Immobilized tyrosine ammonia-lyase genetically engineered microbial cell and preparation and application thereof

Abstract

The present application relates to the technical field of bioengineering, and in particular to a kind of immobilized tyrosine ammonia lyase genetically engineered bacteria microbial cell and its preparation and application.The wet bacteria body is mixed with Gly-NaOH buffer solution first, to obtain bacterial suspension;The wet bacteria body is the wet bacteria body of tyrosine ammonia lyase containing engineering bacteria;Then hydroxylated modified diatomite is mixed with bacterial suspension, to obtain mixture;Finally, PEI aqueous solution is added in the mixture for once crosslinking treatment, then glutaraldehyde is added for twice crosslinking treatment, and the immobilized tyrosine ammonia lyase genetically engineered bacteria microbial cell is obtained after treatment.The immobilized tyrosine ammonia lyase genetically engineered bacteria microbial cell is recycled in the present application, and the substrate conversion rate is more than 70% when repeated reaction is 20 batches, and the catalyst still retains more than 70% of initial enzyme activity.The immobilized catalyst has the industrial application potential of biocatalytic production of p-coumaric acid.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an immobilized tyrosine ammonia-lysin genetically engineered microbial cell and its preparation and application. Background Technology

[0002] p-Coumaric acid, also known as hydroxycinnamic acid, possesses antioxidant, anti-inflammatory, and cardiovascular disease prevention properties and is a precursor to many important compounds. In recent years, due to the booming development of the semiconductor industry, p-coumaric acid has also played a crucial role, serving as a key raw material for photoresists. The production of p-coumaric acid mainly involves three methods: plant extraction, chemical synthesis, and microbial synthesis. Plant-derived p-coumaric acid is unsuitable for large-scale industrial production due to its long cultivation cycle and susceptibility to seasonal climate changes; chemical synthesis suffers from harsh reaction conditions and significant pollution. Microbial production, however, offers advantages such as low cost, high controllability, and environmental friendliness, demonstrating significant development potential and application value. In vivo, p-coumaric acid can be generated from phenylalanine under the catalysis of phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H), or directly from tyrosine under the catalysis of tyrosine ammonia-lyase (TAL). C4H is a cytochrome P450 enzyme that is difficult to express functionally through prokaryotic expression systems, increasing the difficulty of catalysis. In contrast, the deamination of tyrosine to coumaric acid under the catalysis of TAL is simpler.

[0003] Tyrosine ammonia-lyases can exhibit high catalytic performance under suitable reaction conditions. However, in industrial production, the activity of free enzymes decreases significantly, they exhibit poor stability, are not reusable, and are difficult to separate, greatly reducing their application in actual production. To overcome these drawbacks of free enzymes in practical production, they can be immobilized. Immobilized enzymes (cells) are enzyme cells fixed into highly active, water-insoluble, granular immobilized formulations using physical and chemical methods, enabling continuous, automated, and low-cost production. For example, Sts-TAL, discovered in Streptomyces sp. NRRL F-4489, showed only a 58.6% conversion rate for a substrate concentration of 4.92 g / L. (Peiwu C, Weihong Z, YongQ, et al. Characterization of two new aromatic amino acid lyases from actinomycetes for highly efficient production of p-coumaric acid. [J]. Bioprocess and biosystems engineering, 2020, 43 (7): 1287-1298.).

[0004] In recent years, the increasing demand for photoresists and skincare products has spurred the booming development of the coumaric acid industry, leading to a significant need for immobilized tyrosine ammonia-lyase preparations. However, the market for immobilized tyrosine ammonia-lyase preparations is still in its early stages. Therefore, researching a low-cost, high-performance, and industrially scalable immobilization method is of great practical significance. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an immobilized tyrosine ammonia-lyase genetically engineered microbial cell and its preparation and application.

[0006] The objective of this invention can be achieved through the following technical solutions: The first objective of this invention is to provide a method for preparing immobilized tyrosine ammonia-lysin-derived genetically engineered bacterial cells, comprising the following steps: (S1) Mix the wet bacterial cells with Gly-NaOH buffer to obtain a bacterial suspension; The wet bacterial cell is a wet bacterial cell of engineered bacteria containing tyrosine ammonia-lysin; (S2) The modified diatomaceous earth is mixed with the bacterial suspension prepared in step (S1) to obtain a mixture; The modified diatomaceous earth is hydroxylated modified diatomaceous earth; (S3) Add PEI aqueous solution to the mixture prepared in step (S2) for a first cross-linking treatment, then add glutaraldehyde for a second cross-linking treatment, and the post-treatment yields immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells.

[0007] In one embodiment of the present invention, in step (S1), the ratio of the wet bacterial cells to Gly-NaOH buffer is 1 g: 80~120 mL; The engineered bacteria containing tyrosine ammonia-lysin is recombinant Escherichia coli. E. coli BL21(DE3) / pET-28a(+)-FC.

[0008] In one embodiment of the present invention, the recombinant Escherichia coli E. coli BL21(DE3) / pET-28a(+)-FC was prepared by the following method: The gene with the nucleotide sequence shown in SEQ ID NO.1 was added to plasmid pET-28a(+), which was then introduced into the host to obtain recombinant Escherichia coli. E. coli BL21(DE3) / pET-28a(+)-FC.

[0009] In one embodiment of the present invention, in step (S2), the ratio of modified diatomaceous earth to bacterial suspension is 1 g: 100~200 mL; During the mixing process, the temperature is 20~40 ℃, the rotation speed is 200~1000 rpm, and the time is 1~30 min; The modified diatomaceous earth was prepared by the following method: Diatomaceous earth was mixed with sulfuric acid and oxidized to obtain oxidized diatomaceous earth. The oxidized diatomaceous earth was then slurried and calcined to obtain modified diatomaceous earth.

[0010] Diatomaceous earth, primarily composed of silicon dioxide, is a stable and inexpensive natural adsorbent widely used in wastewater treatment, air purification, and the immobilization of biocatalysts due to its numerous orderly arranged pores and large specific surface area. The hydroxyl functional groups on the diatomaceous earth surface play a major role in adsorption. To further enhance its adsorption performance and stability, we modify diatomaceous earth through acid washing, oxidant oxidation, and pore expansion. This modification increases the number of oxygen-containing groups on the diatomaceous earth surface, improving its hydrophilicity, and also increases its specific surface area and pore volume ratio, thereby increasing its adsorption capacity. In essence, the modification of diatomaceous earth increases the number of surface functional groups or alters its chemical bonds, significantly enhancing its adsorption capacity.

[0011] In one embodiment of the present invention, the volume ratio of diatomaceous earth to sulfuric acid is 1:1~3; During the oxidation process, the temperature is 25~60 ℃ and the time is 2~4 h; During the roasting process, the temperature is 400~600 ℃ and the time is 2~4 h.

[0012] In one embodiment of the present invention, in step (S3), the final concentration of PEI is 0.06~0.1%; The final concentration of glutaraldehyde is 0.1% to 0.25%. During a single crosslinking process, the temperature is 25~40 ℃, the rotation speed is 100~260 rpm, and the time is 0.25~1.5 h; During the secondary crosslinking process, the temperature is 25~40 ℃, the rotation speed is 100~260 rpm, and the time is 0.25~1.5 h.

[0013] The second objective of this invention is to provide an immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cell, prepared by the method described above.

[0014] A third objective of this invention is to provide the application of immobilized tyrosine ammonia-lyase genetically engineered microbial cells in the preparation of p-coumaric acid.

[0015] A fourth objective of this invention is to provide a method for preparing p-coumaric acid, comprising the following steps: L-tyrosine solution was mixed with microbial cell solution of immobilized tyrosine ammonia-lysin-derived genetically engineered bacteria and reacted, followed by post-treatment to obtain p-coumaric acid.

[0016] In one embodiment of the present invention, the concentration ratio of the L-tyrosine solution to the immobilized tyrosine ammonia-lysin genetically engineered bacterial cell solution is 1:0.5~2:; During the reaction, the temperature was room temperature and the time was 6~120 h; The post-processing is as follows: After the reaction was completed, the immobilized tyrosine ammonia-lyase genetically engineered microbial cells were washed twice, and the supernatants from each batch were combined. The pH was adjusted to about 4, and crystals were grown. The mixture was then filtered and vacuum dried to obtain p-coumaric acid.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for immobilizing genetically engineered tyrosine ammonia-lyase-containing microbial cells using modified diatomaceous earth as a carrier, achieving a total enzyme activity recovery of >78% in the resulting immobilized tyrosine ammonia-lyase-containing microbial cells. Immobilized *Escherichia coli* is also used. E. coliBL21(DE3) / pET-28a(+)-FC cells were used as a biocatalyst for the batch-catalyzed production of coumaric acid. With a substrate concentration of 10 g / L and a reaction time of 6 h, the product yield was 100% complete conversion. The immobilized tyrosine ammonia-lyase genetically engineered microbial cells were recovered and reused for 20 batches, with substrate conversion rates consistently exceeding 70%, and the catalyst retaining over 70% of its initial enzyme activity. This immobilized catalyst demonstrates potential for industrial application in the biocatalytic production of coumaric acid. Attached Figure Description

[0018] Figure 1 Figure showing the temperature stability results of free cells and microbial cells with immobilized tyrosine ammonia-lysin-derived genetically engineered bacteria. Figure 2 Figure showing the pH stability results of free cells and genetically engineered bacteria with immobilized tyrosine ammonia-lysing enzymes. Figure 3 Schematic diagram of a batch of microbial cells for repeated use of genetically engineered bacteria with immobilized tyrosine ammonia-lysin; Figure 4 This is the liquid phase spectrum of p-coumaric acid. Detailed Implementation

[0019] This invention provides a method for preparing immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells, comprising the following steps: (S1) Mix the wet bacterial cells with Gly-NaOH buffer to obtain a bacterial suspension; The wet bacterial cell is a wet bacterial cell of engineered bacteria containing tyrosine ammonia-lysin; (S2) The modified diatomaceous earth is mixed with the bacterial suspension prepared in step (S1) to obtain a mixture; The modified diatomaceous earth is hydroxylated modified diatomaceous earth; (S3) Add PEI aqueous solution to the mixture prepared in step (S2) for a first cross-linking treatment, then add glutaraldehyde for a second cross-linking treatment, and the post-treatment yields immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells.

[0020] Further, in step (S1), the ratio of the wet bacterial cells to Gly-NaOH buffer is 1 g: 80~120 mL; The engineered bacteria containing tyrosine ammonia-lysin is recombinant Escherichia coli. E. coli BL21(DE3) / pET-28a(+)-FC.

[0021] Furthermore, the recombinant Escherichia coli E. coli BL21(DE3) / pET-28a(+)-FC was prepared by the following method: The gene with the nucleotide sequence shown in SEQ ID NO.1 was added to plasmid pET-28a(+), which was then introduced into the host to obtain recombinant Escherichia coli. E. coli BL21(DE3) / pET-28a(+)-FC.

[0022] Furthermore, in step (S2), the ratio of modified diatomaceous earth to bacterial suspension is 1 g: 100~200 mL; During the mixing process, the temperature is 20~40 ℃, the rotation speed is 200~1000 rpm, and the time is 1~30 min; The modified diatomaceous earth was prepared by the following method: Diatomaceous earth was mixed with sulfuric acid and oxidized to obtain oxidized diatomaceous earth. The oxidized diatomaceous earth was then slurried and calcined to obtain modified diatomaceous earth.

[0023] Diatomaceous earth, primarily composed of silicon dioxide, is a stable and inexpensive natural adsorbent widely used in wastewater treatment, air purification, and the immobilization of biocatalysts due to its numerous orderly arranged pores and large specific surface area. The hydroxyl functional groups on the diatomaceous earth surface play a major role in adsorption. To further enhance its adsorption performance and stability, we modify diatomaceous earth through acid washing, oxidant oxidation, and pore expansion. This modification increases the number of oxygen-containing groups on the diatomaceous earth surface, improving its hydrophilicity, and also increases its specific surface area and pore volume ratio, thereby increasing its adsorption capacity. In essence, the modification of diatomaceous earth increases the number of surface functional groups or alters its chemical bonds, significantly enhancing its adsorption capacity.

[0024] Furthermore, the volume ratio of diatomaceous earth to sulfuric acid is 1:1~3; During the oxidation process, the temperature is 25~60 ℃ and the time is 2~4 h; During the roasting process, the temperature is 400~600 ℃ and the time is 2~4 h.

[0025] Furthermore, in step (S3), the final concentration of PEI is 0.06~0.1%; The final concentration of glutaraldehyde is 0.1% to 0.25%. During a single crosslinking process, the temperature is 25~40 ℃, the rotation speed is 100~260 rpm, and the time is 0.25~1.5 h; During the secondary crosslinking process, the temperature is 25~40 ℃, the rotation speed is 100~260 rpm, and the time is 0.25~1.5 h.

[0026] This invention provides an immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cell, which is prepared by the above method.

[0027] This invention provides the application of immobilized tyrosine ammonia-lyase genetically engineered microbial cells in the preparation of p-coumaric acid.

[0028] This invention provides a method for preparing p-coumaric acid, comprising the following steps: L-tyrosine solution was mixed with microbial cell solution of immobilized tyrosine ammonia-lysin-derived genetically engineered bacteria and reacted, followed by post-treatment to obtain p-coumaric acid.

[0029] Furthermore, the concentration ratio of the L-tyrosine solution to the immobilized tyrosine ammonia-lysin-derived engineered bacterial cell solution is 1:0.5~2:; During the reaction, the temperature was room temperature and the time was 6~120 h; The post-processing is as follows: After the reaction was completed, the immobilized tyrosine ammonia-lyase genetically engineered microbial cells were washed twice, and the supernatants from each batch were combined. The pH was adjusted to about 4, and crystals were grown. The mixture was then filtered and vacuum dried to obtain p-coumaric acid.

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0031] In the following examples, enzyme activity recovery rate refers to the percentage of total enzyme activity after immobilization to total enzyme activity before immobilization, which is used to measure the degree of enzyme loss during operation; unless otherwise specified, all reagents used are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0032] Example 1 This embodiment provides a method for preparing modified diatomaceous earth, as detailed below: Take an appropriate amount of natural diatomaceous earth and add it to 2 times the volume of H2SO4 (concentration 60% (v / v)). Sonicate at 60 ℃ for 3 h to increase the oxygen content on the surface of the diatomaceous earth. Cool to room temperature, filter, and wash with deionized water until neutral to obtain oxidized diatomaceous earth. Add an equal volume of water to the oxidized diatomaceous earth prepared above and stir to form a slurry. Then place it in a muffle furnace and calcine at 500 °C for 3 h. Then filter it with qualitative filter paper, dry it (60 °C, overnight), and then sieve it through a 300-mesh sieve to prepare modified diatomaceous earth. Dry and store it for later use.

[0033] Example 2 This embodiment provides recombinant Escherichia coli. E. coliThe construction, preparation of wet cells, and performance determination of BL21(DE3) / pET-28a(+)-FC are detailed below: (1) Recombinant Escherichia coli E. coli Construction of BL21(DE3) / pET-28a(+)-FC: columnar flavonoids Flavobacterium columnare The tyrosine ammonia-lyase gene (nucleotide sequence shown in SEQ ID NO. 1) was inserted into the pET-28a(+) plasmid (insertion sites were BamHI and HindIII, synthesized by Shanghai Jierui Biotechnology Co., Ltd.) to prepare the recombinant plasmid pET-28a(+)-FC. The recombinant plasmid pET-28a(+)-FC was introduced into the host. E. coli Recombinant Escherichia coli was obtained from BL21(DE3). E. coli BL21(DE3) / pET-28a(+)-FC.

[0034] The sequence of SEQ ID NO.1 is as follows (5'-3'): (2) Preparation of wet mycelium Recombinant E. coli E. coli BL21(DE3) / pET-28a(+)-FC was inoculated into an agar slant containing 50 μg / mL kanamycin and cultured at 37 ℃ for 12 h to obtain slant cells; The slant culture medium consisted of 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, and 15 g / L agar powder, with deionized water as the solvent. The slant culture cells were inoculated into a seed culture medium containing 50 μg / mL kanamycin and cultured at 37 ℃ and 200 rpm for 12 h to obtain the seed culture. The seed culture medium consisted of 10 g / L peptone, 10 g / L sodium chloride, and 5 g / L yeast extract, with deionized water as the solvent. The seed culture was then inoculated into the fermentation medium at a volume concentration of 1%, and cultured at 37 ℃ and 220 rpm until OD reached. 600 When the concentration reaches 0.6~0.8, add 0.05 mM IPTG to the final concentration, induce for 20 h at 18 ℃ and 220 rpm, centrifuge at 4 ℃ and 12000 rpm for 10 min, discard the supernatant, wash three times with 8 g / L sodium chloride aqueous solution to obtain wet cells, and store at -20 ℃ for later use. The fermentation medium consisted of 10 g / L peptone, 10 g / L sodium chloride, and 5 g / L yeast extract, with deionized water as the solvent.

[0035] Example 3 This embodiment provides a method for preparing genetically engineered microbial cells immobilized with tyrosine ammonia-lysin, including the following steps: Prepare a Gly-NaOH buffer solution (100 mM) with a pH of 10 using distilled water. Weigh 0.2 g of the wet bacterial cells prepared in Example 2 and add them to 18.6 mL of the Gly-NaOH buffer solution (100 mM) with a pH of 10. Stir thoroughly to obtain a bacterial suspension.

[0036] Accurately weigh 0.1 g of natural diatomaceous earth and 0.1 g of the modified diatomaceous earth prepared in Example 1, and add them to the above bacterial suspension for mixing. Stir in a water bath at 25 ℃ and 200 rpm for 30 min. Then add 0.6 mL of PEI aqueous solution with a mass concentration of 3.33% (final concentration 0.1%) and stir in a water bath at 25 ℃ and 200 rpm for crosslinking for 0.5 h. Then add 0.8 mL of glutaraldehyde aqueous solution with a mass concentration of 2.5% (final concentration 0.1%) and stir in a water bath at 25 ℃ and 200 rpm for crosslinking for 0.5 h. The immobilization is then complete. Filter to remove the supernatant. Wash the resulting filter cake twice with Gly-NaOH buffer (pH 10, 100 mM). After removing excess water by filtration, obtain the immobilized tyrosine ammonia-lyase genetically engineered bacterial cells and store them at 4 ℃ for later use.

[0037] The immobilized enzyme activity is defined as the amount of enzyme required to catalyze the reaction of L-tyrosine as a substrate for 15 min at 45 ℃ and 1000 rpm to generate 1 μmol of coumaric acid per minute, which is defined as one unit of enzyme activity (U).

[0038] Detection method of the product: The liquid chromatography column was a reversed-phase C18 column (Diamonsil plus, 4.6 mm*250 mm*5 μm); the mobile phase (gradient elution) was 25% acetonitrile and 75% liquid water (containing 0.1% phosphoric acid); the flow rate was 1 min / mL; the column temperature was 25 ℃; the detection wavelength of L-tyrosine was 280 nm; the retention time was 2.778 min; the detection wavelength of p-coumaric acid was 310 nm; the retention time was 8.942 min.

[0039] In this embodiment, the enzyme activity recovery rate of the obtained immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells is shown in Table 1.

[0040] Table 1. Enzyme activity recovery rate of immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells. Example 4 This embodiment provides a method for preparing genetically engineered microbial cells immobilized with tyrosine ammonia-lysin, including the following steps: Prepare a Gly-NaOH buffer solution (100 mM) with a pH of 10 using distilled water. Weigh 0.2 g of the wet bacterial cells prepared in Example 2 and add them to 18.6 mL of the Gly-NaOH buffer solution (100 mM) with a pH of 10. Stir thoroughly to obtain a bacterial suspension.

[0041] Add 0.1 g of the modified diatomaceous earth prepared in Example 1 to the above bacterial suspension and mix. Stir in a water bath at 25 °C and 200 rpm for 30 min. Then add 0.6 mL of PEI aqueous solution with a mass concentration of 3.33% (final concentration 0.1%) and stir in a water bath at 25 °C and 200 rpm for crosslinking for 0.5 h. Then add glutaraldehyde aqueous solution with a mass concentration of 2.5% (final concentrations of 0.02%, 0.04%, 0.06%, 0.08%, 0.10%, 0.15%, 0.2%, 0.25%, and 0.3%) and stir in a water bath at 25 °C and 200 rpm for crosslinking for 0.5 h. The immobilization is then complete. Filter to remove the supernatant. Wash the resulting filter cake twice with Gly-NaOH buffer (pH 10, 100 mM). After removing excess water by filtration, obtain the immobilized tyrosine ammonia-lyase genetically engineered bacterial cells and store them at 4 °C for later use.

[0042] In this embodiment, the enzyme activity recovery rate of the obtained immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells is shown in Table 2.

[0043] Table 2 Enzyme activity recovery rate of immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells Example 5 This embodiment provides a method for preparing genetically engineered microbial cells immobilized with tyrosine ammonia-lysin, including the following steps: Prepare a Gly-NaOH buffer solution (100 mM) with a pH of 10 using distilled water. Weigh 0.2 g of the wet bacterial cells prepared in Example 2 and add them to 18.6 mL of the Gly-NaOH buffer solution (100 mM) with a pH of 10. Stir thoroughly to obtain a bacterial suspension.

[0044] 0.1 g of the modified diatomaceous earth prepared in Example 1 was added to the above bacterial suspension and mixed. The mixture was stirred in a water bath at 25 °C and 200 rpm for 30 min. Then, PEI aqueous solution (final concentrations of 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.15%, and 0.2%, respectively) was added and crosslinked in a water bath at 25 °C and 200 rpm for 0.5 h. Then, 0.8 mL of 5% glutaraldehyde aqueous solution (final concentration 0.2%) was added and crosslinked in a water bath at 25 °C and 200 rpm for 0.5 h, at which point the immobilization was complete. The supernatant was removed by filtration, and the resulting filter cake was washed twice with Gly-NaOH buffer (pH 10, 100 mM). After removing excess water by filtration, the immobilized tyrosine ammonia-lyase genetically engineered bacterial cells were obtained and stored at 4 °C for later use.

[0045] In this embodiment, the enzyme activity recovery rate of the obtained immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells is shown in Table 3.

[0046] Table 3 Enzyme activity recovery rate of immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells Example 6 This embodiment provides a method for preparing genetically engineered microbial cells immobilized with tyrosine ammonia-lysin, including the following steps: Prepare a Gly-NaOH buffer solution (100 mM) with a pH of 10 using distilled water. Weigh 0.2 g of the wet bacterial cells prepared in Example 2 and add them to 18.6 mL of the Gly-NaOH buffer solution (100 mM) with a pH of 10. Stir thoroughly to obtain a bacterial suspension.

[0047] Accurately weigh 0.1 g of the modified diatomaceous earth prepared in Example 1 and add it to the above bacterial suspension for mixing. Stir in a water bath at 25 ℃ and 200 rpm for 30 min. Then add 3.33% PEI aqueous solution (final concentration 0.08%) and stir in a water bath at 25 ℃ for crosslinking for 0.5 h (rotation speeds of 100, 140, 180, 200, 220, and 260 rpm, respectively). Then add 0.8 mL of 5% glutaraldehyde aqueous solution (final concentration 0.2%) and stir in a water bath at 25 ℃ and 200 rpm for crosslinking for 0.5 h. Immobilization is then complete. Filter to remove the supernatant. Wash the resulting filter cake twice with Gly-NaOH buffer (pH 10, 100 mM). After removing excess water by filtration, obtain immobilized tyrosine ammonia-lyase genetically engineered bacterial cells and store them at 4 ℃ for later use.

[0048] In this embodiment, the enzyme activity recovery rate of the obtained immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells is shown in Table 4.

[0049] Table 4 Enzyme activity recovery rate of immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells Example 7 This embodiment provides a method for preparing genetically engineered microbial cells immobilized with tyrosine ammonia-lysin, including the following steps: Prepare a Gly-NaOH buffer solution (100 mM) with a pH of 10 using distilled water. Weigh 0.2 g of the wet bacterial cells prepared in Example 2 and add them to 18.6 mL of the Gly-NaOH buffer solution (100 mM) with a pH of 10. Stir thoroughly to obtain a bacterial suspension.

[0050] Accurately weigh 0.1 g of the modified diatomaceous earth prepared in Example 1 and add it to the above bacterial suspension for mixing. Stir in a water bath at 25 ℃ and 200 rpm for 30 min. Then add 3.33% PEI aqueous solution (final concentration 0.08%) and stir in a water bath at 25 ℃ and 220 rpm for crosslinking for 0.5 h. Then add 0.8 mL of 5% glutaraldehyde aqueous solution (final concentration 0.2%) and stir in a water bath at 25 ℃ for crosslinking for 0.5 h (rotation speeds of 100, 140, 180, 200, 220, and 260 rpm respectively). Immobilization is then complete. Filter to remove the supernatant. Wash the resulting filter cake twice with Gly-NaOH buffer (pH 10, 100 mM). After removing excess water by filtration, obtain immobilized tyrosine ammonia-lyase genetically engineered bacterial cells and store them at 4 ℃ for later use.

[0051] In this embodiment, the enzyme activity recovery rate of the obtained immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells is shown in Table 5.

[0052] Table 5 Enzyme activity recovery rate of immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells Example 8 This embodiment provides a method for preparing genetically engineered microbial cells immobilized with tyrosine ammonia-lysin, including the following steps: Prepare a Gly-NaOH buffer solution (100 mM) with a pH of 10 using distilled water. Weigh 0.2 g of the wet bacterial cells prepared in Example 2 and add them to 18.6 mL of the Gly-NaOH buffer solution (100 mM) with a pH of 10. Stir thoroughly to obtain a bacterial suspension.

[0053] Accurately weigh 0.1 g of the modified diatomaceous earth prepared in Example 1 and add it to the above bacterial suspension for mixing. Stir in a water bath at 25 ℃ and 200 rpm for 30 min. Then add 3.33% PEI aqueous solution (final concentration 0.08%) and stir in a water bath at 25 ℃ and 220 rpm for crosslinking (times of 0.25, 0.5, 0.75, 1, 1.25, and 1.5 h, respectively). Then add 0.8 mL of 5% glutaraldehyde aqueous solution (final concentration 0.2%) and stir in a water bath at 25 ℃ and 200 rpm for crosslinking for 0.5 h, at which point immobilization is complete. Filter to remove the supernatant. Wash the resulting filter cake twice with Gly-NaOH buffer (pH 10, 100 mM). After removing excess water by filtration, obtain immobilized tyrosine ammonia-lyase genetically engineered bacterial cells and store them at 4 ℃ for later use.

[0054] In this embodiment, the enzyme activity recovery rate of the obtained immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells is shown in Table 6.

[0055] Table 6 Enzyme activity recovery rate of immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells Example 9 This embodiment provides a method for preparing genetically engineered microbial cells immobilized with tyrosine ammonia-lysin, including the following steps: Prepare a Gly-NaOH buffer solution (100 mM) with a pH of 10 using distilled water. Weigh 0.2 g of the wet bacterial cells prepared in Example 2 and add them to 18.6 mL of the Gly-NaOH buffer solution (100 mM) with a pH of 10. Stir thoroughly to obtain a bacterial suspension.

[0056] Accurately weigh 0.1 g of the modified diatomaceous earth prepared in Example 1 and add it to the above bacterial suspension for mixing. Stir in a water bath at 25 ℃ and 200 rpm for 30 min. Then add 3.33% PEI aqueous solution (final concentration 0.08%) and stir in a water bath at 25 ℃ and 220 rpm for crosslinking for 0.75 h. Then add 0.8 mL of 5% glutaraldehyde aqueous solution (final concentration 0.2%) and stir in a water bath at 25 ℃ and 200 rpm for crosslinking (times of 0.25, 0.5, 0.75, 1, 1.25, and 1.5 h, respectively). Immobilization is then complete. Filter to remove the supernatant. Wash the resulting filter cake twice with Gly-NaOH buffer (pH 10, 100 mM). After removing excess water by filtration, obtain immobilized tyrosine ammonia-lyase genetically engineered bacterial cells and store them at 4 ℃ for later use.

[0057] In this embodiment, the enzyme activity recovery rate of the obtained immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells is shown in Table 7.

[0058] Table 7 Enzyme activity recovery rate of immobilized tyrosine ammonia-lysin-derived genetically engineered microbial cells Example 10 This embodiment provides free cells (referring to recombinant E. coli). E. coli The thermostability determination of BL21(DE3) / pET-28a(+)-FC (unimmobilized, the same below) or immobilized tyrosine ammonia-lyase genetically engineered microbial cells (hereinafter referred to as "immobilized cells") is as follows: Take 0.1 mg of free cells and immobilized cells (containing 0.1 mg of free cells), and incubate them in Gly-NaOH buffer (100 mM) at pH 10 at 50 ℃, 55 ℃, and 60 ℃ for 3 h, 6 h, 12 h, and 24 h, respectively. The activity before incubation is taken as 100%, and the residual enzyme activity after incubation is calculated by comparing it with the activity before incubation, so as to examine the temperature stability of free cells and immobilized cells.

[0059] The results are as follows Figure 1 As shown, through Figure 1 It can be observed that the residual enzyme activity of free cells after incubation at 60 °C for 24 h was 5.6%, while that of immobilized cells under the same conditions was 53.5%, indicating that the enzyme activity of immobilized cells was significantly improved.

[0060] Example 11 This embodiment provides a method for measuring the pH stability of free or immobilized cells, as detailed below: 0.1 mg of free cells and immobilized cells (containing 0.1 mg of free cells) were placed in buffer systems of 100 mM phosphate (PB) buffer (pH 6, 7, 8), 100 mM Tris-HCl buffer (pH 8, 9), and 100 mM Gly-NaOH buffer (pH 9, 9.5, 10, 10.5, 11), respectively. After incubation at 50 °C for 2 h, the residual enzyme activity was calculated by comparing the activity before incubation with the activity before incubation, with the activity before incubation as 100%, to examine the pH stability of free cells and immobilized cells.

[0061] The results are as follows Figure 2 As shown, through Figure 2 It can be observed that under acidic and neutral conditions, immobilized cells exhibit significantly higher stability than free cells. Under Gly-NaOH (pH 10) conditions, immobilized cells retain 80% of residual enzyme activity, representing the lowest inactivation rate at the optimal reaction pH. Under PB (pH 6) conditions, immobilized cells also retain 58% of residual enzyme activity, while free cells retain only 20% of residual enzyme activity.

[0062] Example 12 This embodiment provides a determination of the reusability of immobilized cells, as detailed below: Immobilized cells (containing 60 mg of free cells) were collected and added to 6 mL of 100 mM Gly-NaOH buffer (pH 10) containing 60 mg of L-tyrosine. After reacting for 6 h, the supernatant was collected by centrifugation. The immobilized cells were washed twice with 100 mM Gly-NaOH buffer (pH 10). The immobilized cells were then resuspended in 6 mL of 100 mM Gly-NaOH buffer (pH 10) containing 60 mg of L-tyrosine and reacted for 6 h. The transformation rate was then detected.

[0063] The results are as follows Figure 3 As shown, through Figure 3 It can be observed that the immobilized cells can still achieve a substrate conversion rate of 100% after 9 cycles, and the substrate conversion rate can still reach more than 70% after 20 cycles, which shows that the immobilized cells have excellent and stable reusability.

[0064] Example 13 This embodiment provides a method for preparing coumaric acid, as detailed below: L-tyrosine was dissolved in Gly-NaOH buffer (pH=10, 100 mM) to serve as the substrate solution; Immobilized cells (containing 200 mg of free cells) were suspended in Gly-NaOH buffer (pH=10, 100 mM) as the immobilized cell solution. The substrate solution and the immobilized cell solution were mixed to obtain a mixture (the concentration of the substrate was 10 g / L and the concentration of the immobilized cells was 10 g / L). The reaction temperature was 25 °C, and 8 batches were reacted (each batch reacted for 6 h). After the reaction, the immobilized cells were washed twice with Gly-NaOH buffer (pH=10, 100 mM). The supernatants of each batch were combined, and the pH was adjusted to about 4 with 6 M HCl at 4 °C for crystal growth. After filtration and vacuum drying (50 °C, overnight), the crystals were collected to obtain a pale yellow powder solid: p-coumaric acid, with a yield of 82.3%.

[0065] High-performance liquid chromatography analysis of coumaric acid revealed a purity greater than 98%. Figure 4 ).

[0066] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for preparing immobilized tyrosine ammonia-lysine-enzyme genetically engineered microbial cells, characterized in that, Includes the following steps: (S1) Mix the wet bacterial cells with Gly-NaOH buffer to obtain a bacterial suspension; The wet bacterial cell is a wet bacterial cell of engineered bacteria containing tyrosine ammonia-lysin; (S2) The modified diatomaceous earth is mixed with the bacterial suspension prepared in step (S1) to obtain a mixture; The modified diatomaceous earth is hydroxylated modified diatomaceous earth; (S3) Add PEI aqueous solution to the mixture prepared in step (S2) for a first cross-linking treatment, then add glutaraldehyde for a second cross-linking treatment, and the post-treatment yields immobilized tyrosine ammonia-lyase genetically engineered bacterial microbial cells.

2. The method for preparing immobilized tyrosine ammonia-lysine-enzyme genetically engineered microbial cells according to claim 1, characterized in that, In step (S1), the ratio of the wet bacterial cells to Gly-NaOH buffer is 1 g: 80~120 mL; The engineered bacteria containing tyrosine ammonia-lysin is recombinant Escherichia coli. E. coli BL21(DE3) / pET-28a(+)-FC.

3. The method for preparing immobilized tyrosine ammonia-lysine-enzyme genetically engineered microbial cells according to claim 2, characterized in that, The recombinant Escherichia coli E. coli BL21(DE3) / pET-28a(+)-FC was prepared by the following method: The gene with the nucleotide sequence shown in SEQ ID NO.1 was added to the pET-28a(+) plasmid and introduced into the host to obtain recombinant Escherichia coli. E. coli BL21(DE3) / pET-28a(+)-FC.

4. The method for preparing immobilized tyrosine ammonia-lysine-enzyme genetically engineered microbial cells according to claim 1, characterized in that, In step (S2), the ratio of modified diatomaceous earth to bacterial suspension is 1 g: 100~200 mL; During the mixing process, the temperature is 20~40 ℃, the rotation speed is 200~1000 rpm, and the time is 1~30 min; The modified diatomaceous earth was prepared by the following method: Diatomaceous earth was mixed with sulfuric acid and oxidized to obtain oxidized diatomaceous earth. The oxidized diatomaceous earth was then slurried and calcined to obtain modified diatomaceous earth.

5. The method for preparing immobilized tyrosine ammonia-lysin-derived engineered bacterial microbial cells according to claim 4, characterized in that, The volume ratio of diatomaceous earth to sulfuric acid is 1:1~3; During the oxidation process, the temperature is 25~60 ℃ and the time is 2~4 h; During the roasting process, the temperature is 400~600 ℃ and the time is 2~4 h.

6. The method for preparing immobilized tyrosine ammonia-lysin-derived engineered microbial cells according to claim 1, characterized in that, In step (S3), the final concentration of PEI is 0.06~0.1%; The final concentration of glutaraldehyde is 0.1% to 0.25%. During a single crosslinking process, the temperature is 25~40 ℃, the rotation speed is 100~260 rpm, and the time is 0.25~1.5 h; During the secondary crosslinking process, the temperature is 25~40 ℃, the rotation speed is 100~260 rpm, and the time is 0.25~1.5 h.

7. A genetically engineered microbial cell containing immobilized tyrosine ammonia-lyase, characterized in that, It is prepared by any of the methods described in claims 1 to 6.

8. The application of the immobilized tyrosine ammonia-lyase genetically engineered microbial cells as described in claim 7 in the preparation of p-coumaric acid.

9. A method for preparing p-coumaric acid, characterized in that, Includes the following steps: L-tyrosine solution was mixed with microbial cell solution of immobilized tyrosine ammonia-lysin-derived genetically engineered bacteria and reacted, followed by post-treatment to obtain p-coumaric acid.

10. A method for preparing p-coumaric acid according to claim 9, characterized in that, The concentration ratio of the L-tyrosine solution to the immobilized tyrosine ammonia-lysin-derived engineered bacterial cell solution is 1:0.5~2:; During the reaction, the temperature was room temperature and the time was 6~120 h; The post-processing is as follows: After the reaction was completed, the immobilized tyrosine ammonia-lyase genetically engineered microbial cells were washed twice, the supernatants from each batch were combined, the pH was adjusted to allow crystal growth, and then the mixture was filtered and vacuum dried sequentially to obtain p-coumaric acid.

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