generate corresponding alkane or olefin under the irradiation of blue light, only simple light power is needed, and the coupled lipase catalysis technology has outstanding advantages in the aspects of improving yield, reducing catalysis cost, reducing pollution emission and the like, and is one of the most promising directions in the fields of green chemistry and catalysis. The enzyme catalysis reaction and the decarboxylation reaction of the fatty acid are the research hotspots which are currently concerned globally, and are important ways for realizing high-value utilization of oil-replacing fuel and waste resources.
The fatty acid light decarboxylase is a biological enzyme only depending on photocatalysis, and the reaction process must have light, so the light transmission of the system is a key factor for limiting the application of the biological enzyme, and the traditional immobilized carrier catalytic systems have poor light transmission and limit the immobilization of the fatty acid light decarboxylase, so an immobilization method for effectively recycling the enzyme is not reported. The lipase matched with the cascade reaction is interfacial enzyme, a large interfacial area is needed to exert the effective catalytic efficiency, most of the existing catalytic systems are difficult to simultaneously meet the catalytic requirements (high light transmittance and catalytic interface) of two special enzymes, and the traditional thinking is urgently needed to be broken out, and a novel catalytic system matched with the lipase is constructed.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a method for catalyzing the hydrolysis and decarboxylation of oil by a cell-like multiphase emulsion photo-enzyme system, lipase and fatty acid photo-decarboxylase CvFAP are simultaneously added into the multiphase emulsion system, co-expression is carried out in a host system for co-immobilization, the lipase catalyzes deep hydrolysis of glyceride, the fatty acid photo-decarboxylase CvFAP catalyzes the decarboxylation of free fatty acid, natural/waste oil is converted into alkane (alkene) hydrocarbon, and a new thought is provided for developing a new generation of biofuel synthesis technology.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for catalyzing grease hydrolysis decarboxylation by a cell-like heterogeneous emulsion photo-enzyme system comprises the following steps:
(1) preparation of a cell-like multiphase emulsion System
Adding soluble salt, polymer and/or hydrophilic alcohol, fatty acid light decarboxylase and grease into lipase liquid to prepare a cell-like multiphase emulsion system;
or mixing soluble salt, polymer and/or hydrophilic alcohol, fatty acid light decarboxylase and fatty acid uniformly to prepare a cell-like multiphase emulsion system;
(2) carrying out enzyme catalysis reaction on a cell-like multiphase emulsion system under the irradiation of blue light under the stirring condition, adding ethyl acetate for extraction after the reaction is finished, standing or centrifuging until the reaction is divided into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer from top to bottom, and collecting an upper liquid product which is a product after hydrolysis and decarboxylation.
Preferably, the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the grease, the fatty acid light decarboxylase and the lipase in the step (1) is (0.1-20): (0.1-20): (0.1-10): (0.001-20): 1;
or the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the fatty acid light decarboxylase and the fatty acid in the step (1) is (0.1-20): (0.1-20): (0.002-20): 1.
preferably, the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the grease, the fatty acid light decarboxylase and the lipase in the step (1) is (0.2-10): (0.2-10): (0.2-4): (0.005-15): 1;
or the mass ratio of the soluble salt, the polymer and/or the hydrophilic alcohol, the fatty acid light decarboxylase and the fatty acid in the step (1) is (0.2-10): (0.2-10): (0.005-12): 1.
preferably, the polymer and/or hydrophilic alcohol in step (1) is one or more of polyethylene glycol, polypropylene glycol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, or ethylene glycol.
Preferably, the polymer and/or hydrophilic alcohol of step (1) is polyethylene glycol 400 or polyethylene glycol 600.
Preferably, the soluble salt in step (1) is one or more of sodium citrate, sodium chloride, sodium sulfate, ammonium sulfate, sodium carbonate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
Preferably, the oil and fat in the step (1) is one or more of edible oil, non-edible oil and waste oil and fat.
Preferably, the lipase liquid in the step (1) is also added with phospholipid, and the mass ratio of the phospholipid to the lipase liquid is (0.1-10): 1.
preferably, the pH value of the multi-phase emulsion system in the step (1) is 6.5-10.5; the reaction conditions of the step (2) are as follows: the temperature is 30-37 ℃, and the reaction time is 2-48 h.
Preferably, the lipase in the step (1) is lipase AYS with the concentration of 0.005g/mL-0.25 g/mL; the fatty acid light decarboxylase is artificially fermented whole cells or simply purified crude enzyme or purified pure enzyme, and the concentration is 0.0002g/mL-1.0 g/mL.
The invention has the following beneficial effects:
in the process of exploring and researching a multi-liquid-phase system, the catalytic efficiency of the multi-liquid-phase system is improved greatly when the hydrophilic solvent forming the multi-liquid-phase system is a polymer or hydrophilic alcohol; when surfactant such as phospholipid is added into the system, the catalytic efficiency can be greatly improved. Through the study on the microstructure, we found that the formation of an emulsion system similar to the structure of biological cells during stirring (as shown in fig. 9) is the key to the high catalytic efficiency. In this configuration, the polymer or hydrophilic alcohol rich phase will coat the hydrophobic phase, suspending in the salt rich phase (FIG. 9a), forming a structure similar to that of the cell in which the cytoplasm coats the nucleus, while the phospholipid forms a membrane-like structure coating the polymer or hydrophilic alcohol rich phase forming a structure similar to that of the cell membrane. In this configuration, the biological enzymes are concentrated in a "cytoplasm-like" polymer or hydrophilic alcohol rich phase, which is more catalytically active and more stable.
A catalytic reaction system similar to a biological liquid environment, namely a 'cell-like multiphase emulsion' multi-enzyme catalytic system, is found and constructed, and is a newly developed enzyme catalytic system. The transfer rate of the reaction substance between each phase is greatly reduced due to the low surface tension, so that the transfer rate is improved; meanwhile, a biological cell-like environment can be provided for the biological enzyme, the biological stability of the enzyme is effectively maintained, the catalytic activity of the enzyme is maintained, the recycling of the enzyme is facilitated, the relative activity of the enzyme after 7 batches is maintained at 65%, and the production cost is greatly reduced. And the separation of the product and the substrate can be realized by utilizing the property difference between the product and the substrate, thereby facilitating the subsequent process treatment. The catalyst and the product can be distributed in different phases by regulating and controlling parameters, so that the product inhibition effect is weakened, the reaction rate is accelerated, the forward progress of the reaction is promoted, and the conversion rate can reach 98.9 percent. The hydrolysis conversion rate of the emulsion system is higher than that of the traditional system, and the oil and water in the traditional system can be emulsified to influence the light transmittance of the system, so that the conversion rate is influenced.
The combined use of the 'cell-like multiphase emulsion' multienzyme catalytic system and the cascade catalytic technology provides scientific basis for further solving the problems of low efficiency, high carbon emission, high energy consumption and the like which are urgently to be solved in the fields of energy regeneration and high-valued utilization of waste resources, and provides basic data support for expanding the application of the cascade catalytic technology of the enzyme.
Drawings
FIG. 1(a) shows the results of GC detection of the hydrolyzed product of the emulsion system of example 1; FIG. 1(b) shows the results of GC analysis of the product of the conventional system of example 1.
FIG. 2(a) shows the results of GC detection of the hydrolyzed product of the emulsion system of example 2; FIG. 2(b) shows the results of GC analysis of the product of the conventional system of example 2.
FIG. 3(a) shows the results of GC detection of the hydrolyzed product of the emulsion system of example 3; FIG. 3(b) shows the GC analysis of the product of the conventional system of example 3.
FIG. 4(a) is the GC test result of the hydrolyzed product of the phospholipid-added emulsion system of example 4; FIG. 4(b) shows the results of GC detection of the hydrolyzed emulsion; FIG. 4(c) shows the GC detection results of the hydrolyzed ionic liquid based multi-liquid phase emulsion system of example 4.
FIG. 5(a) shows the results of GC detection of the hydrolyzed product of the emulsion system of example 5; FIG. 5(b) shows the GC analysis of the product of the conventional system of example 5.
FIG. 6(a) shows the results of GC detection of the hydrolyzed product of the emulsion system of example 6; FIG. 6(b) shows the GC analysis of the product of the conventional system of example 6.
FIG. 7(a) shows the results of GC detection of the hydrolyzed product of the emulsion system of example 7; FIG. 7(b) shows the GC analysis of the product of the hydrolysis of the conventional system of example 7.
FIG. 8(a) shows the GC assay results for the first batch of hydrolysis products of example 8, and FIG. 8(b) shows the GC assay results for the third batch of hydrolysis products of example 8.
FIG. 9 is the microstructure of the emulsion system, FIG. 9a is the microscopic observation result of the system, the green color is the phospholipid layer, the oil phase is wrapped in the middle, and the enzyme is at the interface; FIG. 9b shows the oil in red, and the background in blue; FIG. 9c yellow shows the product of the upper phase with unreacted oil, blue shows the intermediate Phase (PEG), and the white membrane at the interface is phospholipid and lipase. Two test tubes are respectively added with different concentrations of phase-forming salt, the left side is low concentration, and the right side is high concentration.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Lipase AYS (Lipase AYS) is available from Amano wild enzyme preparation, commercial Co., Ltd.
The gene sequence of the fatty acid light decarboxylase is as follows:
TTGGTTTAAGCAGCCGGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTAAGCAGCAACGGTAGCCGGTGCAGCAGCGCTAGCACCGATGGTCGCTTTGCCGGTCAGTAAAGCAGCCGCACGTTCAGCAATCATAACTACCGGAGCACCAGTCTGACCGCCCGGAATTTTAGGTACTACAGAAGCGTCCACGACACGCAGACCTTCCACACCATGAACACGCAGCTGGTTATCAACAACGCTAGAGCTATCACCTGCGTTACCCATTTTGCAGGTACCAGTGATTGCGTTGCTAGAGTGAATGCTACGACGGATATATTCATCGATCTGGTCATCAGATACAACACCGGAACCTGGGAATAGTTCACCGTCCAGGTATTCAGACAGCGCAGAGGAACGTGCCACGTCACGCGCCCAGTGGATACCTTTACGCAGGGTAGCCAGGTCCGCGCCGTCTTTATCGGTCAGGTAGCCCGGAGACAGTTTCGGCGGAGCGAACGGATCAGCAGATTTCAGACCAACGGAACCAGTAGACTGCGGACGGCAGGCGATCAGCTGCATAGTGATACCAGAAGGCCATTTAAGACCCTGAGACTGGAATTTTGCGAAACGCACGTAGGTGGATACGCCGTCCGGGTCCAGTGCCATGCCCGGCACGAAACGTACTTGTAAATCGGGCAATGCCTGACCGGCGGTGCGAACGAAGGCACCACGGTCACAACCGGTGCTAGTCAGGCCACCACGACCGCCCAACAGGTAGCTCGCGATCGCGCGTTTGCGAATCTGGCCTTTTTCGTTATAAATGTGGTCGCTGATAGCGATACCATCGTATTTTTCTTTAACCGGTGCTGCAGTCAGGCATGCCGGCTGATCCTGCAGGTTCTGACCAACACCGGGCCAGATTGGACACACCGGGATACCGAATTCCTTCAGTTCAGCAGACGGACCAACACTGAGTGTTTCAGTAGAACGGGGTGTTGCACCGCACAGCGCACATGATCACTTCACCACCCGGCGCAGTTCGCAGACGACGTTCGCGGTCGGACGTCGGTGCTGACTTCAACACCCAGGTGCTGGTGCTTTACTGGCGGCCTGGTCGATGTTAACC
the gene is transferred into escherichia coli, and the method comprises the following steps:
1) taking commercial competent cell TOP 10, and placing on ice until the cell is dissolved; the CvFAP plasmid was also chilled in ice, 10. mu.L of plasmid was mixed well with 100. mu.L of TOP 10 competent cells and placed on ice for 30 min.
(2) And (3) thermally shocking the mixture subjected to ice bath at 42 ℃ for 90 seconds, and rapidly placing the mixture on ice for 2-3 minutes.
(3) The heat-shocked cells were added to 0.5mL of LB medium and activated for 1 hour at 37 ℃ and 200rpm in a shaker.
(4) Centrifuging the activated cells, removing the supernatant, adding a small amount of LB culture medium to resuspend the cells, coating the cells on a solid LB resistant plate, making a mark, inversely placing the plate in a constant-temperature incubator at 37 ℃ for culturing for 12 hours, and transferring the plate into a test tube for fermentation.
120 g of Escherichia coli whole cell is crushed and extracted, and then concentrated to obtain about 50mL of protein solution (crude enzyme solution), wherein the concentration of the crude enzyme solution is 14mg/mL (determined by protein concentration), the light decarboxylase accounts for about 10 percent, and about 70mg of the light decarboxylase, namely about 0.58mg of the light decarboxylase is contained in each gram of Escherichia coli whole cell.
The amount of enzyme required to consume 1umol of fatty acid per minute at 37 ℃ is defined as 1 decarboxylase activity unit U.
X is decarboxylase activity U/g; c is fatty acid concentration mM; v is the volume mL of the fatty acid solution; m is the amount of enzyme or the amount of whole cells g.
According to calculation, the crude enzyme liquid enzyme activity is 347U/g, and the whole cell enzyme activity of the escherichia coli is 808U/g.
Example 1
0.05g of lipase AYS is put into a transparent glass bottle, 0.24g of dipotassium phosphate, 1mL (0.975g) of Tris-HCl (100mM, pH 8.5) solution is mixed uniformly, 1.5mL (about 0.6g) of escherichia coli whole cell (containing CvFAP) is added for mixing uniformly, 0.233g of polyethylene glycol 400 and 0.05g of waste cooking oil are added, the mixture is put into a sandwich beaker wound with an LED lamp device capable of generating blue light and placed on a constant-temperature stirrer with the rotating speed of 1000rpm, and the reaction is controlled at 30 ℃ for 12 hours. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, wherein free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction are mainly enriched in the upper liquid layer after hydrolysis, collecting the upper liquid layer product as a product, and measuring the obtained product by GC (shown in figure 1a), wherein the conversion rate can reach 98.9%, and the product is mainly alkane of C11-C17.
Adding lipase AYS and Tris-HCl buffer solution with the same mass into a blank glass bottle, uniformly mixing 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP), adding 0.05g of waste cooking oil, reacting for 12 hours under the same condition, adding ethyl acetate with the volume 2 times that of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3 minutes, dividing the mixture into two layers, namely an upper liquid layer and a lower liquid layer, enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction into the upper liquid layer, and collecting the product of the upper liquid layer, namely the product. The product obtained, as determined by GC (FIG. 1b), had a conversion of only 37%.
Example 2
0.05g of lipase AYS is placed in a transparent glass bottle, 0.30g of sodium sulfate, 1mL (0.975g) of Tris-HCl (100mM, pH 8.5) solution is mixed, 1.5mL (about 0.6g) of Escherichia coli whole cell (containing CvFAP) is added after mixing, 0.279g of polyethylene glycol 600 and 0.05g of camellia oil are added, the mixture is placed in a sandwich beaker wound with an LED lamp device capable of generating blue light and placed on a constant temperature stirrer with the rotating speed of 1000rpm, and reaction is controlled at 30 ℃ for 12 hours. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, wherein free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction are mainly enriched in the upper liquid layer after hydrolysis, collecting the upper liquid layer product, namely a product, and measuring the obtained product by GC (shown in figure 2a), wherein the conversion rate can reach 95%, and the product is mainly alkane of C11-C17.
Adding lipase AYS and Tris-HCl buffer solution with the same mass into a blank glass bottle, uniformly mixing 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP), adding 0.05g of camellia oil, reacting for 12h under the same condition, adding ethyl acetate with the volume 2 times that of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into two layers, namely an upper liquid layer and a lower liquid layer, enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction into the upper liquid layer, and collecting the product of the upper liquid layer, namely the product. The product obtained, as determined by GC (FIG. 2b), had a conversion of only 76%.
Example 3
0.05g of lipase AYS is put into a transparent glass bottle, 0.24g of dipotassium phosphate and 1mL (0.975g) of Tris-HCl (100mM, pH 8.5) solution are mixed uniformly, 1.5mL (about 0.6g) of escherichia coli whole cell (containing CvFAP) is added into the mixture to be mixed uniformly, 0.285g of polyethylene glycol 400 and 0.05g of soybean oil are added into a sandwich beaker wound with an LED lamp device capable of generating blue light, the beaker is placed on a constant-temperature stirrer with the rotating speed of 1000rpm, and the reaction is controlled at 30 ℃ for 12 hours. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, mainly enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction in the upper liquid layer after hydrolysis, and collecting the product of the upper liquid layer, namely the product. The conversion rate of the obtained product can reach 82 percent through GC (shown in figure 3a), and the product is mainly C11-C17 alkane.
Adding lipase AYS and Tris-HCl buffer solution with the same mass into a blank glass bottle, uniformly mixing 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP), adding 0.05g of soybean oil, reacting for 12h under the same condition, adding ethyl acetate with the volume 2 times that of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into two layers, namely an upper liquid layer and a lower liquid layer, enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction into the upper liquid layer, and collecting the product of the upper liquid layer, namely the product. The product obtained was determined by GC (FIG. 3b) to have a conversion of only 53%.
Example 4
Putting 0.05g of lipase AYS into a transparent glass bottle, putting 0.24g of dipotassium phosphate, 0.5mL (0.487g) of Tris-HCl (100mM, pH 8.5) solution and 0.05g of phospholipid, uniformly mixing, adding 0.487g of crude enzyme solution (containing CvFAP), uniformly mixing, adding 0.285g of polyethylene glycol 400 and 0.05g of waste cooking oil, putting into a sandwich beaker wound with an LED lamp device capable of generating blue light, placing on a constant-temperature stirrer with the rotating speed of 1000rpm, and reacting for 12 hours at the temperature of 30 ℃. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, wherein free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction are mainly enriched in the upper liquid layer after hydrolysis, collecting the upper liquid layer product, namely a product, and measuring the obtained product by GC (shown in figure 4a), wherein the conversion rate can reach 65%, and the product is mainly alkane of C11-C17.
And adding Tris-HCl buffer solution, dipotassium phosphate, lipase AYS and crude enzyme liquid (containing CvFAP) with the same mass into a blank glass bottle, uniformly mixing, adding 0.285g of polyethylene glycol 400 and waste cooking oil, filling into an interlayer beaker wound with an LED lamp device capable of generating blue light, placing on a constant-temperature stirrer with the rotating speed of 1000rpm, and reacting for 12 hours at the temperature of 30 ℃. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm, centrifuging for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, mainly enriching the free fatty acid, the long-chain alkane generated by decarboxylation and the ester not participating in the reaction in the upper liquid layer after hydrolysis, collecting the upper liquid layer product, namely the product, and measuring the obtained product by GC (shown in figure 4b), wherein the conversion rate is about 55%, and the product is mainly the alkane of C11-C17.
Adding lipase AYS, dipotassium hydrogen phosphate, Tris-HCl buffer solution and 0.487g of crude enzyme solution (containing CvFAP) into another blank glass bottle, mixing uniformly, and adding [ BMIM ]]BF4And 0.05g of waste cooking oil to form an ionic liquid based multiple liquid phase emulsion system, placing the system under the same condition for reaction for 12 hours, adding ethyl acetate with the volume 2 times of that of the system for extraction, setting the rotation speed of 12000rpm for centrifugation for 3 minutes, dividing the system into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer, mainly enriching long-chain alkane generated by hydrolysis and decarboxylation and ester not participating in the reaction in the upper liquid layer, and collecting the product of the upper liquid layer, namely the product. The product obtained was determined by GC (fig. 4c) with a conversion of only 35%. Other conditions being the same, [ BMIM ]]BF4Replacement by [ BMIM]The conversion rate is only 32.7 percent when Br is added
Example 5
0.05g of lipase AYS is put into a transparent glass bottle, 0.24g of sodium sulfate and 1mL (0.975g) of Tris-HCl (100mM, pH 8.5) solution are mixed, 1.5mL (about 0.6g) of Escherichia coli whole cell (containing CvFAP) is added after mixing, 0.285g of polyethylene glycol 600 and 0.05g of olive oil are added after mixing, the mixture is put into a sandwich beaker wound with an LED lamp device capable of generating blue light and placed on a constant temperature stirrer with the rotating speed of 1000rpm, and the reaction is controlled at 30 ℃ for 12 hours. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, mainly enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction in the upper liquid layer after hydrolysis, and collecting the product of the upper liquid layer, namely the product. The GC determination of the obtained product (figure 5a) shows that the conversion rate can reach 86 percent, and the product is mainly C11-C17 alkane.
Adding lipase AYS and Tris-HCl buffer solution with the same mass into a blank glass bottle, uniformly mixing 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP), adding 0.05g of olive oil, reacting for 12h under the same condition, adding ethyl acetate with the volume 2 times that of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into two layers, namely an upper liquid layer and a lower liquid layer, enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction into the upper liquid layer, and collecting the product of the upper liquid layer, namely the product. The product obtained, as determined by GC (FIG. 5b), had a conversion of only 40%.
Example 6
0.05g of lipase AYS is put into a transparent glass bottle, 0.24g of dipotassium phosphate, 1mL (0.975g) of Tris-HCl (100mM, pH 8.5) solution is mixed uniformly, 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP) are added, 0.285g of polyethylene glycol 600 and 0.05g of soybean oil are added after mixing uniformly, the mixture is put into a sandwich beaker wound with an LED lamp device capable of generating blue light and placed on a constant-temperature stirrer with the rotating speed of 1000rpm, and the reaction is controlled at 30 ℃ for 12 hours. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, wherein free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction are mainly enriched in the upper liquid layer after hydrolysis, collecting the upper liquid layer product, namely a product, and measuring the obtained product by GC (shown in figure 6a), wherein the conversion rate can reach 92%, and the product is mainly alkane of C11-C17.
Adding lipase AYS and Tris-HCl buffer solution with the same mass into a blank glass bottle, uniformly mixing 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP), adding 0.05g of soybean oil, reacting for 12h under the same condition, adding ethyl acetate with the volume 2 times that of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into two layers, namely an upper liquid layer and a lower liquid layer, enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction into the upper liquid layer, and collecting the product of the upper liquid layer, namely the product. The product obtained was determined by GC (FIG. 6b) with a conversion of only 34%.
Example 7
In a clear glass bottle, 0.24g of dipotassium hydrogen phosphate, 1mL (0.975g) of a Tris-HCl (100mM, pH 8.5) solution was added, after mixing, 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP) were added, after mixing, 0.285g of polyethylene glycol 400 and 0.05g of oleic acid were added, and after mixing, the mixture was placed in a sandwich beaker wrapped with an LED lamp device capable of producing blue light, placed on a constant temperature stirrer rotating at 1000rpm, and reacted at 30 ℃ for 12 hours. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, wherein free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction are mainly enriched in the upper liquid layer after hydrolysis, collecting the upper liquid layer product as a product, and measuring the obtained product by GC (shown in figure 7a), wherein the conversion rate can reach 100%, and the product is mainly alkane of C11-C17.
Adding Tris-HCl buffer solution with the same mass into a blank glass bottle, uniformly mixing 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP), adding 0.05g of oleic acid, reacting for 12h under the same condition, adding ethyl acetate with the volume 2 times that of the system, extracting, setting the rotating speed of 12000rpm, centrifuging for 3min, dividing into two layers, namely an upper liquid layer and a lower liquid layer, enriching the free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction into the upper liquid layer, and collecting the product of the upper liquid layer, namely the product. The product obtained was determined by GC (FIG. 7b) to have a conversion of only 56%.
Example 8
In a clear glass bottle, 0.24g of dipotassium hydrogen phosphate, 1mL (0.975g) of a Tris-HCl (100mM, pH 8.5) solution, after mixing, 1.5mL (about 0.6g) of escherichia coli whole cells (containing CvFAP) were added, after mixing, 0.285g of polyethylene glycol 400 and 0.05g of soybean oil were added, and the mixture was put into a sandwich beaker wrapped with an LED lamp device capable of producing blue light, placed on a constant temperature stirrer rotating at 1000rpm, and reacted at 30 ℃ for 4 hours. After the reaction is finished, adding ethyl acetate 2 times of the volume of the system for extraction, setting the rotating speed of 12000rpm for centrifugation for 3min, dividing the mixture into three layers, namely an upper liquid layer, a middle liquid layer and a lower liquid layer in sequence, mainly enriching free fatty acid, long-chain alkane generated by decarboxylation and ester which does not participate in the reaction in the upper liquid layer after hydrolysis, supplementing 0.05g of soybean oil again after the upper phase is completely removed, and placing the mixture in the same reaction environment for reaction for 4 h. Repeat 7 batches. The upper liquid layer of each batch was subjected to GC assay to calculate relative enzyme activity. Taking the activity of the first batch as 100%, and the activities of the subsequent 6 batches are respectively as follows: 95%, 86%, 72%, 70%, 68%, 65%, the relative activity of the enzyme was also maintained at 65% after 7 batches.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<120> method for catalyzing hydrolysis decarboxylation of grease by using cell-like multiphase emulsion photo-enzyme system
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1123
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttggtttaag cagccgggat ctcagtggtg gtggtggtgg tgctcgagtg cggccgcaag 60
cttttaagca gcaacggtag ccggtgcagc agcgctagca ccgatggtcg ctttgccggt 120
cagtaaagca gccgcacgtt cagcaatcat aactaccgga gcaccagtct gaccgcccgg 180
aattttaggt actacagaag cgtccacgac acgcagacct tccacaccat gaacacgcag 240
ctggttatca acaacgctag agctatcacc tgcgttaccc attttgcagg taccagtgat 300
tgcgttgcta gagtgaatgc tacgacggat atattcatcg atctggtcat cagatacaac 360
accggaacct gggaatagtt caccgtccag gtattcagac agcgcagagg aacgtgccac 420
gtcacgcgcc cagtggatac ctttacgcag ggtagccagg tccgcgccgt ctttatcggt 480
caggtagccc ggagacagtt tcggcggagc gaacggatca gcagatttca gaccaacgga 540
accagtagac tgcggacggc aggcgatcag ctgcatagtg ataccagaag gccatttaag 600
accctgagac tggaattttg cgaaacgcac gtaggtggat acgccgtccg ggtccagtgc 660
catgcccggc acgaaacgta cttgtaaatc gggcaatgcc tgaccggcgg tgcgaacgaa 720
ggcaccacgg tcacaaccgg tgctagtcag gccaccacga ccgcccaaca ggtagctcgc 780
gatcgcgcgt ttgcgaatct ggcctttttc gttataaatg tggtcgctga tagcgatacc 840
atcgtatttt tctttaaccg gtgctgcagt caggcatgcc ggctgatcct gcaggttctg 900
accaacaccg ggccagattg gacacaccgg gataccgaat tccttcagtt cagcagacgg 960
accaacactg agtgtttcag tagaacgggg tgttgcaccg cacagcgcac atgatcactt 1020
caccacccgg cgcagttcgc agacgacgtt cgcggtcgga cgtcggtgct gacttcaaca 1080
cccaggtgct ggtgctttac tggcggcctg gtcgatgtta acc 1123