CN115181738B - Novel organic adhesive for embedding oleaphilic strain, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of yeast embedding, and particularly relates to novel organic adhesive for embedding an oleophilic strain, and a preparation method and application thereof. Novel organic gums for the entrapment of oleaginous strains include organic gums and entrapped yeast cells; the organic adhesive comprises an alkyl methacrylate monomer, a cross-linking agent and an initiator; the alkyl methacrylate monomer is C12-C18 alkyl methyl methacrylate. The organic adhesive has excellent oil adsorption capacity; can ensure that the oleaginous microorganisms exist in an oil layer in water-oil mixture and can continuously degrade petroleum. The invention can preserve yeast for a long time, and encrypt the strain carrying DNA information for the second time, thus realizing accurate information transmission.

Description

Novel organic glue for embedding oleophilic strains and its preparation method and application

Technical Field

The present invention belongs to the field of yeast embedding, and specifically relates to a novel organic glue for embedding oleophilic bacterial strains and a preparation method and application thereof.

Background technique

Immobilized cell technology is to fix or locate free cells in a limited spatial area by physical or chemical means, and keep their inherent catalytic activity. Immobilized biological cells can continue to proliferate, metabolize and die, and their activity remains stable. Immobilized biological cells can continue to proliferate, dormant and die, and their activity remains stable. In addition to maintaining their original recognition, binding and catalytic activities, supporting and ensuring the survival of cells/microorganisms and protecting them from harsh environments, immobilized biological cells also have the characteristics of high cell density, fast reaction rate, strong toxicity resistance, high stability, easy product separation, and continuous operation, which can greatly improve production capacity. Cell immobilization technology is often used in various biomedical and industrial applications, including probiotic delivery, biocatalysts, functional active materials, etc.

At present, cell immobilization technology plays an increasingly important role in people's lives. For example, in the purification of urban sewage, the composition of urban sewage is becoming more and more complex, which puts forward higher requirements for treatment technology. The turbidity of urban sewage is high, which is easy to affect the mass transfer of fixed bacteria. In addition, the chemical composition of urban sewage is complex, which is very destructive to the stability of fixed carriers. Zeng Yu et al. used agar, sodium alginate, sodium alginate plus activated carbon powder, polyvinyl alcohol, and polyvinyl alcohol plus activated carbon powder as carriers to embed and immobilize photosynthetic bacteria to treat urban sewage. The static test results show that the bacteria embedded with calcium alginate also show good activity. After 96 hours of sewage treatment, the COD removal rate reached more than 90%.

In addition, cell immobilization technology is also of great significance in the food production industry. Immobilized cells made by the thermosetting method of bacteria have achieved continuous production of fructose syrup. Chihata et al. used immobilized polyacrylamide hydrogel to immobilize Escherichia coli that can produce aspartase to produce L-aspartic acid, reducing production costs by 40%. Tanabe Pharmaceutical Factory in Japan used K-carrageenan gel to embed bacteria and used glutaraldehyde and hexamethylenediamine for hardening. The half-life of the immobilized bacteria obtained was 630 days, and its production capacity was 14 times higher than that of bacteria embedded in polyacrylamide gel. Biological microcapsules are an emerging cell immobilization technology. Entering the 21st century, the application of microcapsule technology in the food industry has developed rapidly. The application of microcapsule technology in the food industry has greatly promoted the transformation of the food industry from primary processing of agricultural products to advanced products. The combination of microcapsule technology with ultrafine grinding technology, biotechnology, membrane technology and hot pressure reaction technology has shown a bright prospect for the development and application of high-tech in the food industry. Cell immobilization technology is frequently used in various biomedical and industrial applications, including probiotic delivery, biocatalysts, artificial organs, and functional active materials. This strategy can serve as a support to ensure the survival of cells/microorganisms, protect them from harsh environments, improve their stability and reusability, make them easy to operate, and increase their density in bioreactors, thereby significantly improving their functionality.

So far, scientists have explored a variety of organic and inorganic encapsulation materials, including hydrogels, polypamine, polyelectrolytes, polyelectrolyte/oxidized carbon nanotube hybrids, silica, and calcium phosphate/carbonate. They are all hydrophilic for several reasons. First, mature technology makes it easy to prepare biocompatible and non-toxic hydrophilic materials, which can ensure the survival of encapsulated cells/microorganisms. Second, all cells and most microorganisms live in an aqueous environment, and hydrophilic materials can ensure the effective mass transfer of water-soluble molecules. Third, the design of biocompatible hydrophobic encapsulation materials remains a challenge. Therefore, hydrophilic materials, especially hydrogels, seem to be the current "gold standard" for encapsulating cells/microorganisms.

However, oleaginous microorganisms are essential for industrial applications. Oleaginous microorganisms such as microalgae, bacteria, fungi, and yeast can synthesize more than 20% lipids based on the cell dry weight within the cell and can even synthesize up to 70% lipids based on the cell dry weight.

First, they can degrade greasy food waste and petroleum. Oil-loving microorganisms can directly utilize cheap renewable resources such as straw-like agricultural and forestry waste, agricultural and sideline product processing waste, industrial waste and wastewater, and convert them into microbial diesel through fermentation. For example, Yarrowia lipolytica is an oil-loving yeast that can absorb various oily substances, including oil waste, animal fat waste and alkanes; Pseudomonas isolated from petroleum can degrade petroleum hydrocarbons into simple non-toxic compounds; the acyl carrier protein of marine oil-producing microorganisms, as a very conservative transport protein, can serve as an acyl donor in fatty acid metabolism and is an indispensable cofactor. The study of the structure and function of acyl carrier protein provides a basis for guiding the application and transformation of fatty acid synthesis pathways (screening of marine oil-producing microorganisms and cloning of acyl carrier protein genes); in addition, studies have found that metal ions regulate the activity of key enzymes of oil-producing microorganisms by affecting the morphology and growth of oil-producing microorganisms, and the osmotic pressure of metal ions affects the accumulation of oil in oil-producing microorganisms and affects their yield.

Second, oil-producing microorganisms can be used for fermentation and biomanufacturing. For example, Li Jianzheng et al. used waste molasses as raw material and used Rhodotorula glutinosus as the starting strain to ferment and produce oil; Xu et al. reported that the engineered Yarrowia lipophila can be used as a yeast biorefining platform to produce fuel-like molecules and oily chemicals; Zhou et al. found that Candidatusmethanoliparum can convert long-chain alkanes into methane. Therefore, it is urgent to develop hydrophobic, lipophilic and biocompatible encapsulation materials.

Third, oleaginous microorganisms have great potential in synthesizing a variety of intracellular carbon compounds. They can synthesize triacylglycerols (TAGs) that can be converted into biodiesel and jet fuel; they can also synthesize drugs such as steroid hormones and eicosanoids; and produce various industrial products such as polymers, wax esters, and hydroxy fatty acids. In addition, the fatty acid composition, triacylglycerol type, and secondary metabolites of oleaginous microorganisms have the same characteristics as their counterparts in plants. It is reported that the intracellular lipids synthesized by oleaginous microorganisms not only serve as energy storage materials, but also play an important role in cell biogenesis and the integrity of cell membranes.

Therefore, it is urgent to develop encapsulation materials with hydrophobicity, lipophilicity and biocompatibility to promote the application of these oil-containing microorganisms. In addition, as biological samples, the long-term preservation of microorganisms is of great significance, and cryopreservation is currently recognized as one of the most effective long-term preservation technologies for strains. Therefore, encapsulation materials that can freeze oleophilic strains for a long time have important application prospects. Organogels are hydrophobic materials composed of three-dimensional polymer networks that are permeated with oil or non-polar organic solvents. They have many promising properties, including lipophilicity and designability, showing potential candidates as oil-containing microbial encapsulation. However, since their preparation usually requires the use of toxic organic solvents, non-biocompatible organogel agents and harsh preparation conditions, their biocompatibility is insufficient. Therefore, the development of biocompatible organogels needs to avoid these harmful factors.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide a new organic glue for embedding oleophilic strains and its preparation method and application.

To achieve the above purpose, the technical solution adopted by the present invention is:

A novel organic glue for embedding oleophilic bacterial strains comprises an organic glue and embedded yeast cells; the organic glue comprises an alkyl methacrylate monomer, a crosslinking agent and an initiator; wherein the alkyl methacrylate monomer is an alkyl methyl methacrylate containing a long carbon side chain; and the yeast cells are oleophilic bacterial strains.

The monomer is C12-C18 alkyl methyl methacrylate.

The cross-linking degree of the organic glue is 0.5-5%.

Preferably, the cross-linking degree of the organic glue is 1-2%.

The cross-linking agent is EDGMA or TEGDMA.

The initiator is IRGACURE 819 or IRGACURE TPO. Preferably, the molar ratio of the initiator to the monomer is 0.2:100.

The present invention also includes a method for preparing the novel organic glue for embedding oleophilic strains, comprising the following steps:

S1: dissolving the initiator in the alkyl methacrylate monomer solution and mixing it evenly by ultrasonication; adding the crosslinking agent to the mixed solution and dispersing the crosslinking agent by shaking to obtain a gel precursor mixture;

S2: Yeast cells in the gel precursor mixture solution, mix them well;

S3: The mixture obtained in step S2 is transferred between two glass sheets separated by polytetrafluoroethylene; the glass sheets are placed under a xenon lamp for irradiation, and the precursor mixture forms an organic glue embedding yeast cells through photopolymerization.

The present invention also includes an application of the novel organic glue for embedding and preserving oil-loving strains and storing and transporting yeast containing DNA information.

The oleophilic strain is Yarrowia lipolytica.

The present invention also includes a method for preparing the novel organic glue for embedding oleophilic strains, comprising the following steps:

S1: dissolving the initiator in the alkyl methacrylate monomer solution and mixing it evenly by ultrasonication; adding the crosslinking agent to the mixed solution and dissolving the crosslinking agent by shaking to obtain a gel precursor mixture;

S2: Yeast cells are added to the gel precursor mixture solution and mixed; preferably, about 1×10 ⁸ yeast cells are added to 600 μL of the gel precursor solution;

S3: The mixed mixture obtained in step S2 is transferred between two glass sheets separated by polytetrafluoroethylene; the glass sheets are placed under a xenon lamp for irradiation, and the precursor mixture forms an organic glue embedding the yeast cells through photopolymerization.

The present invention also includes an application of the novel organic glue for embedding oleophilic strains and storing and transporting yeast containing DNA information. The oleophilic strains include but are not limited to Yarrowia lipolytica.

Compared with the prior art, the present invention has the following beneficial effects:

The novel organic glue used for embedding oleophilic strains overcomes the deficiency that all existing embedding materials are hydrophilic materials, and has biocompatibility, hydrophobicity and the ability to absorb organic solvents.

The organic glue is prepared by a simple and mild preparation method, using methacrylic acid molecules that are non-toxic to yeast as monomers, using an optimized ratio, and the preparation process hardly causes obvious death of the embedded organisms.

The organic glue of the present invention has excellent oil adsorption capacity, can ensure that the embedded oleophilic yeast obtains sufficient carbon source to support growth, can ensure that the oleophilic microorganisms exist in the oil layer of the water-oil mixture, and can continuously degrade oil.

The present invention utilizes multidisciplinary cross-cutting technologies such as synthetic biology and biomaterials to provide some highly biocompatible organic glues as carriers of oleophilic strains, and can also secondary encrypt strains carrying DNA information to achieve accurate transmission of information.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Organic glue preparation process diagram;

Figure 2: Schematic diagram of the hydrophobicity of organic glue;

Figure 3: Absorption performance of organic glue for organic molecules;

Figure 4: Schematic diagram of water contact angle and oil contact angle of organic glue;

Figure 5: Morphological images of yeast grown in different media for 48 hours;

Figure 6: Survival rate of yeast embedded in organic gel;

Figure 7: Survival rate of organic gel cryopreservation;

Figure 8: Schematic diagram of organic glue information storage.

Detailed ways

In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

Example 1-15: Design and preparation of organic glue: As shown in Figure 1, five different alkyl methacrylates (dodecyl/tridecyl/tetradecyl/hexadecyl/octadecyl methyl methacrylate), EDGMA and IRGACURE819 are used as monomers, crosslinkers and photoinitiators respectively; 0.2wt% of the initiator (IRGACURE 819) is dissolved in 600mL of the monomer solution and ultrasonically treated at 30°C for 10min. After ultrasonication, different doses of crosslinkers are added to the solution, and the crosslinkers are dissolved in the above solution by shaking. The crosslinking density (i.e., the molar ratio of the crosslinker to the monomer) is 1%, 1.5% and 2%, respectively. The mixed gel precursor solution is transferred between two glass sheets (thickness of 1.5mm) separated by polytetrafluoroethylene (PTFE). It is placed under the irradiation of a xenon lamp (LAB-SQLAR500, 2kW/ ^m2 ), and the precursor solution forms an organic glue by photopolymerization. The different illumination times are adjusted to 10min, corresponding to Table 1. Calculate the monomer reaction rates for different cross-linking degrees and different reaction times.

Table 1

Cut the organic glue under different light exposure time into a disc shape with a diameter of 8mm and a thickness of 1.5mm, and measure the dry weight of the completely dried sample. Then, soak the dried sample in chloroform for 48 hours to reach absorption equilibrium. Take out the sample, gently place it on weighing paper, and then place it in a fume hood for 24 hours to allow the chloroform in the organic glue to evaporate completely.

Weigh the mass of the final dried organic glue and calculate the monomer reaction rate.

Monomer reaction rate (%) = 1-(Wa-Wb)/Wa×100%; wherein Wa is the weight of the initial dry gel; Wa is the weight of the final dry gel.

test:

1. Characterization of hydrophobicity and absorption properties of organic glue

The organogel (corresponding to formula 1 in Table 1) is placed in the oil phase, the hydrogel is placed in the water phase, and then the organogel is pressed or pulled into the water phase, and the path trajectory of the organogel is recorded (Figure 2).

The oil absorption performance of the organogel was determined by weighing method. The organogel used in this experiment corresponds to the different formulations in Table 1. The organogel was punched into a disk with a diameter of 8 mm and a thickness of 1.5 mm, and the dry weight of the completely dried sample was measured. Subsequently, the dried sample was soaked in soybean oil for 72 hours to reach absorption equilibrium. Finally, the sample was taken out, the excess oil was gently removed, and the wet weight was measured at room temperature. The oil absorption rate was calculated as follows:

Oil absorption rate (%) = (Wa-Wb)/Wb×100%

Where, Wb is the weight of dry gel; Wa is the weight of wet gel.

The same method was used to measure the absorption capacity of organogels (corresponding to 1, 4, 7, 10, and 13 in Table 1) for different organic solvents, including methanol, ACN, ethanol, DMF, tetrahydrofuran, DMSO, DCM, n-hexane, toluene, and tetrahydrofuran (Figure 3). As can be seen from Figure 3, for polar molecules that are miscible with water, the absorption performance of organogels is very low. For non-polar organic solvents that are insoluble in water, organogels have excellent absorption capacity.

2. Organic glue contact angle test. We tested the water contact angle and oil contact angle of the prepared organic glue. The organic glue used corresponds to the different embodiments in Table 1. We used water and soybean oil to test the contact angle. When the water droplet or oil droplet touches the surface of the organic glue, a photo is captured, and then the angle is measured (Figure 4). As shown in Figure 4, the contact angles are all less than 45°, and the water contact angles are all greater than 106°, indicating the hydrophobic and lipophilic properties of the organic glue.

3. Monomer toxicity test. Five different monomers were added to YPO medium (3.7% v/v) to prepare composite medium. Yarrowia lipolytica seed solution was cultured in YPO medium and inoculated into five different composite mediums and YPO medium at a final concentration of OD ₆₀₀ = 0.2. The bacterial solution was cultured at 30°C and 200rpm. The morphology of yeast after 48 hours of culture was observed under a microscope. (Figure 5) It can be seen from Figure 5 that the five monomers have no obvious effect on the growth of yeast and will not affect the morphology of yeast.

4. Organic gel embedding of Yarrowia esterolyticus. About 1×10 ⁸ yeast cells were added to 600 μL of gel precursor solutions of different examples and mixed. The precursor solution containing yeast cells was transferred to a glass slide mold and then irradiated under a xenon lamp (LAB-SQLAR500, 2 kW/m ² ) to obtain an organic gel containing yeast cells.

Fluorescent yeast (genome modified with red fluorescent protein gene) was used to prepare fluorescent samples for microscopic observation. The organisms involved in other identification experiments were all Yarrowia lipolytica (ATCC 201249) without further genetic modification. In a clean mortar, the prepared organic gel containing yeast cells was chopped up and repeatedly rinsed with 1M1 PBS solution to extract the yeast in the organic gel. The obtained PBS solution containing yeast was stained using a 7012 live-dead staining kit. After staining for 30 minutes, the survival state of the yeast cells was observed under a fluorescence microscope. As shown in Figure 6, when the amount of cross-linking agent was between 1.5 and 2, the organic gel had a higher yeast survival rate (Table 2 shows the specific survival rate data), among which the organic gel prepared by Example 2 had the highest yeast survival rate. After the gel-making process, the yeast survival rate could reach 96%.

Table 2

5. Organic glue is used for cryopreservation of yeast. Organic glue containing yeast cells was prepared according to the method of the embodiment. Five kinds of organic glue were obtained from five different monomers. Since the cell metabolic rate decreases with the decrease of temperature, a metabolic stagnation state can be achieved. Therefore, cryopreservation is currently recognized as one of the most effective long-term preservation technologies for strains.

Five types of organic gels containing yeast were directly placed in a -80℃ or -196℃ system. YPO medium was used as a control group to freeze the yeast. After long-term freezing, the organic gels and the control group were taken out, the organic gels were chopped up, and the yeast after freeze-thaw was extracted with PBS solution. The yeast extracted from the organic gels and the control group was tested for survival rate by live-dead staining. The results are shown in Figure 7.

6. Organic glue is used for storage and transportation of yeast containing DNA information. Organic glue containing yeast cells was prepared according to the method of the embodiment. Five kinds of organic glue were obtained from five different monomers. Since the monomers contain side chains of different lengths, the organic glue has different phase transition properties. The organic glue prepared by C12-C14 does not undergo phase transition with temperature change, while the organic glue prepared by C16 and C18 has reduced transparency and phase transition with decreasing temperature. As can be seen from Figure 8, in the process of DNA being used as information storage, the preservation of DNA information is an important link. The experiment shows that the organic glue of the present application can be used for the storage and transportation of yeast containing DNA information.

The organic gels of Example 8 and Example 11 are used for exemplary illustration; Gel ₁₆ (Example 11) with phase change properties is selected as a carrier of yeast cells containing DNA information, and Gel ₁₄ (Example 8) without phase change properties is used to store other yeast cells that do not contain DNA information. During transportation, there is no difference between the two gels, and the information can be well protected. When it is necessary to extract information, the organic gel is placed in a 4°C refrigerator for about 2 minutes, and the organic gel containing the target information can be easily selected, and then the effective DNA information can be extracted. At the same time, the experiment shows that after 4 days of storage, the strain extracted from the organic gel is Yarrowia lipolytica (Figure 8c), indicating that after the organic gel is stored, the strain will not be contaminated, ensuring the safety of storage.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (3)

1. A novel organic glue for embedding oleophilic bacterial strains, characterized in that it comprises an organic glue and embedded yeast cells; The organic glue comprises an alkyl methacrylate monomer, a crosslinking agent and an initiator; wherein the alkyl methacrylate monomer is an alkyl methyl methacrylate containing a long carbon side chain; The yeast cell is an oleophilic strain; the oleophilic strain is Yarrowia lipolytica; The alkyl methacrylate monomer is C12 alkyl methyl methacrylate, and the degree of crosslinking is 1%, 1.5% or 2%; Or the alkyl methacrylate monomer is C13 alkyl methyl methacrylate, and the degree of crosslinking is 1%, 1.5% or 2%; Or the alkyl methacrylate monomer is C14 alkyl methyl methacrylate, and the degree of crosslinking is 1% or 1.5%; Or the alkyl methacrylate monomer is C16 alkyl methyl methacrylate, and the degree of crosslinking is 1%, 1.5% or 2%; Or the alkyl methacrylate monomer is C18 alkyl methyl methacrylate, and the degree of crosslinking is 1% or 1.5%; The cross-linking agent is EDGMA; The initiator is IRGACURE 819. 2. A method for preparing the novel organic glue for embedding oleophilic strains according to claim 1, characterized in that it comprises the following steps: S1: dissolving the initiator in the alkyl methacrylate monomer solution and uniformly mixing by ultrasonication; adding the crosslinking agent to the mixed solution and dispersing the crosslinking agent by shaking to obtain a gel precursor mixture; S2: Yeast cells in the gel precursor mixture solution, mix them well; S3: The mixture obtained in step S2 is transferred between two glass slides separated by polytetrafluoroethylene; the glass slides are placed under a xenon lamp for irradiation, and the precursor mixture forms an organic glue embedding yeast cells through photopolymerization. 3. An application of the novel organic glue according to claim 1, characterized in that it is used for embedding and preserving oil-loving strains and storing and transporting yeast containing DNA information.