CN107446813B - Device for producing 2-PE through continuous conversion and method for producing 2-PE through continuous conversion - Google Patents
Device for producing 2-PE through continuous conversion and method for producing 2-PE through continuous conversion Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
- C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/10—Separation or concentration of fermentation products
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/12—Purification
Abstract
The invention discloses a device for producing 2-PE by continuous conversion, which is characterized by comprising a No. 1 reaction tank 3, a conversion liquid conveying pump 5, a microfiltration membrane unit 6, a resin column device 12, a column passing liquid storage tank 9, a column passing liquid conveying pump 10, a sterile filter 11 and a plurality of valves. The invention also discloses a method for producing 2-PE by utilizing the yeast resting cells and the device for continuous transformation. The invention can realize industrialized continuous production of 2-PE, yeast resting cells have little growth in the transformation process, a batch of yeast resting cells can be prepared for continuous use for more than 10 batches, each batch can be operated for more than 15 days continuously, and the transformation rate is not obviously reduced; the continuous 2-PE conversion process does not need to be converted under aseptic conditions, the operation is very simple and convenient, the yield is high, the single-tank continuous conversion is carried out for 5 days, and the yield of 2-PE reaches more than 21 g/L. The two tanks are continuously transformed for 15 days, the accumulated output can reach more than 90g/L, the byproducts are few, the production cost of the product is greatly reduced, and the method has obvious advantages in future industrial application.
Description
Technical Field
The invention belongs to the field of biotechnology for preparing 2-PE, and particularly relates to a device for producing 2-PE by continuously converting resting cells of yeast; the invention also relates to a method for producing 2-PE by using the yeast resting cells to realize continuous bioconversion through a product in-situ transfer technology.
Background
2-PE (2-phenethyl alcohol) is an aromatic alcohol with rose smell, widely existing in nature, is a main component of the aroma of many foods, is also a natural flavor substance of various fermented foods, has great demand in daily chemical products and food industry, and is also an important pharmaceutical intermediate and fine chemical intermediate. At present, 2-PE is basically produced by adopting a chemical synthesis method, but raw materials benzene or styrene which are used mostly belong to cancerogenic substances, and the products mostly contain some bad byproducts which are difficult to remove, so that the application range of the products is greatly limited. And 2-PE is extracted from natural plants, so that the problem that the large-scale industrial production cannot be met due to the fact that the concentration of 2-PE in the plants is too low and the cost is high. The use of yeast to catalyze L-phenylalanine (L-Phe) to produce 2-PE is an effective way for industrially producing natural 2-PE, but the concentration of 2-PE synthesized by a bioconversion method is low, usually 0.5-2.5 g/L due to the toxic effect of products and ethanol on yeast cells. In recent years, researchers (such as Chen Jian, etc., university of Jiangnan) select and breed 2-PE strains with high yield by means of recombinant escherichia coli over-expressing phenylpyruvate decarboxylase (KDC) and Alcohol Dehydrogenase (ADHI) escherichia coli engineering bacteria, and the highest yield is only 130mg/L at present; wang Chengtao and the like are coupled and overexpressed through an aminotransferase gene (AR 08) and a decarboxylase gene (AR 010) of the saccharomyces cerevisiae to construct high-yield 2-PE saccharomyces cerevisiae engineering bacteria, the yield is improved compared with that of the strain, but the problem of product inhibition cannot be effectively solved. The toxicity of the product and ethanol to yeast is the bottleneck in the bioconversion process to 2-PE.
At present, the product 2-PE is continuously recovered from fermentation liquor by an in-situ separation technology (ISPR), so that the concentration of the product in the fermentation liquor can be maintained at a lower level, the inhibition of the product is avoided, and the method is a main method for improving the total yield of the 2-PE. For in situ transfer 2-The PE is mainly extracted by water/oil two-phase extraction, water/ionic liquid two-phase extraction, and supercritical CO 2 Extraction techniques), adsorption techniques (such as macroporous resin adsorption, composite resin or novel media adsorption), and pervaporation-permeation extraction techniques with membrane separation. Stark et al, which uses oleic acid as an extractant to carry out Saccharomyces cerevisiae Giv2009 for two-phase batch fed-batch culture, wherein the concentration of 2-PE in the oleic acid is 24.0g/L, and the total yield of 2-PE reaches 12.6g/L; MMW.Eschmann et al used polypropylene glycol 1200 as extractant during yeast cell culture with 2-PE yields of 26.5g/L and 0.3g/L in the organic and aqueous phases, respectively, with a total yield of 10.2g/L. Although the 2-PE is effectively extracted by using an organic solvent, the method has toxic effect on cells, or has the problems of complex product separation, high cost and the like caused by high viscosity, is difficult to apply in actual production, and cannot continuously operate. The 2-PE is transferred from the water phase in situ by adopting the resin adsorption technology, namely, the 2-PE is adsorbed and extracted by utilizing the pore diameter and the polarity of the resin, and the volume yield of the 2-PE can be greatly improved by adding a certain amount of macroporous resin into the fermentation liquor. Guan Ang adopts macroporous resin FD0816 as a medium of an in-situ transfer product, the final concentration of 2-PE reaches 12.8g/L, and 232% is improved compared with a fermentation system without resin; wang et al used FD0816 resin as extractant to semi-continuously culture yeast S.cerevisiae GivR-Uv3 to obtain optimal cell growth rate and highest 2-PE productivity, when 2-PE content in culture medium is higher than 2.7g/L, new resin is needed to be replaced, and final accumulated yield is up to 32.5g/L by continuously separating and replacing resin in conversion solution from fermentation solution; lu Shaoming A batch fermentation conversion test is carried out on a 50L fermentation tank by taking macroporous resin as a product adsorbent, the 2-PE content in the fermentation liquor reaches more than 3.0g/L, growing cells are adopted to put resin into the fermentation liquor for ISPR bioconversion, although higher yield is obtained, the resin needs to be sterilized, repeated high-temperature sterilization can influence the adsorption efficiency of the resin, otherwise, the resin is easy to pollute and cause the conversion to be difficult to continuously carry out, in addition, the resin is easy to break under the action of stirring shearing force, and the thallus cells are difficult to separate from the resin, the recovery workload of the resin from the fermentation tank is large, the pollution and the impurities are easy to pollute, and are difficult to operate in practical production, and the problems lead to fine growthThe ISPR technology of the cells in the actual production process is difficult to realize. In addition, since the culture solution contains a large amount of sugar, protein powder, yeast powder and other organic matters, and a large amount of byproducts such as ethanol and the like are generated in the cell growth and metabolism process, the organic matters and the metabolic byproducts can increase the difficulty of product purification.
Technical proposal
The invention aims to solve the technical problems of complex components, more byproducts, easy pollution, difficult separation of thalli and resin, complex operation, large discharge amount of organic wastewater and the like of a growing cell conversion solution by an ISPR technology of yeast resting cell bioconversion-microfiltration membrane unit filtration-resin column serial adsorption product.
The invention solves the technical problems by the following technical proposal,
the device for producing 2-PE by continuous conversion is characterized by comprising a No. 1 reaction tank, a conversion liquid delivery pump, a microfiltration membrane unit, a resin column device, a column passing liquid storage tank, a column passing liquid delivery pump, a sterile filter and a plurality of valves;
the No. 1 reaction tank is a reaction vessel for producing 2-PE through continuous conversion, and is internally provided with a stirring paddle, an air breather, a pH value monitoring device and a temperature control device;
the input end of the conversion liquid conveying pump is connected to the No. 1 reaction tank through a valve, and the conversion liquid in the No. 1 reaction tank is conveyed to the microfiltration membrane unit;
the microfiltration membrane unit filters the conversion liquid input by the No. 1 reaction tank, the obtained concentrated liquid is conveyed back to the No. 1 reaction tank through a valve, and the obtained clear liquid is conveyed to the resin column device through the valve;
the resin column device is an adsorption resin column for adsorbing 2-PE in the clear liquid, and the liquid after passing through the column is conveyed to a column passing liquid storage tank through an outlet end of the resin column device and a valve;
the column passing liquid storage tank collects liquid after column passing;
the input end and the output end of the column passing liquid conveying pump are respectively connected to a column passing liquid storage tank and an aseptic filter through valves, and column passing liquid is returned to the No. 1 reaction tank through the aseptic filter;
the bacteria-free filter filters and removes bacteria in the collected liquid from the post liquid storage tank.
As optimization, a 2# reaction tank is also arranged, and the 2# reaction tank is connected with the 1# reaction tank in parallel and is respectively connected with the input end of the conversion liquid conveying pump and the sterile filter through a valve;
the input end of the conversion liquid conveying pump is connected to the 1# reaction tank and the 2# reaction tank through valves, and the output end of the conversion liquid conveying pump is connected with the microfiltration membrane unit;
the microfiltration membrane unit filters the conversion liquid input by the No. 1 reaction tank and the No. 2 reaction tank, the obtained concentrated liquid is respectively conveyed back to the respective reaction tank through a valve, and the obtained clear liquid is conveyed to the resin column device through the valve; the column passing liquid of the resin column passing device returns to the respective reaction tanks through a column passing liquid storage tank, a column passing liquid conveying pump and a sterile filter;
as optimization, a material supplementing tank and a material supplementing pump are also arranged, buffer solution is arranged in the material supplementing tank, and the concentration of yeast resting cells in the No. 1 reaction tank and the No. 2 reaction tank is regulated;
the input end of the feeding pump is connected with a feeding tank, the output end of the feeding pump is respectively connected with a 1# reaction tank and a 2# reaction tank through a valve, and buffer solution in the feeding tank is pumped into the 1# reaction tank and the 2# reaction tank respectively;
as optimization, the microfiltration membrane unit is a flat plate type microfiltration membrane manufactured by KOCH company, the aperture is 0.22um, and the membrane area is 0.2m 2 ；
As an optimization, the resin column device comprises a 1# resin column and a 2# resin column; the 1# resin column and the 2# resin column are arranged in parallel, and valves are respectively arranged at two ends of the 1# resin column and the 2# resin column; the adsorption is convenient to be continued by the other resin column when one resin column is fully adsorbed for desorption operation, and continuous production is not influenced;
as optimization, the resin column device is a macroporous resin column, and the macroporous resin column is immersed by buffer solution, wherein the buffer solution is potassium phosphate solution with the pH value of 5.1 and the concentration of 0.1 mol/L; the macroporous resin is HZ-816 resin produced by Shanghai Huazhu science and technology Co., ltd;
preferably, the filter membrane pore size of the sterile filter is 0.22um.
Another technical problem to be solved by the present invention is to provide a method for producing 2-PE by continuous transformation using yeast resting cells and the above-mentioned apparatus.
A method for producing 2-PE by continuous conversion of a device for producing 2-PE by continuous conversion comprises the following steps,
(1) Proportioning a conversion solution: the concentration of L-Phe (L-phenylalanine) in the conversion solution is 4-10 g/L, the concentration of ethanol is 5-20 g/L, and the concentration of resting cells of yeast is 6-10% (w/w);
(2) Conversion reaction: putting the conversion solution in the proportion in the step (1) into a 1# reaction tank, wherein the conversion temperature is 26-35 ℃, the ventilation rate is 0.1-0.8 vvm, the stirring speed is 100-400 r/min, and the pH value is 4.0-7.0;
(3) Continuous ISPR separation: when the concentration of 2-PE in the conversion liquid in the 1# reaction tank is 2.5-3.50 g/L, starting the microfiltration membrane unit and the resin column device to perform in-situ separation of the product, namely continuous ISPR separation; extracting concentrated solution of the conversion solution in the No. 1 reaction tank after passing through the microfiltration membrane unit, returning the concentrated solution to the No. 1 reaction tank, allowing clear solution to enter a resin column device for product adsorption, and returning column passing solution passing through the resin column device to the No. 1 reaction tank through an online sterile filter;
when the concentration of the 2-PE in the conversion liquid in the 1# reaction tank is lower than 0.5g/L, ending continuous ISPR separation, and continuing the conversion reaction according to the step (2);
(4) When the concentration of the product 2-PE in the conversion solution is 2.5-3.50 g/L again, repeating the step (3) to perform continuous ISPR separation;
(5) Reaction is finished: ending the reaction to terminate the conversion when the conversion rate of the product 2-PE in the conversion liquid in the reaction tank 1# is detected to be lower than 0.15 g/(L.h) or the specified reaction time is reached; and carrying out continuous ISPR separation on the conversion solution in the No. 1 reaction tank according to the step (3);
(6) Eluting to obtain 2-PE: eluting 2-PE adsorbed in the resin column device with ethanol at a flow rate of 3.5-4.5m 3 /(m 3 (column capacity). H), collecting the eluting ethanol solution containing the product 2-PE, and measuring the content of the 2-PE in the eluting ethanol solution.
After the step (5) is optimized, centrifuging the conversion solution in the No. 1 reaction tank at 5000r/min for 10min, respectively collecting thalli and clear solution, weighing thalli, adding an equivalent amount of potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1mol/L, uniformly mixing to obtain a yeast resting cell bacterial solution, and storing in a refrigerator at the temperature of 4 ℃ for later use;
as optimization, the yeast resting cell bacterial liquid obtained after the step (5) is subjected to the next batch of conversion reaction according to the steps (1) - (6);
as optimization, in the conversion reaction process of the step (2), a small amount of budding reproduction is still carried out on the yeast resting cells, the cell concentration is required to be monitored, when the concentration of the yeast resting cells is higher than 10% (w/w), partial yeast resting cells are shunted into a No. 2 reaction tank, an equal volume of buffer solution is added into the No. 1 reaction tank, the concentration of L-Phe in the conversion solution in the No. 1 reaction tank is controlled to be 4-10 g/L, the concentration of ethanol is controlled to be 5-20 g/L, and the concentration of the yeast resting cells is controlled to be 6-10% (w/w), and then the reaction is continuously carried out according to the steps (2) - (6); the buffer solution is potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1 mol/L;
as optimization, after a certain amount of yeast resting cells are collected in a 2# reaction tank, adding a proper amount of buffer solution, L-Phe and ethanol to prepare a conversion solution, and controlling the concentration of the L-Phe in the conversion solution to be 4-10 g/L, the concentration of the ethanol to be 5-20 g/L and the concentration of the yeast resting cells to be 6-10% (w/w); continuing the conversion reaction according to the steps (2) - (6), and realizing the production process of simultaneous continuous conversion and adsorption of the No. 1 reaction tank and the No. 2 reaction tank; the buffer solution is potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1 mol/L;
in the step (6), the 2-PE adsorbed in the resin column device is eluted by 1.5-2.5 times of ethanol, and the concentration of the 2-PE in the eluted ethanol solution is 25-80 g/L.
All the operation processes of the invention are automatically carried out, no separate sterile operation is needed, the yeast resting cells are repeatedly used for more than 10 batches according to the operation, and the conversion rate is not obviously reduced.
The invention can realize industrialized continuous production of 2-PE, yeast resting cells have little growth in the transformation process, a batch of yeast resting cells can be prepared for continuous use for more than 10 batches, each batch can be operated for more than 15 days continuously, and the transformation rate is not obviously reduced; the continuous 2-PE conversion process does not need to be converted under aseptic conditions, the operation is very simple and convenient, the yield is high, the single-tank continuous conversion is carried out for 5 days, and the yield of 2-PE reaches more than 21 g/L. The two tanks are continuously transformed for 15 days, the accumulated output can reach more than 90g/L, the byproducts are few, the production cost of the product is greatly reduced, and the method has obvious advantages in future industrial application.
Drawings
Fig. 1 is a schematic structural diagram of embodiment 1 of the present invention.
Detailed Description
The present invention will be further described in detail with reference to the drawings and examples.
Example 1
A reaction device for producing 2-phenethyl alcohol by continuous conversion comprises a feed tank 1, a feed pump 2, a No. 1 reaction tank 3, a No. 2 reaction tank 4, a conversion liquid delivery pump 5, a microfiltration membrane unit 6, a resin column device 12, a column passing liquid storage tank 9, a column passing liquid delivery pump 10, a sterile filter 11 and a plurality of valves 31, 32, 33, 34, 41, 42, 43, 44, 71, 72, 81 and 82,
buffer solution is filled in the material supplementing tank 1, and the concentration of yeast resting cells in the No. 1 reaction tank 3 and the No. 2 reaction tank 4 is regulated; the buffer solution is potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1 mol/L;
the input end of the feed supplement pump 2 is connected with the feed supplement tank 1, the output end of the feed supplement pump is respectively connected with the 1# reaction tank 3 and the 2# reaction tank 4 through a valve, and the buffer in the feed supplement tank 1 is pumped into the 1# reaction tank 3 and the 2# reaction tank 4;
the reaction tanks 1# 3 and 2# 4 are reaction vessels for producing 2-PE through continuous conversion, and are internally provided with stirring paddles, a ventilation device, a pH value monitoring device and a temperature control device;
the input end of the conversion liquid conveying pump 5 is respectively connected to the 1# reaction tank 3 and the 2# reaction tank 4 through valves 34 and 44, and the output end of the conversion liquid conveying pump is connected with the microfiltration membrane unit 6; transferring the conversion liquid in the No. 1 reaction tank 3 and the No. 2 reaction tank 4 to a microfiltration membrane unit 6;
the microfiltration membrane unit 6 filters the conversion liquid input by the No. 1 reaction tank 3 and the No. 2 reaction tank 4, the obtained concentrated liquid is respectively conveyed back to the respective reaction tanks through valves 31 and 41, and the obtained concentrated liquid is obtainedIs fed to the resin column device 12 through valves 71, 81; the microfiltration membrane unit 6 is a flat plate type microfiltration membrane manufactured by KOCH company in the United states, the aperture is 0.22um, and the membrane area is 0.2m 2 ；
The resin column device 12 is an adsorption resin column, and adsorbs 2-PE in the clear liquid, and the column passing liquid passing through the resin column device 12 is conveyed to a column passing liquid storage tank 9 through an outlet end of the resin column device 12 and valves 72 and 82; the resin column device 12 is a macroporous resin column, 900g of macroporous resin is selected for column packing, 600mL of buffer solution is used for immersing the macroporous resin, and the buffer solution is potassium phosphate solution with the pH value of 5.1 and the concentration of 0.1 mol/L; the macroporous resin is HZ-816 resin produced by Shanghai Huazhu science and technology Co., ltd;
the resin column device 12 includes a 1# resin column 7 and a 2# resin column 8; the 1# resin column 7 and the 2# resin column 8 are arranged in parallel, and the two ends of the 1# resin column 7 and the 2# resin column 8 are respectively connected with valves 71, 72, 81 and 82; from the viewpoint of continuous operation and convenience, when one resin column is saturated in adsorption, ethanol can be used for eluting products, and the other resin column can continuously participate in adsorption operation at the moment, so that the continuous operation of the reaction is not influenced;
the column passing liquid storage tank 9 collects liquid after column passing;
the input end and the output end of the column passing liquid conveying pump 10 are respectively connected to a column passing liquid storage tank 9 and a sterile filter 11, and column passing liquid is returned to the respective reaction tanks through the sterile filter 11;
the bacteria-free filter 11 filters and removes bacteria in the collected liquid in the post liquid storage tank 9; the filter membrane pore size of the sterile filter 11 is 0.22um.
EXAMPLE 2 Yeast resting cell culture
The resting cell culture of yeast comprises the following steps,
(1) Picking 2 rings of lawn from the inclined plane strain stored in a refrigerator, inoculating the lawn to a 250mL triangular flask filled with 60mL of cell proliferation culture solution, and oscillating for 18h at a temperature of 28 ℃ in a shaking table of 180 r/min; the cell proliferation culture medium comprises the following components: 30g/L glucose, 5g/L peptone, 3g/L yeast powder, 3g/L malt extract, pH value of 5.8-6.2, and sterilizing at 115 ℃ for 20min; the inclined plane strain in the embodiment of the invention is an inclined plane strain of a yeast resting cell prepared by Chinese invention patent CN2015105496188, and the yeast resting cell is named in Chinese: saccharomyces cerevisiae SH003, english naming: saccharomyces cerevisiae SH003 and the preservation number CCTCC NO: m2015389;
(2) Inoculating the liquid prepared in the step (1) into a 5L fermentation tank filled with 3L proliferation culture solution, stirring at 28 ℃ at a rotation speed of 200r/min, introducing air, culturing for 20-24h, and culturing at an aeration rate of 0.3vvm (vvm: air volume/culture volume/min), so as to perform cell proliferation culture, wherein the pH is reduced from 5.5 to 4.95 in the culturing process;
(3) After the culture is finished, centrifugally collecting the bacterial cells, centrifuging for 10min at a centrifugal speed of 5000r/min, discarding the supernatant, and collecting 231g of wet bacterial cells;
(4) Centrifugally washing thalli twice by using buffer solution, and centrifuging for 10min at a centrifugal speed of 5000 r/min; the buffer solution is sodium phosphate solution with the pH value of 6.8 and the concentration of 0.1 mol/L;
(5) Then the strain is suspended in potassium phosphate buffer with pH value of 5.1 and concentration of 0.1mol/L, and yeast resting cell liquid with strain concentration of 8% (w/w) is prepared, so as to obtain 2.7L of yeast resting cell strain suspension. Example 3 Single vessel continuous ISPR separation assay
A method for producing 2-PE by continuous conversion in a single reaction vessel by resting cells of yeast and the apparatus described in example 1, comprising the steps of,
(1) 2.7L of the yeast resting cell bacterial suspension prepared in the example 2 is placed in a 1# reaction tank 3 of the device of the example 1, and the 1# reaction tank 3 is a 5L reaction vessel;
(2) Proportioning a conversion solution: 27g of substrate L-Phe (L-phenylalanine) and 54g of ethanol were added to reaction tank 1# 3; the concentration of L-Phe in the transformation liquid is 10g/L, the concentration of ethanol is 20g/L, and the concentration of resting cells of yeast is 8% (w/w);
(3) The microfiltration membrane unit 6 is a flat plate type microfiltration membrane manufactured by KOCH company, and has a pore diameter of 0.22um and a membrane area of 0.2m 2 Cleaning for later use;
(4) Weighing 900g of pretreated macroporous resin, loading the pretreated macroporous resin into a resin column device 12, adding 600mL of buffer solution into the resin column device 12 to immerse the macroporous resin, wherein the buffer solution is potassium phosphate solution with the pH value of 5.1 and the concentration of 0.1mol/L, and the resin column device 12 is reserved; the macroporous resin is HZ-816 resin produced by Shanghai Huazhu science and technology Co., ltd;
(5) Conversion reaction: placing the conversion solution in the No. 1 reaction tank 3 at 26 ℃, stirring at a rotation speed of 200r/min and an aeration rate of 0.1-0.25vvm, sampling and monitoring the concentration of the substrate L-Phe, the ethanol and the 2-PE after the conversion reaction is carried out for 12h, and when the concentration of the substrate L-Phe is lower than 4g/L or the concentration of the ethanol is lower than 5g/L, timely supplementing the substrate L-Phe or the ethanol, and controlling the concentration of the L-Phe in the conversion solution to be 4-10 g/L and the concentration of the ethanol to be 5-20 g/L;
(6) Continuous ISPR separation: when the conversion reaction is carried out for 24 hours, the concentration of 2-PE in the mixed solution in the 1# reaction tank 3 is measured to be 3.21g/L, the concentrated solution obtained after the mixed solution passes through the microfiltration membrane unit 6 is directly returned to the 1# reaction tank 3, and clear solution passes through the resin column device 12 to carry out product adsorption and then is pumped back to the 1# reaction tank 3 after passing through the 0.22um sterile filter 11; ending the continuous ISPR separation when the concentration of 2-PE in the No. 1 reaction tank 3 is lower than 0.5 g/L; the continuous ISPR separation refers to a process of continuously recovering a product from the mixed liquor/conversion liquor through the micro-filtration membrane unit 6 and the resin column/resin column device 12;
(7) Operating according to the steps (5) - (6), and operating the continuous ISPR separation respectively when the conversion reaction is carried out for 24 hours, 46 hours and 70 hours, wherein the operation time is 1.5-2.5 hours each time; adding substrate L-Phe and ethanol respectively for 18h, 30h, 42h, 60h and 78h, and adding substrate L-Phe 50g and ethanol 270g;
(8) In the conversion reaction process, sampling and measuring the concentration of 2-PE in the mixed solution in the reaction container every 6 hours, and detecting that the concentration increase of 2-PE is more than 0.9g/L every 6 hours, namely, the conversion rate is more than 0.15 g/(L.h), and continuing the reaction; stopping the reaction when the conversion rate is less than 0.15 g/(L.h); in the embodiment, when the conversion reaction is carried out for 90 hours, the conversion rate of the product 2-PE in the mixed solution in the reaction vessel is lower than 0.15 g/(L.h), and the conversion reaction is finished;
(9) Reaction is finished: after the reaction is finished, carrying out continuous ISPR separation on the mixed solution in the 1# reaction tank 3 according to the step (6), centrifuging the converted solution after the continuous ISPR separation for 10min at 5000r/min, collecting 291g of thalli, adding 291mL of potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1mol/L, uniformly mixing to prepare a yeast resting cell mixed solution, and storing in a refrigerator at 4 ℃ for later use;
(10) Eluting to obtain 2-PE: eluting 2-PE in the resin column apparatus 12 with ethanol at a flow rate of 3.5-4.5m 3 /(m 3 (column capacity). H), 1.58L of eluting ethanol solution is collected together, the content of 2-PE in the eluting ethanol solution is 27.2658g/L, 43.08g of 2-PE product is obtained together, the concentration of 2-PE in the folded reaction solution (2.7L) is 15.95g/L, and the molar conversion rate of the substrate L-Phe is 75.6%.
Molar conversion (%) of substrate L-phe=molar amount of product 2-PE obtained +.molar amount of substrate L-Phe added +.100%
Example 4 test of 2-phenethyl alcohol production by continuous 120h conversion in the apparatus described in example 1
A method for producing 2-PE by continuous transformation of yeast resting cells and the apparatus described in example 1, comprising the steps of,
(1) The yeast resting cells are obtained from the yeast resting cell mixed solution stored in the refrigerator in the step (9) of the example 3, and the yeast resting cells are obtained after filtration and separation, and the yeast resting cells are reused for the 8 th time;
(2) Taking 300g of yeast resting cells obtained in the step (1), adding 2.7L of potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1mol/L, preparing 3.0L of yeast resting cell liquid with the thallus concentration of 10% (w/w), and placing the yeast resting cell liquid into a 1# reaction tank 3 of the reaction device described in the example 1, wherein the 1# reaction tank 3 is a 5L reaction container;
(3) Proportioning a conversion solution: then, 30g of substrate L-Phe and 60g of ethanol are added into the 1# reaction tank 3; the concentration of L-Phe in the mixed solution is 10g/L, the concentration of ethanol is 20g/L, and the concentration of resting cells of yeast is 10% (w/w);
(4) The preparation of the resin column device 12 and the microfiltration membrane unit 6 was the same as in steps (3) and (4) in example 3, and was fixed to the microfiltration membrane unit 6 and the resin column device 12 of the reaction apparatus described in example 1, respectively;
(5) Conversion reaction in reaction tank 1 #: the temperature is 28 ℃, the stirring speed is 2000r/min, the ventilation rate is 0.1-0.3vvm, sampling is started to monitor the concentration of the substrate L-Phe, the concentration of the ethanol and the concentration of the 2-PE after the conversion reaction is carried out for 12 hours, when the concentration of the substrate L-Phe is lower than 4g/L or the concentration of the ethanol is lower than 5g/L, the substrate L-Phe and the ethanol are timely added, the concentration of the L-Phe in the conversion solution is controlled to be 4-10 g/L and the concentration of the ethanol is controlled to be 5-20 g/L, and the other operations are the same as the step (5) in the embodiment 3;
(6) 1# reaction tank 3 continuous ISPR separation: when the conversion reaction is carried out for 20 hours, the concentration of 2-PE in the conversion liquid in the No. 1 reaction tank 3 is 3.25g/L, continuous ISPR separation is started, the concentrated liquid of the conversion liquid in the No. 1 reaction tank 3 after passing through the microfiltration membrane unit 6 is directly returned to the No. 1 reaction tank 3, and clear liquid is pumped into the No. 1 reaction tank 3 after passing through the resin column device 12 for product adsorption and then passing through the sterile filter 11; ending the continuous ISPR separation when the concentration of 2-PE in the No. 1 reaction tank 3 is lower than 0.5 g/L; the procedure is as in step (6) of example 3;
(7) Operating according to the steps (5) - (6), and operating the continuous ISPR separation respectively when the conversion reaction is carried out for 20h, 42h, 64h, 84h and 106h, wherein the operation time is 1.5-2.5 h each time; adding substrates L-Phe and ethanol respectively at 18h, 30h, 42h, 64h, 84h, 96h and 110h, controlling the concentration of L-Phe in the conversion liquid to be 4-10 g/L and the concentration of ethanol to be 5-20 g/L, adding 60g of L-Phe and 485g of ethanol together, finishing the conversion reaction from a 1# reaction tank 3, and adding 90g of L-Phe and 549g of ethanol together in the 1# reaction tank 3 in an accumulated way;
(8) Conversion reaction in 2# reaction tank 4: taking 20mL of conversion liquid from the 1# reaction tank 3 before each continuous ISPR separation, centrifuging for 10min at 5000r/min, weighing, when the concentration of yeast resting cells is higher than 10% (w/w), diverting a part of concentrated liquid obtained by passing through the microfiltration membrane unit 6 from the conversion liquid in the 1# reaction tank 3 to the 2# reaction tank 4 in a proper amount during the continuous ISPR separation, supplementing an equal amount of potassium phosphate buffer with the pH value of 5.1 and the concentration of 0.1mol/L to the 1# reaction tank 3, supplementing an appropriate amount of potassium phosphate buffer with the pH value of 5.1 and the concentration of 0.1mol/L to the 2# reaction tank 4, and controlling the concentration of yeast resting cells in the 1# reaction tank 3 and the 2# reaction tank 4 to be 6-10% (w/w);
in the embodiment, when the conversion reaction of the 1# reaction tank 3 is carried out for 20 hours, 42 hours, 64 hours, 84 hours and 106 hours, the concentrated solution of the 1# reaction tank 3 passing through the microfiltration membrane unit 6 is split into 300mL, 400mL and 300mL to the 2# reaction tank 4, potassium phosphate buffer with the concentration of 0.1mol/L (pH of 5.1) is respectively added to the 1# reaction tank 3 and the 2# reaction tank 4, the concentration of yeast resting cells in the 1# reaction tank 3 and the 2# reaction tank 4 is controlled to be 6-10% (w/w), substrate L-Ph and ethanol are added to the 2# reaction tank 4, the concentration of L-Phe in the conversion solution in the 2# reaction tank 4 is controlled to be 4-10 g/L, the concentration of ethanol is controlled to be 5-20 g/L, and the 2# reaction tank 4 carries out the conversion reaction under the same conditions of the step (5) and the 1# reaction tank 3;
(9) 2# reaction tank 4 continuous ISPR separation: the 2# reactor 4 performs continuous ISPR separation according to the method described in step (6); when the conversion reaction is carried out for 20 hours, the concentration of 2-PE in the conversion liquid in the 2# reaction tank 4 is 3.30g/L, continuous ISPR separation is started, the concentrated liquid of the conversion liquid in the 2# reaction tank 4 after passing through the microfiltration membrane unit 6 is directly returned to the 2# reaction tank 4, and clear liquid is pumped into the 2# reaction tank 4 after passing through the resin column device 12 for product adsorption and then passing through the sterile filter 11; ending the continuous ISPR separation when the concentration of 2-PE in the 2# reaction tank 4 is lower than 0.5 g/L;
(10) The 2# reaction tank 4 is operated according to the steps (8) - (9), 18g of L-Phe and 85g of ethanol are added into the 2# reaction tank 4;
(11) Reaction is finished: after the reaction in the 1# reaction tank 3 is finished for 120 hours, the conversion solution in the 1# reaction tank 3 and the conversion solution in the 2# reaction tank 4 are subjected to continuous ISPR separation according to the method in the step (6), the conversion solution after the continuous ISPR separation is centrifuged for 10 minutes at 5000r/min, 484g of thalli are collected together, and an equal weight of potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1mol/L is added for uniform mixing and is put into a refrigerator at the temperature of 4 ℃ for standby;
(12) Eluting to obtain 2-PE: eluting 2-PE in the resin column apparatus 12 with ethanol at a flow rate of 3.5-4.5m 3 /(m 3 (column volume). H), 1.75L of eluting ethanol solution was collected together, and the content of 36.184g/L of 2-PE was measured, and 63.32g of 2-PE was obtained in total, and the concentration of 2-PE in the reaction solution (3.0L) was 21.1g/L, and the molar conversion of the substrate L-Phe was 79.23%.
Example 5 test of continuous conversion of 2-phenethyl alcohol produced in the apparatus described in example 1
A method for producing 2-PE by continuous transformation of yeast resting cells and the apparatus described in example 1, comprising the steps of,
(1) The yeast resting cells are obtained from the yeast resting cell mixed solution stored in the refrigerator in the step (11) of the example 4, and the yeast resting cells are obtained after filtration and separation, and the yeast resting cells are reused for the 10 th time;
(2) Using the reaction apparatus of example 1, wherein the reaction tank 1# 3 and the reaction tank 2# 4 are both 5L reaction tanks, and the yeast resting cell liquid 3.0L having a cell concentration of 10% (w/w) was prepared in the reaction tank 1# 3, and the preparation method and the substrate addition concentration were the same as those in steps (1) to (3) of example 4; the reaction tank 2 is cleaned for standby;
(3) The preparation of the resin column device 12 and the microfiltration membrane unit 6 was the same as in step (4) of example 4, and was fixed to the microfiltration membrane unit 6, the # 1 resin column 7 and the # 2 resin column 8 of the reaction apparatus described in example 1, respectively;
(4) Conversion reaction in reaction tank 1 #: the temperature is 30 ℃, the stirring speed is 250r/min, the ventilation rate is 0.1-0.25vvm, sampling is started to monitor the concentration of the substrate L-Phe, the concentration of the ethanol and the concentration of the 2-PE after the conversion reaction is carried out for 12 hours, when the concentration of the substrate L-Phe is lower than 4g/L or the concentration of the ethanol is lower than 5g/L, the substrate L-Phe and the ethanol are timely added, the concentration of the L-Phe in the conversion solution is controlled to be 4-10 g/L and the concentration of the ethanol is controlled to be 5-20 g/L, and the other operations are the same as the step (5) in the example 4;
(5) 1# reaction tank 3 continuous ISPR separation: when the conversion reaction is carried out for 24 hours, the concentration of 2-PE in the conversion liquid in the 1# reaction tank 3 is 3.88g/L, continuous ISPR separation is started, the concentrated liquid of the conversion liquid in the 1# reaction tank 3 after passing through the microfiltration membrane unit 6 is directly returned to the 1# reaction tank 3, and clear liquid is pumped into the 1# reaction tank 3 after passing through the 1# resin column 7 of the resin column device 12 for product adsorption and then passing through the sterile filter 11; ending the continuous ISPR separation when the concentration of 2-PE in the No. 1 reaction tank 3 is lower than 0.5 g/L; step (6) was carried out in the same manner as in example 4;
(6) Operating as in step (7) of example 4, the 1# reactor 3 was operated for a continuous ISPR separation every 20 to 24 hours, with a run time of 1.5 to 3.0 hours each time; measuring the contents of L-Phe, 2-PE and ethanol in a conversion solution in a 1# reaction tank 3 every 12 hours, adding a proper amount of substrate L-Phe and ethanol, and controlling the concentration of L-Phe in the conversion solution to be 4-10 g/L and the concentration of ethanol to be 5-20 g/L; after the adsorption saturation of the No. 1 resin column 7, transferring the clear liquid to the No. 2 resin column 8 for adsorption, eluting the No. 1 resin column 7, after the adsorption saturation of the No. 2 resin column 8, transferring the clear liquid to the No. 1 resin column 7 for adsorption, eluting the No. 2 resin column 8, and using the No. 1 resin column 7 and the No. 2 resin column 8 alternately according to the method, so as to ensure continuous production;
(7) Conversion reaction in 2# reaction tank 4: before the end of each continuous ISPR separation, 300-600mL of concentrated solution obtained after the conversion solution in the No. 1 reaction tank 3 passes through the microfiltration membrane unit 6 is shunted in the No. 2 reaction tank 4, meanwhile, the same volume of potassium phosphate buffer with the pH value of 5.1 and the concentration of 0.1mol/L is supplemented in the No. 1 reaction tank 3, a proper amount of potassium phosphate buffer with the pH value of 5.1 and the concentration of 0.1mol/L is also supplemented in the No. 2 reaction tank 4, the concentration of yeast resting cells in the No. 1 reaction tank 3 and the No. 2 reaction tank 4 is controlled to be 6-10% (w/w), a proper amount of substrate L-Phe and ethanol are respectively supplemented, and the conversion reaction is continued after the concentration of L-Phe in the conversion solution is controlled to be 4-10 g/L and the concentration of ethanol is controlled to be 5-20 g/L; the method comprises the steps of carrying out a first treatment on the surface of the After the buffer solution and the concentrated solution split by the No. 1 reaction tank 3 are continuously fed into the No. 2 reaction tank 4 for 4 times, the volume of the concentrated solution reaches 2.87L, and the No. 2 reaction tank 4 and the No. 1 reaction tank 3 are converted under the same conditions according to the steps (4) - (6);
(8) Reaction in reaction tank 1# 3 is completed: in the conversion reaction process, sampling and measuring the concentration of 2-PE in the mixed solution in the reaction container every 5 hours, and detecting that the concentration increase of 2-PE is more than 0.75g/L every 5 hours, namely, the conversion rate is more than 0.15 g/(L.h), and continuing the reaction; stopping the reaction when the conversion rate is less than 0.15 g/(L.h); in the embodiment, when the conversion reaction of the 1# reaction tank 3 is carried out for 100 hours, the conversion rate of 2-PE in the conversion solution in the 1# reaction tank 3 is detected to be lower than 0.15 g/(L.h), the conversion reaction of the 1# reaction tank 3 is finished, the conversion solution in the 1# reaction tank 3 is subjected to continuous ISPR separation according to the method of the step (5), yeast resting cells are obtained by centrifugal separation of the conversion solution after the continuous ISPR separation, the separated yeast resting cells are resuspended in potassium phosphate buffer with the pH value of 5.1 and the concentration of 0.1mol/L and are prepared into a yeast resting cell solution with the initial volume of 1.5-2.0L and the concentration of 6-8% (w/w), and then the yeast resting cell solution is placed in the 1# reaction tank 3 to start a new round of conversion according to the steps (4) - (6); placing the redundant yeast resting cells in a refrigerator at 4 ℃ for standby;
(9) Along with the increase of the concentration of the yeast resting cells in the 2# reaction tank 4, when the concentration of the yeast resting cells in the 2# reaction tank 4 conversion solution is higher than 10% (w/w), before each continuous ISPR separation is finished, 300-600mL of concentrated solution obtained by separating the conversion solution in the 2# reaction tank 4 by the microfiltration membrane unit 6 is shunted in the 1# reaction tank 3, meanwhile, the same volume of potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1mol/L is supplemented in the 2# reaction tank 4, a proper amount of potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1mol/L is also supplemented in the 1# reaction tank 3, a proper amount of substrate L-Phe and ethanol are further supplemented, the concentration of the yeast resting cells in the 1# reaction tank 3 and the 2# reaction tank 4 is controlled to be 6-10% (w/w), the L-Phe 4g/L and the ethanol 5g/L to 20g/L, and the conversion reaction is continuously carried out, and the steps (4) - (6) are carried out;
(10) Reaction in reaction tank # 24 is completed: in the conversion reaction process, sampling and measuring the concentration of 2-PE in the mixed solution in the reaction container every 5 hours, and detecting that the concentration increase of 2-PE is more than 0.75g/L every 5 hours, namely, the conversion rate is more than 0.15 g/(L.h), and continuing the reaction; stopping the reaction when the conversion rate is less than 0.15 g/(L.h); in this example, when the conversion reaction in the 2# reaction tank 4 is 100 hours, it is detected that the conversion rate of 2-PE in the conversion solution in the 2# reaction tank 4 is lower than 0.15 g/(L.h), the 2# reaction tank 4 ends the conversion reaction, the conversion solution in the 2# reaction tank 4 is subjected to continuous ISPR separation according to the method described in step (5), the conversion solution after continuous ISPR separation is subjected to centrifugal separation to obtain yeast resting cells, the separated yeast resting cells are resuspended in potassium phosphate buffer with a pH value of 5.1 and a concentration of 0.1mol/L and prepared into an initial volume of 1.5-2.0L of 6-8% (w/w) yeast resting cell solution, and then the yeast resting cells are placed in the 2# reaction tank 4 to start a new round of conversion according to steps (4) - (6); placing the redundant yeast resting cells in a refrigerator at 4 ℃ for standby; other operations are the same as the step (8);
(11) Starting from the step (2), repeating the steps (4) - (10) to continuously perform ISPR operation for 15 days, and adding 482g of L-Phe substrate and 630g of ethanol into the 1# reaction tank 3 and the 2# reaction tank 4;
(12) Reaction is finished: after the reaction is finished for 15 days, carrying out continuous ISPR separation on the conversion liquid of the No. 1 reaction tank 3 and the No. 2 reaction tank 4 according to the method in the step (5), centrifuging the conversion liquid after the continuous ISPR separation for 10min at 5000r/min, collecting 944g of resting yeast cells altogether, collecting resting yeast cells required by the next batch of conversion, adding 0.1mol/L potassium phosphate buffer solution with the pH value of 5.1 mol/L and the concentration of 0.1mol/L, uniformly mixing, refrigerating and preserving, and taking the rest resting yeast cells as bacterial proteins for feed;
(13) Eluting to obtain 2-PE: eluting 2-PE in the resin column apparatus 12 with ethanol at a flow rate of 3.5-4.5m 3 /(m 3 (column volume). H), eluting with a common eluent of 6.84L (including the eluent obtained by eluting the saturated resin in step (6), elutingThe ethanol solution had a 2-PE content of 41.096g/L, which yielded 281.1g of 2-PE product, and the initial 3L conversion of 96g/L of 2-PE, and the molar conversion of substrate L-Phe was 78.74% (based on the reaction substrate used in combination of 1# reactor 3 and 2# reactor 4).
The resting cells of the yeast are preserved in China Center for Type Culture Collection (CCTCC) of university of Wuhan, china at a period of 2015, 6 months and 19 days, and the preservation number is CCTCC NO: m2015389, deposit name: saccharomyces cerevisiae SH003, saccharomyces cerevisiae SH003; but is not limited to the preservation number cctccc NO: m2015389, including other biological cells capable of 2-PE transformation.
The invention can effectively solve various problems of growing cells in the bioconversion process, such as easy pollution, difficult separation of resin and cells, low adsorption rate of products after resin sterilization, incapability of continuous operation, complex operation, large wastewater discharge and the like.
The invention adopts a continuous ISPR separation technology of in-situ separation of yeast resting cell combined products to synthesize 2-PE, and the continuous ISPR separation technology comprises the steps of separating thalli cells by a microfiltration membrane unit 6 and adsorbing products by a resin column device 12, thereby realizing continuous bioconversion of the yeast resting cells into 2-PE. The invention has simple operation, no need of running under aseptic condition, very simple components of the conversion solution, repeated use of the resting cells of the yeast for 10 times, no obvious reduction of the conversion capability during 15 days of continuous conversion of each batch, high 2-PE yield and substrate conversion rate, more than 20g/L and 79 percent of yield and conversion rate of single-tank batch for 5 days of continuous conversion, 15 days of continuous conversion of two tanks, 96g/L and 78 percent of accumulated 2-PE yield and 78 percent of substrate conversion rate. Because the resting cells also maintain a certain reproductive capacity in the transformation process, the resting cells can be used for multiple times when being prepared, and compared with the transformation process of cells to be cultured in each batch of production cells, the method has the advantages of greatly simplifying the operation process and reducing the production cost. In addition, the produced surplus thallus can also be used as thallus protein for feed, thus increasing the use value.
The invention is also a clean production process, the conventional growing cells need to be subjected to cell culture every time of transformation, and the transformation liquid contains a large amount of sugar, organic nitrogen and transformation byproducts, so that the discharge amount of organic wastewater is large, and each batch of transformation liquid needs to be sterilized, so that the energy consumption is large. The preparation method can continuously produce resting cells for at least 3 months, namely, one tank of organic wastewater is discharged every 3-6 months, and the discharge amount of the organic wastewater is very small, so that the transformation process of the resting cells is a clean and environment-friendly production process, sterilization is not needed in the production process, and the energy consumption is greatly reduced.
The product 2-PE of the invention is totally adsorbed on resin, and the 2-PE crude product with the concentration of 25-80g/L can be obtained through ethanol elution, so that the biological conversion and the product separation are carried out simultaneously, and the subsequent extraction and refining process is simplified.
Claims (10)
1. The device for producing 2-PE by continuous conversion is characterized by comprising a No. 1 reaction tank, a conversion liquid delivery pump, a microfiltration membrane unit, a resin column device, a column passing liquid storage tank, a column passing liquid delivery pump, a sterile filter and a plurality of valves;
the No. 1 reaction tank is a reaction vessel for producing 2-PE through continuous conversion, and is internally provided with a stirring paddle, an air breather, a pH value monitoring device and a temperature control device;
the input end of the conversion liquid conveying pump is connected to the No. 1 reaction tank through a valve, and the conversion liquid in the No. 1 reaction tank is conveyed to the microfiltration membrane unit;
the microfiltration membrane unit filters the conversion liquid input by the No. 1 reaction tank, the obtained concentrated liquid is conveyed back to the No. 1 reaction tank through a valve, and the obtained clear liquid is conveyed to the resin column device through the valve;
the microfiltration membrane unit is a flat plate type microfiltration membrane produced by KOCH company in the United states, the aperture is 0.22 mu m, and the membrane area is 0.2m 2 ；
The resin column device is an adsorption resin column for adsorbing 2-PE in the clear liquid, and the liquid after passing through the column is conveyed to a column passing liquid storage tank through an outlet end of the resin column device and a valve;
the column passing liquid storage tank collects liquid after column passing;
the input end and the output end of the column passing liquid conveying pump are respectively connected to a column passing liquid storage tank and an aseptic filter through valves, and column passing liquid is returned to the No. 1 reaction tank through the aseptic filter;
the bacteria-free filter filters and removes bacteria in the collected liquid from the post liquid storage tank.
2. The device for producing 2-PE by continuous conversion according to claim 1, wherein a 2# reaction tank is also provided, and the 2# reaction tank is arranged in parallel with the 1# reaction tank and is respectively connected with the input end of a conversion liquid conveying pump and a sterile filter through a valve;
the input end of the conversion liquid conveying pump is connected to the 1# reaction tank and the 2# reaction tank through valves, and the output end of the conversion liquid conveying pump is connected with the microfiltration membrane unit;
the microfiltration membrane unit filters the conversion liquid input by the No. 1 reaction tank and the No. 2 reaction tank, the obtained concentrated liquid is respectively conveyed back to the respective reaction tank through a valve, and the obtained clear liquid is conveyed to the resin column device through the valve; the column passing liquid of the resin column passing device is returned to the respective reaction tanks through a column passing liquid storage tank, a column passing liquid conveying pump and a sterile filter.
3. The device for producing 2-PE by continuous conversion according to claim 2, further comprising a feed tank and a feed pump, wherein the feed tank is filled with buffer solution, and the concentration of yeast resting cells in the No. 1 reaction tank and the No. 2 reaction tank is adjusted;
the input of feed pump is connected with the feed tank, and its output passes through the valve and connects 1# retort and 2# retort respectively, pumps into 1# retort and 2# retort respectively with the buffer solution in the feed tank.
4. The apparatus for continuous conversion production of 2-PE according to claim 1, wherein the resin column apparatus comprises a 1# resin column and a 2# resin column; the 1# resin column and the 2# resin column are arranged in parallel, and valves are respectively arranged at two ends of the 1# resin column and the 2# resin column.
5. The apparatus for producing 2-PE by continuous conversion according to claim 1, wherein the resin column apparatus is a macroporous resin column, and the macroporous resin column is immersed by a buffer solution, and the buffer solution is a potassium phosphate solution with a pH value of 5.1 and a concentration of 0.1 mol/L; the macroporous resin is HZ-816 resin produced by Shanghai Huazhu science and technology Co.
6. A process for the continuous conversion of 2-PE comprising the steps of,
(1) Proportioning a conversion solution: the concentration of L-Phe (L-phenylalanine) in the conversion solution is 4-10 g/L, the concentration of ethanol is 5-20 g/L, and the concentration of resting cells of yeast is 6-10% w/w;
(2) Conversion reaction: putting the conversion solution in the proportion in the step (1) into a 1# reaction tank, wherein the conversion temperature is 26-35 ℃, the ventilation rate is 0.1-0.8 vvm, the stirring speed is 100-400 r/min, and the pH value is 4.0-7.0;
(3) Continuous ISPR separation: when the concentration of 2-PE in the conversion liquid in the 1# reaction tank is 2.5-3.50 g/L, starting the microfiltration membrane unit and the resin column device to perform in-situ separation of the product, namely continuous ISPR separation; extracting concentrated solution of the conversion solution in the No. 1 reaction tank after passing through the microfiltration membrane unit, returning the concentrated solution to the No. 1 reaction tank, allowing clear solution to enter a resin column device for product adsorption, and returning column passing solution passing through the resin column device to the No. 1 reaction tank through an online sterile filter;
when the concentration of the 2-PE in the conversion liquid in the 1# reaction tank is lower than 0.5g/L, ending continuous ISPR separation, and continuing the conversion reaction according to the step (2);
(4) When the concentration of the product 2-PE in the conversion solution is 2.5-3.50 g/L again, repeating the step (3) to perform continuous ISPR separation;
(5) Reaction is finished: ending the reaction to terminate the conversion when the conversion rate of the product 2-PE in the conversion liquid in the reaction tank 1# is detected to be lower than 0.15 g/(L.h) or the specified reaction time is reached; and carrying out continuous ISPR separation on the conversion solution in the No. 1 reaction tank according to the step (3);
(6) Eluting to obtain 2-PE: eluting 2-PE adsorbed in the resin column device with ethanol at a flow rate of 3.5-4.5m 3 /(m 3 (column capacity). H), collecting the eluting ethanol solution containing the product 2-PE, and measuring the content of the 2-PE in the eluting ethanol solution.
7. The method for producing 2-PE by continuous conversion according to claim 6, wherein after the step (5), the conversion solution in the reaction tank # 1 is centrifuged at 5000r/min for 10min, thalli and clear solution are respectively collected, the thalli are weighed, and then an equal amount of potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1mol/L is added for uniform mixing to obtain yeast resting cell bacterial solution, and the yeast resting cell bacterial solution is stored in a refrigerator at the temperature of 4 ℃ for standby.
8. The method for producing 2-PE by continuous conversion according to claim 6, wherein during the conversion reaction in step (2), the yeast resting cells still have a small amount of budding and need to be monitored for cell concentration, when the concentration of the yeast resting cells is higher than 10% w/w, a part of the yeast resting cells is shunted into a 2# reaction tank, and an equal volume of buffer solution is added into the 1# reaction tank, and the reaction is continued according to steps (2) - (6) after the concentration of L-Phe in the conversion solution in the 1# reaction tank is controlled to be 4-10 g/L, the concentration of ethanol is controlled to be 5-20 g/L, and the concentration of the yeast resting cells is controlled to be 6-10% w/w; the buffer solution is potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1 mol/L.
9. The method for producing 2-PE by continuous conversion according to claim 8, wherein after a certain amount of yeast resting cells are collected in the 2# reaction tank, a proper amount of buffer solution, L-Phe and ethanol are added to prepare a conversion solution, and the concentration of L-Phe in the conversion solution is controlled to be 4-10 g/L, the concentration of ethanol is controlled to be 5-20 g/L, and the concentration of yeast resting cells is controlled to be 6-10% w/w; continuing the conversion reaction according to the steps (2) - (6); the buffer solution is potassium phosphate buffer solution with the pH value of 5.1 and the concentration of 0.1 mol/L.
10. The method for producing 2-PE by continuous conversion according to claim 6, wherein in the step (6), 2-PE adsorbed in the resin column apparatus is eluted with 1.5 to 2.5 times of ethanol, and the concentration of 2-PE in the eluted ethanol is 25 to 80g/L.
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