CN112852797A - Immobilized yeast cell microsphere and preparation method and application thereof - Google Patents

Immobilized yeast cell microsphere and preparation method and application thereof Download PDF

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CN112852797A
CN112852797A CN201911192271.0A CN201911192271A CN112852797A CN 112852797 A CN112852797 A CN 112852797A CN 201911192271 A CN201911192271 A CN 201911192271A CN 112852797 A CN112852797 A CN 112852797A
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immobilized yeast
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单雨瑶
王竞辉
张雅萍
杨付伟
王坤
黎源
孔令晓
王庆义
周芸
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Wanhua Chemical Group Co Ltd
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Abstract

The invention discloses an immobilized yeast cell microsphere and a preparation method and application thereof. The invention discloses an immobilized yeast cell microsphere which is a saccule-shaped structure, wherein the shell is an embedding material, the interior of the microsphere is a core material containing yeast cells, and the embedding material is a product obtained by crosslinking glyceraldehyde and a polyglycine-serine block copolymer. The immobilized yeast cell microspheres provided by the invention have high embedding efficiency and small cell activity loss, can realize rapid separation of fermentation products when being used for fermenting and producing the phenethyl alcohol, and can be recycled, so that the production efficiency of the phenethyl alcohol is greatly increased, the production cost is reduced, and the immobilized yeast cell microspheres have wide application prospects in the field of fermentation.

Description

Immobilized yeast cell microsphere and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to an immobilized yeast cell microsphere, and a preparation method and application thereof.
Background
The cell immobilization technology is characterized in that microbial cells are physically or chemically immobilized on a carrier, the carrier has certain hydrophilicity but is insoluble in water, after the thalli are immobilized, normal physiological metabolism can be carried out in a specific area on the carrier, the catalytic and preparation activities of the thalli are maintained, the thalli can be recycled for multiple times, and the thalli can also maintain high stability in continuous operation. Therefore, the method is an important means for reducing the production cost, controlling the reaction process, preventing the contamination of bacteria in the fermentation process and quickly separating the product from the bacteria. The immobilized cell technology is developed on the basis of immobilized enzymes, the natural environment in cells is kept to the greatest extent, each cell is a small natural reactor, and the trouble caused by complicated steps such as cell crushing, enzyme liquid extraction and the like is reduced.
Since the fifth and sixty years of the last century, the immobilized cell technology has been continuously developed and widely used in various fields such as food, environment, and pharmaceutical industry, and more researchers pay attention to and actively explore new immobilization materials and immobilization methods due to its excellent characteristics such as convenience and reusability. Methods for immobilizing cells can be roughly classified into the following three methods:
an adsorption method: it is divided into two methods, physical adsorption and chemical adsorption. The physical adsorption method is to entrap cells in a carrier by utilizing the macroporous property of an immobilization material, or to adsorb cells to the surface of a carrier by utilizing the electrostatic attraction between the material and the surface of a microorganism. Common adsorbing materials include silica gel, quartz sand, activated carbon and the like. The chemisorption method mainly uses some active groups (for example, -NH) on the surface of the microorganism2, -COOH, etc.) and active groups on the surface of the immobilized material form ionic bonds or covalent bonds to achieve the purpose of mutual connection. Materials commonly used are carboxymethyl cellulose, TEAE cellulose, and the like. The cell immobilization method by the adsorption method is simple to operate and mild in action condition, but has higher requirements on the carrier, and the recovery rate is low due to weak adsorption force when the whole cells are adsorbed by acting force.
Embedding method: the embedding method is currently the most common immobilization method. Means that whole cells are embedded within specific microspheres or in lattice spaces by a suitable carrier material. The mixed system of the cells and the embedding material is generally uniformly dispersed by stirring. Common embedding materials include trehalose, agar, polyvinyl alcohol (PVA), chitosan, and the like. The embedding method for immobilizing the cells has the advantages of simple operation, less loss of cell activity in the process, wide application range and the like.
A crosslinking method: refers to a method for fixing cells by covalent bonding between cells without using a carrier and using a common cross-linking agent (such as glutaraldehyde). However, since the process of covalent bonding has a large loss of cell activity, it is generally used in combination with other methods for immobilizing cells, such as an entrapment-crosslinking method, an adsorption-crosslinking method, and the like.
The phenethyl alcohol is a natural essence substance, but the chemically synthesized phenethyl alcohol has the defects of large toxicity of raw materials, more byproducts and the like, and the yeast cells can convert phenylalanine into the phenethyl alcohol, so the fermentation method is a safe and efficient method for producing the phenethyl alcohol. However, like other alcohols, high-concentration phenethyl alcohol has certain toxicity to microbial cells and has an inhibitory effect on yield in the later fermentation period. Therefore, the toxicity inhibition of the phenethyl alcohol to the yeast cells must be overcome in the process of high-yield phenethyl alcohol production. In view of fermentation technology, the development of a technology for recovering phenethyl alcohol from a fermentation culture medium is a very effective method for continuously separating products from fermentation liquor and relieving inhibition. However, the major barrier of the phenylethanol separation technology is that the thalli needs to be separated from the fermentation liquor, the efficiency of separating the thalli by adopting pervaporation or centrifugation and other modes is low, or the energy consumption is high, so that the phenylethanol fermentation is greatly limitedThe development of fermentation technology. Therefore, many studies have been made to develop a novel method for separating a cell from a fermentation broth, and patent CN109455828A discloses the use of magnetic Fe for preparing immobilized cells3O4And adding diatomite to adsorb cells and increasing the embedding efficiency. However, the immobilization principle of the invention is solidification, the material strength is low, the particle size of the diatomite is about 3-5 microns, which is much smaller than the particle size of yeast cells 6-7 microns, the adsorption effect is not good, and the final embedding efficiency and the final product yield are affected. There is therefore a need for improved solutions to increase the efficiency of encapsulation and the performance of immobilized cells.
Disclosure of Invention
The invention aims to solve the technical problems that the existing immobilized yeast cells are low in embedding efficiency (generally only 80%), large in cell activity loss, difficult in the final fermentation product phenethyl alcohol recovery process, large in energy consumption and the like.
In order to solve the technical problems, the invention provides an immobilized yeast cell microsphere which is a saccule-shaped structure, wherein the shell is an embedding material, and the interior of the immobilized yeast cell microsphere is a core material containing yeast cells.
In some embodiments, in the above-described immobilized yeast cell microspheres, the embedding material is a product of cross-linking glyceraldehyde and polyglycine-serine block copolymer.
In some embodiments, the immobilized yeast cell microsphere described in any of the above, wherein the molecular weight of the polyglycine-serine block copolymer is 20kDa-100kDa, the block copolymer can be prepared by solid phase synthesis methods conventional in the art, based on the principle that amino groups and carboxyl groups on amino acids can undergo dehydration condensation reaction to form amide bonds, thereby forming a long-chain amino acid copolymer.
In some embodiments, in any of the above-described immobilized yeast cell microspheres, the inner core material of the immobilized yeast cell microspheres further comprises an adsorbent and magnetic particles;
the adsorbent is preferably Al2O3Porous particles of said Al2O3Granules of porous particlesThe diameter is preferably 6-10 microns, and the average diameter is 7 microns;
the magnetic particles are preferably Fe3O4Magnetic nanoparticles.
In some embodiments, the immobilized yeast cell microspheres of any one of the above, comprising or consisting of: the embedding material, yeast cells, the adsorbent and the magnetic particles can be prepared from, for example, a crosslinked product of a polyglycine-serine block copolymer and glyceraldehyde, yeast cells, Al2O3Porous particles and Fe3O4The magnetic nano-particles are in a sacculus-shaped structure, the shell is an embedding material, and the inner core material consists of yeast cells, an adsorbent and magnetic particles.
In some embodiments, in any of the above-mentioned immobilized yeast cell microspheres, the yeast cells can be cultured by using a conventional culture medium suitable for culturing yeast cells, and the culture solution is centrifuged and the cells are washed with sterile water to obtain yeast cells, for example, YPD culture medium can be used, shake culture is performed for 16-36 h at 30 ℃ and 250rpm, the culture solution is centrifuged for 5-15 min at 6000-8000 rpm, after centrifugation, the upper layer culture medium is discarded to obtain cells, and the cells are washed with sterile water for 2-4 times to obtain clean yeast cells for immobilization.
In some embodiments, the immobilized yeast cell microsphere of any one of the above, wherein the Fe is present in the immobilized yeast cell microsphere3O4The magnetic nano-particles are prepared according to a chemical coprecipitation method: weighing a certain amount of FeCl which is a trivalent iron salt3·6H2O and FeCl which is a divalent iron salt2·4H2Dissolving O in ultrapure water, dropwise adding NaOH aqueous solution into the iron salt solution under stirring, detecting the pH value of the solution, stopping dropwise adding the NaOH aqueous solution when the pH value is about 10, and externally adding a magnetic field to separate black Fe3O4And (4) cleaning the magnetic nanoparticles with ultrapure water until the pH is neutral, and drying in vacuum.
In some embodiments, in the immobilized yeast cell microspheres, ferric salt FeCl3·6H2O and FeCl which is a divalent iron salt2·4H2The feeding molar ratio of O is 2: 1-3: 1, the concentration of NaOH aqueous solution is 0.1-1 mol/L, and the step should be carried out under the protection of nitrogen in order to prevent oxidation in the process of dropwise adding the alkali solution.
In order to solve the technical problems, the invention also provides a preparation method of any one of the immobilized yeast cell microspheres, which comprises the step of embedding yeast cells by using the polyglycine-serine block copolymer as an embedding agent.
In some embodiments, in the above method, the embedding medium further comprises glyceraldehyde.
In some embodiments, the method of any of the above, further comprising the step of adding the adsorbent and/or the magnetic particles.
In some embodiments, the above method comprises mixing the aqueous solution of the polyglycine-serine block copolymer with yeast cells, adding Fe3O4Magnetic nanoparticles and Al2O3Porous particles are uniformly mixed to obtain a mixed solution; dripping the mixed solution into an equal volume of glyceraldehyde aqueous solution, and crosslinking at 20-30 ℃;
the aqueous solution of the polyglycine-serine block copolymer was prepared by adding a powder of the polyglycine-serine block copolymer to ultrapure water, followed by heating to promote dissolution thereof.
In some embodiments, in any of the above methods, the concentration of the aqueous solution of the polyglycine-serine block copolymer is 2% to 5%, and the molecular weight of the polyglycine-serine block copolymer is preferably 20kDa to 100 kDa; and/or
The concentration of the yeast cells in the mixed solution is 50-150 g/L; and/or
The mixed solution contains Fe3O4The concentration of the magnetic nano particles is 0.5-2 g/L; and/or
The Al in the mixed solution2O3The concentration of the porous particles is 1-5 g/L, and the Al is2O3Porous granuleThe particle size of the particles is preferably 6-10 micrometers, and the average particle size is 7 micrometers; and/or
The concentration of the glyceraldehyde aqueous solution is 5 to 10 percent; and/or
The reaction time of the crosslinking reaction is 10-60 min.
In order to solve the technical problems, the invention also provides a method for producing the phenethyl alcohol by fermentation, which comprises the step of producing the phenethyl alcohol by using any one of the immobilized yeast cell microspheres.
In some embodiments, in the above method for producing phenylethyl alcohol by fermentation, the fermentation medium for the production of phenylethyl alcohol by the fermentation of immobilized yeast cell microspheres is: 2-10 g/L KH yeast powder2PO4 2～10g/L，MgSO4 0.1～0.5g/L，(NH4)2HPO4 2～10g/L，CuCl20.01-0.1 g/L, 20-100 g/L glucose, 5-20 g/L-phenylalanine;
adding 20-200g/L of the immobilized yeast cell microspheres into a fermentation medium under the aseptic condition for fermentation culture.
In some embodiments, the method for the fermentative production of phenylethyl alcohol as described in any of the above, comprising the step of separating the immobilized yeast cell microspheres from the fermentation broth using an applied magnetic field.
In some embodiments, in any of the methods for producing phenylethyl alcohol by fermentation described above, a circulation pipeline is arranged outside a fermentation tank used in the fermentation, the fermentation liquor is externally circulated, and the phenylethyl alcohol is separated from the fermentation liquor by liquid-liquid extraction, for example, the phenylethyl alcohol in the fermentation liquor is extracted by using n-decane.
In some embodiments, in any of the methods for the fermentative production of phenylethyl alcohol described above, the phenylethyl alcohol product can be extracted by: after fermentation is carried out for 36-72 hours, concentrating the immobilized yeast cell microspheres at the bottom of a fermentation tank by using an external magnetic field, pumping out supernatant fermentation liquor by using a pump, filtering by using a filter membrane on a pipeline, feeding the liquor into an extraction tower, and adding n-decane for extraction; after the extraction is finished, the supernatant enters a rectifying tower to refine the phenethyl alcohol, the lower water phase is circulated back to the fermentation tank through another pump, and after the circulation lasts for 2-4 hours, most of the phenethyl alcohol in the fermentation liquid is extracted, so that the effect of continuously separating the phenethyl alcohol is achieved;
the immobilized yeast cell microspheres can be repeatedly used for fermentation after completing a batch of fermentation experiments, and the use times are 10-15 times.
The invention takes the composite material of two substances of glyceraldehyde and polyglycine-serine segmented copolymer as embedding agent and Al2O3Porous particles are adsorbent and Fe3O4The magnetic nano-particles are magnetic particles for preparing the immobilized yeast cell microspheres. Because glyceraldehyde molecules carry aldehyde groups, and glycine side chains carry amino groups, the glyceraldehyde molecules and the aldehyde groups can be subjected to Schiff base reaction to form crosslinking, in order to further strengthen the crosslinking effect, serine is added to a molecular chain of the polyglycine for copolymerization to form a block copolymer, and hydroxyl groups are carried on the serine side chains, so that condensation reaction can be performed with the aldehyde groups to further form crosslinking, the crosslinking efficiency can be improved, the mechanical strength of the microspheres is higher, and the microspheres can be used in the field of mechanical stirring fermentation. And, porous Al with larger grain size is used2O3The solid particles replace diatomite to adsorb cells, so that the embedding efficiency is increased. In addition, the immobilized yeast cell microspheres provided by the invention can realize the rapid separation of fermentation liquor and the microspheres through an external magnetic field, the separated fermentation supernatant can quickly enter the subsequent treatment steps, and the immobilized yeast cell microspheres can be recycled, so that the production efficiency is greatly increased, the production cost is reduced, and the immobilized yeast cell microspheres can be applied to the fermentation fields of various products and bacteria needing to be separated.
The immobilized yeast cell microspheres provided by the invention have high embedding efficiency and small cell activity loss, can realize rapid separation of fermentation products when being used for fermenting and producing the phenethyl alcohol, and can be recycled, so that the production efficiency of the phenethyl alcohol is greatly increased, the production cost is reduced, and the immobilized yeast cell microspheres have wide application prospects in the field of fermentation.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) was purchased from the Guangdong province collection of microorganisms under the accession number GIM 2.213.
Glyceraldehyde is a product of Aladdin reagent (Shanghai) Co., Ltd., product number S138916
The polyglycine-serine block copolymer is a product of gill biochemical (shanghai) ltd.
Al2O3The porous particles are products of Aladdin reagent (Shanghai) Limited, the catalog number of the products is A102005, the trade name is alumina, the particle size is 6-10 micrometers, and the average particle size is 7 micrometers.
In the following examples, "%" represents "g/100 ml" unless otherwise specified.
The embedding efficiency in the following examples was measured as follows: 1mL of uniform mixing solution (containing polyglycine-serine segmented copolymer, yeast and Fe) dropwise added into glyceraldehyde aqueous solution during preparation of immobilized cells3O4Magnetic nanoparticles and Al2O3Porous particles) diluted ten-fold and the number of yeast cells therein was measured by a cell counter and recorded as a. After yeast cells are immobilized, taking 1mL of centrifuged supernatant, diluting ten times, detecting the number of the yeast cells in the supernatant by using a cell counter, and marking the number as B;
the encapsulation efficiency is (A-B)/A.
The contents of L-phenylalanine and phenethyl alcohol in the following examples were measured as follows: performing high performance liquid chromatography by adopting an Agilent ZORBAX SB-C18 analytical chromatographic column, wherein the column temperature is 35 ℃, the PDA detection is performed, and the wavelength is 214 nm; the mobile phase is a mixed solution of 10% methanol and 90% water, and the elution is carried out at equal speed with the flow rate of 0.8 mL/min; the retention time of the L-phenylalanine peak is 4.5min, and the retention time of the phenethyl alcohol is 12.7 min.
Example 1
First, culturing thallus
Preparing YPD culture medium, sterilizing at 121 deg.C for 20min, inoculating Saccharomyces cerevisiae (Saccharomyces cerevisiae) stored at-20 deg.C, and shake culturing at 30 deg.C and 150rpm for 16 h. The culture solution was transferred to a centrifuge tube and centrifuged at 8000rpm for 10min, and then the upper layer of the culture medium was discarded. Adding sterile water to resuspend the yeast cells, centrifuging again, and cleaning for 3-4 times by using the sterile water to obtain the cleaned yeast.
II, Fe3O4Preparation of magnetic nanoparticles
Weighing ferric salt FeCl with the feeding molar ratio of 3:1 on weighing paper3·6H2O and FeCl which is a divalent iron salt2·4H2And O, dissolving in 100mL of ultrapure water to obtain an iron salt solution with the total iron content of 0.4mol/L, and continuously mechanically stirring in a three-mouth bottle. Preparing 1mol/L NaOH aqueous solution, slowly dripping the NaOH aqueous solution into the iron salt solution under the protection of nitrogen, and beginning to generate black Fe in the solution3O4And (4) precipitating. Stopping dripping NaOH aqueous solution when the pH value is about 10, and adding Fe under the condition of external magnetic field3O4Separating magnetic nanoparticles from the solution, repeatedly washing with ultrapure water to neutral pH, and vacuum drying at 50 deg.C for 24 hr to obtain dried Fe3O4Magnetic nanoparticles.
Preparation of immobilized cells
Preparing 3% aqueous solution of polyglycine-serine block copolymer (molecular weight 100kDa), mixing with the cleaned yeast in the first step, and adding the Fe prepared in the second step3O4Magnetic nanoparticles and Al2O3Porous granules (as adsorbent), wherein the final concentration of yeast is 150g/L, Fe3O4Final concentration of magnetic nanoparticles 0.5g/L, Al2O3The final concentration of the porous particles was 1g/L, and the above materials were mixed uniformly by mechanical stirring. The mixture was slowly added dropwise to an equal volume of 10% aqueous glyceraldehyde solution using a needle syringeThe crosslinking reaction was carried out at room temperature (20 ℃) for 20min to immobilize the yeast cells. And transferring the solidified mixed solution into a centrifuge tube, centrifuging at 8000rpm for 10min, removing supernatant, repeatedly washing the lower layer immobilized yeast cell microspheres with ultrapure water for 3-4 times, and storing in PBS (phosphate buffer solution) with pH of 7.4.
The prepared immobilized yeast cell microsphere is of a saccule-shaped structure, the shell is an embedding material, and the inner core material consists of yeast cells, an adsorbent and magnetic particles. The embedding efficiency is 88 percent through detection.
Fourthly, fermenting phenethyl alcohol by immobilized cells
Preparing a basic culture medium and placing the basic culture medium in a fermentation tank, wherein the basic culture medium comprises the following formula: yeast powder 2g/L, KH2PO4 10g/L、MgSO4 0.5g/L、(NH4)2HPO4 8g/L、CuCl20.05g/L and the balance of water. And (3) sterilizing at the high temperature of 121 ℃ for 20min, and then adding the immobilized yeast cell microspheres prepared in the step three into a fermentation tank to enable the final concentration to be 20 g/L. L-phenylalanine of 15g/L final concentration was added as a substrate and 80g/L glucose as a carbon source to the fermenter, and the fermentation culture was carried out at 30 ℃ and 250rpm for phenethyl alcohol. After fermentation for 48h, stopping stirring, and applying a magnetic field at the bottom of the fermentation tank to concentrate the immobilized yeast cell microspheres. Pumping out the supernatant fermentation clear liquid by using a diaphragm pump, filtering the supernatant fermentation clear liquid by using a filter membrane on a pipeline, then feeding the clear liquid into an extraction tower, and adding n-decane for extraction, wherein the volume ratio of the n-decane to the fermentation liquid is 1: 10. And (3) feeding the extracted supernatant into a rectifying tower to refine the phenethyl alcohol, circulating the lower water phase into a fermentation tank through another diaphragm pump, pumping the upper fermentation clear liquid through the diaphragm pump to perform extraction and circulation, turning off the pump after the extraction lasts for 4 hours, stopping the external circulation extraction, removing the external magnetic field to enable the immobilized yeast cell microspheres to be freely dispersed in the fermentation tank, starting glucose supplementation to 80g/L, and continuing to perform fermentation reaction. The immobilized yeast cell microspheres can be used repeatedly for 10 times. The final phenethyl alcohol yield is 1.0-1.5g/L/h and the conversion rate of L-phenylalanine is 82% through HPLC detection.
Example 2
First, culturing thallus
Preparing YPD culture medium, sterilizing at 121 deg.C for 20min, inoculating Saccharomyces cerevisiae stored at-20 deg.C to YPD culture medium, and shake culturing at 30 deg.C and 150rpm for 24 h. The culture solution was transferred to a centrifuge tube and centrifuged at 8000rpm for 10min, and then the upper layer of the culture medium was discarded. Adding sterile water to resuspend the yeast cells, centrifuging again, and cleaning for 3-4 times by using the sterile water to obtain the cleaned yeast.
II, Fe3O4Preparation of magnetic nanoparticles
Weighing ferric salt FeCl with the feeding molar ratio of 2:1 on weighing paper3·6H2O and FeCl which is a divalent iron salt2·4H2And O, dissolving in 100mL of ultrapure water to obtain an iron salt solution with the total iron content of 0.4mol/L, and continuously mechanically stirring in a three-mouth bottle. Preparing 1mol/L NaOH aqueous solution, slowly dripping the NaOH aqueous solution into the iron salt solution under the protection of nitrogen, and beginning to generate black Fe in the solution3O4And (4) precipitating. Stopping dripping NaOH aqueous solution when the pH value is about 10, and adding Fe under the condition of external magnetic field3O4Separating magnetic nanoparticles from the solution, repeatedly washing with ultrapure water to neutral pH, and vacuum drying at 50 deg.C for 24 hr to obtain dried Fe3O4Magnetic nanoparticles.
Preparation of immobilized cells
Preparing 5% aqueous solution of polyglycine-serine block copolymer (molecular weight is 80kDa), mixing with the cleaned yeast in the first step, and adding the Fe prepared in the second step3O4Magnetic nanoparticles and Al2O3Porous granules, wherein the final concentration of yeast is 50g/L, Fe3O4Final concentration of magnetic nanoparticles 1g/L, Al2O3The final concentration of the porous particles was 3g/L, and the above materials were mixed uniformly by mechanical stirring. The mixture was slowly added dropwise to an equal volume of 10% glyceraldehyde aqueous solution using a needle syringe, and crosslinking reaction was performed at room temperature (25 ℃) for 40min to immobilize the yeast cells. Transferring the solidified mixed solution into a centrifuge tube, centrifuging at 8000rpm for 10min, removing supernatant, and collecting the lower layer of immobilized yeastThe cytomicrosphere is repeatedly washed by ultrapure water for 3-4 times and then stored in PBS (phosphate buffer solution) with pH being 7.4.
The detection shows that the embedding efficiency is 97%.
Fourthly, fermenting phenethyl alcohol by immobilized cells
Preparing a basic culture medium and placing the basic culture medium in a fermentation tank, wherein the basic culture medium comprises the following formula: yeast powder 4g/L, KH2PO4 8g/L、MgSO40.4g/L、(NH4)2HPO4 6g/L、CuCl20.08g/L and the balance of water. And (3) sterilizing at the high temperature of 121 ℃ for 20min, and then adding the immobilized yeast cell microspheres prepared in the third step into a fermentation tank to enable the final concentration to be 80 g/L. L-phenylalanine of 20g/L final concentration was added as a substrate and glucose of 100g/L was added as a carbon source to the fermenter, and the fermentation culture was carried out at 30 ℃ and 250 rpm. After fermenting for 36h, stopping stirring, and applying a magnetic field at the bottom of the fermentation tank to concentrate the immobilized yeast cell microspheres. Pumping out the supernatant fermented clear liquid by using a diaphragm pump, filtering the supernatant fermented clear liquid by using a filter membrane on a pipeline, then feeding the clear liquid into an extraction tower, and adding n-decane for extraction, wherein the volume of the n-decane and the fermentation liquid is 1: 10. And (3) feeding the extracted supernatant into a rectifying tower to refine the phenethyl alcohol, circulating the lower water phase into a fermentation tank through another diaphragm pump, pumping the upper fermentation clear liquid through the diaphragm pump to perform extraction and circulation, turning off the pump after the extraction lasts for 2 hours, stopping the external circulation extraction, removing the external magnetic field to enable the immobilized yeast cell microspheres to be freely dispersed in the fermentation tank, starting glucose supplementation to 100g/L, and continuing to perform fermentation reaction. The immobilized yeast cell microspheres can be used repeatedly for 10 times. The final phenethyl alcohol yield is 1.5-2.0g/L/h and the conversion rate of L-phenylalanine is 87% through HPLC detection.
Example 3
First, culturing thallus
Preparing YPD culture medium, sterilizing at 121 deg.C for 20min, inoculating Saccharomyces cerevisiae stored at-20 deg.C to YPD culture medium, and shake culturing at 30 deg.C and 250rpm for 16 h. The culture solution was transferred to a centrifuge tube and centrifuged at 8000rpm for 10min, and then the upper layer of the culture medium was discarded. Adding sterile water to resuspend the yeast cells, centrifuging again, and cleaning for 3-4 times by using the sterile water to obtain the cleaned yeast.
II, Fe3O4Preparation of magnetic nanoparticles
Weighing ferric salt FeCl with the feeding molar ratio of 2:1 on weighing paper3·6H2O and FeCl which is a divalent iron salt2·4H2And O, dissolving in 100mL of ultrapure water to obtain an iron salt solution with the total iron content of 0.4mol/L, and continuously mechanically stirring in a three-mouth bottle. Preparing 0.7mol/L NaOH aqueous solution, slowly dripping the NaOH aqueous solution into the mixed solution of the ferric salt under the protection of nitrogen, and beginning to generate black Fe in the solution3O4And (4) precipitating. Stopping dripping NaOH aqueous solution when the pH value is about 10, and adding Fe under the condition of external magnetic field3O4Separating magnetic nanoparticles from the solution, repeatedly washing with ultrapure water to neutral pH, and vacuum drying at 50 deg.C for 24 hr to obtain dried Fe3O4Magnetic nanoparticles.
Preparation of immobilized cells
Preparing 5% aqueous solution of polyglycine-serine block copolymer (molecular weight is 20kDa), mixing with the cleaned yeast in the first step, and adding the Fe prepared in the second step3O4Magnetic nanoparticles and Al2O3Porous granules, wherein the final concentration of yeast is 150g/L, Fe3O4Final concentration of magnetic nanoparticles 0.5g/L, Al2O3The final concentration of the porous particles was 5g/L, and the above materials were mixed uniformly by mechanical stirring. The mixture was slowly added dropwise to an equal volume of a 5% aqueous solution of glyceraldehyde using a needle syringe, and crosslinking reaction was performed at room temperature (25 ℃) for 60min to immobilize the yeast cells. And transferring the solidified mixed solution into a centrifuge tube, centrifuging at 8000rpm for 10min, removing supernatant, repeatedly washing the lower layer immobilized yeast cell microspheres with ultrapure water for 3-4 times, and storing in PBS (phosphate buffer solution) with pH of 7.4.
The embedding efficiency is 93% through detection.
Fourthly, fermenting phenethyl alcohol by immobilized cells
Preparing basic culture medium, placing it in fermentation tank, basic cultureThe base formula is as follows: yeast powder 6g/L, KH2PO4 8g/L、MgSO40.5g/L、(NH4)2HPO4 2g/L、CuCl20.01g/L and the balance of water. And (3) sterilizing at the high temperature of 121 ℃ for 20min, and then adding the immobilized yeast cell microspheres prepared in the third step into a fermentation tank to enable the final concentration to be 120 g/L. L-phenylalanine of 10g/L final concentration was added as a substrate and 60g/L glucose as a carbon source to the fermenter, and the fermentation culture was carried out at 30 ℃ and 250rpm for phenethyl alcohol. After fermentation for 72h, stopping stirring, and applying a magnetic field at the bottom of the fermentation tank to concentrate the immobilized yeast cell microspheres. Pumping out the supernatant fermentation clear liquid by using a diaphragm pump, filtering the supernatant fermentation clear liquid by using a filter membrane on a pipeline, then feeding the clear liquid into an extraction tower, and adding n-decane for extraction, wherein the volume ratio of the n-decane to the fermentation liquid is 1: 10. And (3) feeding the extracted supernatant into a rectifying tower to refine the phenethyl alcohol, circulating the lower water phase into a fermentation tank through another diaphragm pump, pumping the upper fermentation clear liquid out by the diaphragm pump to perform extraction and circulation, turning off the pump after the extraction lasts for 4 hours, stopping the external circulation extraction, removing the external magnetic field to enable the immobilized yeast cell microspheres to be freely dispersed in the fermentation tank, starting glucose supplementation to 60g/L, and continuing to perform fermentation reaction. The immobilized yeast cell microspheres can be used repeatedly for 10 times. The final phenethyl alcohol yield is 1.2-1.7g/L/h and the conversion rate of L-phenylalanine is 84% by HPLC detection.
Example 4
First, culturing thallus
Preparing YPD culture medium, sterilizing at 121 deg.C for 20min, inoculating Saccharomyces cerevisiae stored at-20 deg.C to YPD culture medium, and shake culturing at 30 deg.C and 200rpm for 24 h. The culture solution was transferred to a centrifuge tube and centrifuged at 8000rpm for 10min, and then the upper layer of the culture medium was discarded. Adding sterile water to resuspend the yeast cells, centrifuging again, and cleaning for 3-4 times by using the sterile water to obtain the cleaned yeast.
II, Fe3O4Preparation of magnetic nanoparticles
Weighing ferric salt FeCl with the feeding molar ratio of 3:1 on weighing paper3·6H2O and FeCl which is a divalent iron salt2·4H2And O, dissolving in 100mL of ultrapure water to obtain an iron salt solution with the total iron content of 0.4mol/L, and continuously mechanically stirring in a three-mouth bottle. Preparing 0.1mol/L NaOH aqueous solution, slowly dripping the NaOH aqueous solution into the iron salt solution under the protection of nitrogen, and beginning to generate black Fe in the solution3O4And (4) precipitating. Stopping dripping NaOH aqueous solution when the pH value is about 10, and adding Fe under the condition of external magnetic field3O4Separating magnetic nanoparticles from the solution, repeatedly washing with ultrapure water to neutral pH, and vacuum drying at 50 deg.C for 24 hr to obtain dried Fe3O4Magnetic nanoparticles.
Preparation of immobilized cells
Preparing 5% aqueous solution of polyglycine-serine block copolymer (molecular weight 100kDa), mixing with the cleaned yeast in the first step, and adding the Fe prepared in the second step3O4Magnetic nanoparticles and Al2O3Porous granules, wherein the final concentration of yeast is 50g/L, Fe3O4Final concentration of magnetic nanoparticles was 2g/L, Al2O3The final concentration of the porous particles was 1g/L, and the above materials were mixed uniformly by mechanical stirring. The mixture was slowly added dropwise to an equal volume of an 8% aqueous solution of glyceraldehyde using a needle syringe, and crosslinking reaction was performed at room temperature (30 ℃) for 10min to immobilize the yeast cells. And transferring the solidified mixed solution into a centrifuge tube, centrifuging at 8000rpm for 10min, removing supernatant, repeatedly washing the lower layer immobilized yeast cell microspheres with ultrapure water for 3-4 times, and storing in PBS (phosphate buffer solution) with pH of 7.4.
The detection shows that the embedding efficiency is 91%.
Fourthly, fermenting phenethyl alcohol by immobilized cells
Preparing a basic culture medium and placing the basic culture medium in a fermentation tank, wherein the basic culture medium comprises the following formula: yeast powder 8g/L, KH2PO4 6g/L、MgSO40.3g/L、(NH4)2HPO4 4g/L、CuCl20.1g/L, and the balance of water. And (3) sterilizing at the high temperature of 121 ℃ for 20min, and then adding the immobilized yeast cell microspheres prepared in the third step into a fermentation tank to enable the final concentration to be 160 g/L. Adding into a fermentation tankThe final concentration of 5g/L phenylalanine as substrate, 20g/L glucose as carbon source, at 30 degrees, 250rpm under benzene ethanol fermentation culture. After fermentation for 48h, stopping stirring, and applying a magnetic field at the bottom of the fermentation tank to concentrate the immobilized yeast cell microspheres. Pumping out the supernatant fermentation clear liquid by using a diaphragm pump, filtering the supernatant fermentation clear liquid by using a filter membrane on a pipeline, then feeding the clear liquid into an extraction tower, and adding n-decane for extraction, wherein the volume ratio of the n-decane to the fermentation liquid is 1: 10. And (3) feeding the extracted supernatant into a rectifying tower to refine the phenethyl alcohol, circulating the lower water phase into a fermentation tank through another diaphragm pump, pumping the upper fermentation clear liquid through the diaphragm pump to perform extraction and circulation, turning off the pump after the extraction lasts for 3 hours, stopping the external circulation extraction, removing the external magnetic field to enable the immobilized yeast cell microspheres to be freely dispersed in the fermentation tank, starting glucose supplementation to 20g/L, and continuing to perform fermentation reaction. The immobilized yeast cell microspheres can be used repeatedly for 15 times. The final phenethyl alcohol yield is 1.2-1.8g/L/h and the conversion rate of L-phenylalanine is 85% through HPLC detection.
Example 5
First, culturing thallus
Preparing YPD culture medium, sterilizing at 121 deg.C for 20min, inoculating Saccharomyces cerevisiae stored at-20 deg.C into YPD culture medium, and shake culturing at 30 deg.C and 250rpm for 36 h. And transferring the culture solution into a centrifuge tube, centrifuging at 8000rpm for 10min, removing the upper culture medium, adding sterile water to resuspend the yeast cells, centrifuging again, and cleaning with sterile water for 3-4 times to obtain the cleaned yeast.
II, Fe3O4Preparation of magnetic nanoparticles
Weighing ferric salt FeCl with the feeding molar ratio of 2:1 on weighing paper3·6H2O and FeCl which is a divalent iron salt2·4H2And O, dissolving in 100mL of ultrapure water to obtain an iron salt solution with the total iron content of 0.4 mol/L. Mechanical stirring was continued in a three-necked flask. Preparing 0.5mol/L NaOH aqueous solution, slowly dripping the NaOH aqueous solution into the iron salt solution under the protection of nitrogen, and beginning to generate black Fe in the solution3O4And (4) precipitating. Stopping dripping NaOH aqueous solution when the pH value is about 10, and externally adding magnetismUnder the condition of field, Fe3O4Separating magnetic nanoparticles from the solution, repeatedly washing with ultrapure water to neutral pH, and vacuum drying at 50 deg.C for 24 hr to obtain dried Fe3O4Magnetic nanoparticles.
Preparation of immobilized cells
Preparing 2% aqueous solution of polyglycine-serine segmented copolymer (molecular weight is 60kDa), mixing with the cleaned yeast in the first step uniformly, and adding the Fe prepared in the second step3O4Magnetic nanoparticles and Al2O3Porous granules, wherein the final concentration of yeast is 100g/L, Fe3O4Final concentration of magnetic nanoparticles 0.5g/L, Al2O3The final concentration of the porous particles was 5g/L, and the above materials were mixed uniformly by mechanical stirring. The mixture was slowly added dropwise to an equal volume of a 5% aqueous solution of glyceraldehyde using a needle syringe, and crosslinking reaction was performed at room temperature (20 ℃) for 60min to immobilize the yeast cells. And transferring the solidified mixed solution into a centrifuge tube, centrifuging at 8000rpm for 10min, removing supernatant, repeatedly washing the lower layer immobilized yeast cell microspheres with ultrapure water for 3-4 times, and storing in PBS (phosphate buffer solution) with pH of 7.4.
The detection shows that the embedding efficiency is 90%.
Fourthly, fermenting phenethyl alcohol by immobilized cells
Preparing a basic culture medium and placing the basic culture medium in a fermentation tank, wherein the basic culture medium comprises the following formula: yeast powder 10g/L, KH2PO4 6g/L、MgSO4 0.2g/L、(NH4)2HPO4 4g/L、CuCl20.1g/L, and the balance of water. And (3) sterilizing at the high temperature of 121 ℃ for 20min, and then adding the immobilized yeast cell microspheres prepared in the third step into a fermentation tank to enable the final concentration to be 180 g/L. L-phenylalanine of 20g/L final concentration was added as a substrate and 50g/L glucose as a carbon source to the fermenter, and the fermentation culture was carried out at 30 ℃ and 250rpm for phenethyl alcohol. After fermentation for 72h, stopping stirring, and applying a magnetic field at the bottom of the fermentation tank to concentrate the immobilized yeast cell microspheres. Pumping out supernatant fermented clear liquid with a diaphragm pump, filtering with a filter membrane on a pipeline, and feeding the clear liquid into an extraction towerAdding n-decane for extraction, wherein the volume ratio of the n-decane to the fermentation liquid is 1: 10. And (3) feeding the extracted supernatant into a rectifying tower to refine the phenethyl alcohol, circulating the lower water phase into a fermentation tank through another diaphragm pump, pumping the upper fermentation clear liquid out by the diaphragm pump to perform extraction and circulation, turning off the pump after the extraction lasts for 2 hours, stopping the external circulation extraction, removing the external magnetic field to enable the immobilized yeast cell microspheres to be freely dispersed in the fermentation tank, starting glucose supplementation to 50g/L, and continuing to perform fermentation reaction. The immobilized yeast cell microspheres can be used repeatedly for 15 times. The final phenethyl alcohol yield is 1.2-2.0g/L/h and the conversion rate of L-phenylalanine is 89% through HPLC detection.
Example 6
First, culturing thallus
Preparing YPD culture medium, sterilizing at 121 deg.C for 20min, inoculating Saccharomyces cerevisiae stored at-20 deg.C to YPD culture medium, and shake culturing at 30 deg.C and 200rpm for 36 h. The culture solution was transferred to a centrifuge tube and centrifuged at 8000rpm for 10min, and then the upper layer of the culture medium was discarded. Adding sterile water to resuspend the yeast cells, centrifuging again, and cleaning for 3-4 times by using the sterile water to obtain the cleaned yeast.
II, Fe3O4Preparation of magnetic nanoparticles
Weighing ferric salt FeCl with the feeding molar ratio of 3:1 on weighing paper3·6H2O and FeCl which is a divalent iron salt2·4H2And O, dissolving in 100mL of ultrapure water to obtain an iron salt solution with the total iron content of 0.4mol/L, and continuously mechanically stirring in a three-mouth bottle. Preparing 0.1mol/L NaOH aqueous solution, slowly dripping the NaOH aqueous solution into the iron salt solution under the protection of nitrogen, and beginning to generate black Fe in the solution3O4And (4) precipitating. Stopping dripping NaOH aqueous solution when the pH value is about 10, and adding Fe under the condition of external magnetic field3O4Separating magnetic nanoparticles from the solution, repeatedly washing with ultrapure water to neutral pH, and vacuum drying at 50 deg.C for 24 hr to obtain dried Fe3O4Magnetic nanoparticles.
Preparation of immobilized cells
Preparing 2% aqueous solution of polyglycine-serine segmented copolymer (molecular weight is 20kDa), mixing with the cleaned yeast in the first step uniformly, and adding the Fe prepared in the second step3O4Magnetic nanoparticles and Al2O3Porous granules, wherein the final concentration of yeast is 150g/L, Fe3O4Final concentration of magnetic nanoparticles was 2g/L, Al2O3The final concentration of the porous particles was 1g/L, and the above materials were mixed uniformly by mechanical stirring. The mixture was slowly added dropwise to an equal volume of 10% glyceraldehyde aqueous solution using a needle syringe, and crosslinking reaction was performed at room temperature (25 ℃) for 60min to immobilize the yeast cells. And transferring the solidified mixed solution into a centrifuge tube, centrifuging at 8000rpm for 30min, removing supernatant, repeatedly washing the lower layer immobilized yeast cell microspheres with ultrapure water for 3-4 times, and storing in PBS (phosphate buffer solution) with pH of 7.4.
The detection shows that the embedding efficiency is 90%.
Fourthly, fermenting phenethyl alcohol by immobilized cells
Preparing a basic culture medium and placing the basic culture medium in a fermentation tank, wherein the basic culture medium comprises the following formula: yeast powder 10g/L, KH2PO4 2g/L、MgSO4 0.1g/L、(NH4)2HPO4 10g/L、CuCl20.08g/L and the balance of water. And (3) sterilizing at the high temperature of 121 ℃ for 20min, and then adding the immobilized yeast cell microspheres prepared in the third step into a fermentation tank to ensure that the final concentration of the immobilized yeast cell microspheres is 200 g/L. L-phenylalanine at a final concentration of 15g/L was added as a substrate and 100g/L glucose as a carbon source to a fermenter, and the fermentation culture was carried out at 30 ℃ and 250rpm for phenethyl alcohol. After fermentation for 72h, stopping stirring, and applying a magnetic field at the bottom of the fermentation tank to concentrate the immobilized yeast cell microspheres. Pumping out the supernatant fermentation clear liquid by using a diaphragm pump, filtering the supernatant fermentation clear liquid by using a filter membrane on a pipeline, then feeding the clear liquid into an extraction tower, and adding n-decane for extraction, wherein the volume ratio of the n-decane to the fermentation liquid is 1: 10. The supernatant after extraction enters a rectifying tower to refine phenethyl alcohol, the lower water phase is circulated back to a fermentation tank through another diaphragm pump, the upper fermentation supernatant is pumped out by the diaphragm pump to carry out the extraction and circulation, the pump is turned off after the duration of 4 hours, and the external circulation is stoppedPerforming ring extraction, removing the external magnetic field to enable the immobilized yeast cell microspheres to be freely dispersed in a fermentation tank, starting glucose feeding to 100g/L, and continuing to perform fermentation reaction. The immobilized yeast cell microspheres can be used repeatedly for 15 times. The final phenethyl alcohol yield is 1.0-1.5g/L/h and the conversion rate of L-phenylalanine is 84% through HPLC detection.

Claims (10)

1. An immobilized yeast cell microsphere is a balloon-shaped structure, the shell is an embedding material, and the interior is a core material containing yeast cells.
2. The immobilized yeast cell microsphere of claim 1, wherein: the embedding material is a product obtained by crosslinking glyceraldehyde and a polyglycine-serine segmented copolymer.
3. The immobilized yeast cell microsphere of claim 1, wherein: the inner core material of the immobilized yeast cell microsphere further comprises an adsorbent and magnetic particles;
the adsorbent is preferably Al2O3Porous particles;
the magnetic particles are preferably Fe3O4Magnetic nanoparticles.
4. The method for producing microspheres of immobilized yeast cells according to any one of claims 1 to 3, comprising the step of embedding yeast cells with the polyglycine-serine block copolymer as an embedding agent.
5. The method of claim 4, wherein: the embedding medium also comprises glyceraldehyde.
6. The method of claim 5, wherein: the method further comprises the step of adding the adsorbent and/or the magnetic particles.
7. The method of claim 6, wherein the step of removing the metal oxide layer comprises removing the metal oxide layer from the metal oxide layerThe method comprises the following steps: the method comprises the steps of uniformly mixing the aqueous solution of the polyglycine-serine block copolymer with yeast cells, and adding Fe3O4Magnetic nanoparticles and Al2O3Porous particles are uniformly mixed to obtain a mixed solution; and dropwise adding the mixed solution into an equal volume of glyceraldehyde aqueous solution, and crosslinking at 20-30 ℃.
8. The method of claim 7, wherein: the concentration of the aqueous solution of the polyglycine-serine segmented copolymer is 2 to 5 percent; and/or
The concentration of the yeast cells in the mixed solution is 50-150 g/L; and/or
The mixed solution contains Fe3O4The concentration of the magnetic nano particles is 0.5-2 g/L; and/or
The Al in the mixed solution2O3The concentration of the porous particles is 1-5 g/L; and/or
The concentration of the glyceraldehyde aqueous solution is 5-10%.
9. A method for the fermentative production of phenylethyl alcohol comprising the step of using immobilized yeast cell microspheres according to any one of claims 1 to 3 for fermentative production of phenylethyl alcohol.
10. The method of claim 9, wherein: the method comprises the step of separating the immobilized yeast cell microspheres from a fermentation broth by using an applied magnetic field.
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Citations (3)

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US5314814A (en) * 1985-11-15 1994-05-24 Gist-Brocades Preparation of novel immobilized biocatalysts
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CN105838703A (en) * 2016-06-07 2016-08-10 华中农业大学 Method for removing patulin in orange juice by utilizing immobilized inactivated yeast cells of magnetic microspheres and application of method

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US5314814A (en) * 1985-11-15 1994-05-24 Gist-Brocades Preparation of novel immobilized biocatalysts
CN101240075A (en) * 2008-03-07 2008-08-13 哈尔滨工业大学 Method for preparing chitosan magnetic micro-sphere and method for immobilizing yeast by using the magnetic micro-sphere
CN105838703A (en) * 2016-06-07 2016-08-10 华中农业大学 Method for removing patulin in orange juice by utilizing immobilized inactivated yeast cells of magnetic microspheres and application of method

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