Method for optimizing fermentation of Thermus thermophilus to produce photolyase at high yield
By employing a low-temperature, high-oxygen, stepped-heating, and light-induced fermentation method, along with nanoparticle assistance, the biomass and photolyase production of thermophilic bacteria were increased. This solved the problems of slow growth rate and limited oxygen mass transfer of thermophilic bacteria at high temperatures, thus achieving efficient photolyase production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-17
- Publication Date
- 2026-04-07
AI Technical Summary
Thermophilic bacteria exhibit slow growth rates, insufficient dissolved oxygen, limited oxygen mass transfer, and low intracellular enzyme expression at high temperatures, resulting in insufficient production of photolyases, which is insufficient to meet the needs of the cosmetics industry.
A low-temperature, high-oxygen, stepwise heating, and light-induced fermentation method was adopted, and nano-TiO2, nano-SiO2, or nanoscale molecular sieves were added to the fermentation broth to enhance dissolved oxygen mass transfer and promote photolyase synthesis.
This significantly increased the biomass and photolyase yield of thermophilic bacteria, solved the problems of insufficient bacterial cell count and limited oxygen mass transfer, and provided an efficient method for preparing photolyase.
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to a Thermus thermophilus optimization method, in particular to a method for optimizing Thermus thermophilus fermentation to produce photolyase in a high yield. BACKGROUND
[0002] Thermus thermophilus is an extreme thermophilic bacterium that can grow efficiently at a temperature of 55-80 DEG C and is widely distributed in hot springs, hydrothermal vents and other high-temperature environments. Its genome, membrane lipid and enzyme system are highly “heat adapted”: the GC content in DNA is as high as 68-72%, tRNA and rRNA contain a large number of GC base pairs; the membrane lipid is mainly composed of long-chain, high-saturation and branched fatty acids, and can maintain a liquid crystal state at high temperature; the protein surface is rich in charged and aromatic amino acids, and the core is more compact, thereby maintaining the conformational stability at high temperature.
[0003] Photolyase is a class of flavoprotein enzymes that depend on visible light and can repair DNA damage caused by ultraviolet light. Thermus thermophilus can breed photolyase with high heat resistance and stability due to its unique living environment. The photolyase belongs to type I CPD-photolyase, has a molecular weight of about 55 kDa, and has a catalytic center of FAD coenzyme. The coenzyme cycle is driven by visible light (350-450 nm), and the Tm value is higher than 90 DEG C. The photolyase can still maintain activity under the conditions of about 75 DEG C, pH 4-10, high salt or oxygen free radicals, and is suitable for being added to sunscreen, after-sun care and medical beauty wound care formulations, and is used for instant reversal of DNA dimers induced by ultraviolet light under blue light or natural light irradiation, reduction of erythema, photoaging and mutation risk.
[0004] However, the current wild Thermus thermophilus has a specific growth rate of only 0.15 h - about 1 / 2 of that of Escherichia coli under the conditions of 55-75 DEG C and 1-2vvm air; the DCW reaches 4 g / L, and then the oxygen supply is limited, and the biomass is easy to accumulate acetic acid, and the highest biomass is about 4.5 g / L; the cell base of the Thermus thermophilus is inherently insufficient, resulting in a small amount of secreted photolyase; the liquid viscosity of the Thermus thermophilus is low at high temperature, and the saturated dissolved oxygen is low (≈3.2 mg / L at 75 DEG C), and the synthesis of the photolyase gene Phr requires high oxygen consumption (≈2.3 g O2 per g enzyme), and the cell is in a micro-aerobic state for a long time, and the enzyme production rate is reduced by more than 40%; the wild Thermus thermophilus also lacks efficient secretion systems such as Sec / SRT, and the photolyase gene Phr must be expressed intracellularly, and the recombinant protein accounting for 1% (w / w) of the total cell protein will trigger inclusion bodies, thereby reducing the proportion of active enzyme, resulting in a small amount of directly secreted photolyase and a yield of <1 mg / L; even if the recombinant expression is carried out by Escherichia coli, the highest yield disclosed at present is only 3.25 mg / L.
[0005] Therefore, the current Thermus thermophilus exists the problems of insufficient cell base, limited oxygen mass transfer and low expression of intracellular enzyme, resulting in low fermentation level, less biomass and small amount of secreted photosynthetic reaction center protein. SUMMARY
[0006] The present application aims to provide a method for optimizing Thermus thermophilus fermentation to produce photosynthetic reaction center protein in large amount.
[0007] The technical scheme of the present application is a method for optimizing Thermus thermophilus fermentation to produce photosynthetic reaction center protein in large amount, comprising the following steps: S1, tank preparation; S2, preparation of culture medium; S3, inoculation: aseptically inoculating the seed liquid of Thermus thermophilus into the culture medium in the fermentation tank for increasing culture; S4, control of parameters of the fermentation tank: 0-6h: controlling the temperature to be 50-60℃, the dissolved oxygen to be 75-85%, and the pH to be 7.2±0.05 by adjusting the air flow; 6-6.5h: linearly increasing the temperature to 70-80℃, and synchronously decreasing the dissolved oxygen to 25-35%; 6.5-12h: controlling the temperature to be 70-80℃, and the dissolved oxygen to be 25-35%, and simultaneously irradiating with blue light with a wavelength of 405nm for 12h; 12-12.5h: rapidly cooling to a temperature of 25-35℃, and stopping aeration to obtain the final fermentation liquid; S5, harvesting: centrifuging the final fermentation liquid, collecting the cells, resuspending the cells with PBS solution, and then sequentially performing heat sinking, ice bath, secondary centrifugation and filtration to obtain the clear enzyme liquid.
[0008] In the aforementioned method for optimizing Thermus thermophilus fermentation to produce photosynthetic reaction center protein in large amount, the composition of the culture medium in step S2 comprises the following components: yeast powder 10-15g / L, peptone 5-10g / L, glycerol 2-8g / L, NaCl 1-6g / L, KH2PO40.5-2g / L, MgSO4·7H2O 0.2-1g / L, FeCl3·6H2O 0.005-0.02g / L, and the pH of the culture medium is 7.0-7.4.
[0009] In the aforementioned method for optimizing Thermus thermophilus fermentation to produce photosynthetic reaction center protein in large amount, the seed liquid of Thermus thermophilus inoculated in step S3 is obtained by culturing Thermus thermophilus in a shake flask, and the Thermus thermophilus uses the strain with the preservation number CCTCC M20252862.
[0010] In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3. 600 In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3.
[0011] In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3.
[0012] In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3.
[0013] In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3.
[0014] In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3.
[0015] In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3. -2 In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3.
[0016] In the method for optimizing the fermentation of Thermus thermophilus to produce high yield of photoreductase, the OD of the fermentation broth at the initial time in step S3 is 0.2-0.3.
[0017] Compared with the prior art, the method has the advantages that: The present application adopts a fermentation method of low-temperature high-oxygen-stepwise heating-light induction to ferment thermophilic thermus, the low-temperature high-oxygen stage (50-60 DEG C, 75-85% high dissolved oxygen) first makes the thermophilic thermus proliferate rapidly, accumulates sufficient biomass and ATP, then the stepwise heating to 70-80 DEG C and the 405nm blue light are turned on, the temperature is suddenly increased and the light is provided, the oxidation stress and the light repair signal are triggered synchronously, the bacteria body synthesizes a large amount of heat shock protein and FAD dependent CPD photolyase, meanwhile, the high dissolved oxygen ensures that the energy required for the cofactor cycle and protein folding is sufficient, so that the yield of photolyase is stepped up on the basis of higher DCW, and finally the dry weight DCW of the bacteria body is 4.9g / L, and the CPD photolyase is 1.3mg / L.
[0018] And further adopt the "nanoparticle-coupling" fermentation process, and the nanometer titanium dioxide, nanometer silicon dioxide or molecular sieve are added in the fermentation broth to significantly improve the dissolved oxygen mass transfer coefficient, so that the dry weight DCW of the bacteria body is greater than or equal to 5.5g / L, and the volume yield of the CPD photolyase is increased to greater than or equal to 5.2mg / L.
[0019] Therefore, the present application solves the three major bottlenecks of "insufficient bacteria base, limited oxygen mass transfer and low intracellular enzyme expression" in the prior art, and can provide the cosmetic industry with low-cost, high-activity and quantitatively added DNA repair raw materials. DETAILED DESCRIPTION
[0020] The present application will be further described below in conjunction with examples, but not as the basis for limiting the present application.
[0021] Example 1: A method for optimizing thermophilic thermus fermentation to produce photolyase, comprising the following steps: S1, tank preparation: prepare a 5L borosilicate glass jacketed fermentation tank, the working volume of the fermentation tank is 3L, and the empty tank is sterilized at 121 DEG C for 20min.
[0022] S2, medium preparation (3L): 3L medium is prepared in the fermentation tank according to the following formula: yeast powder 12g / L, peptone 8g / L, glycerol 5g / L, NaCl 4g / L, KH2PO41g / L, MgSO4·7H2O 0.5g / L, FeCl3·6H2O 0.01g / L; the pH of the medium is 7.2 (5M NaOH solution and 25% H3PO4 solution are used to adjust the pH value). After preparation, the whole fermentation tank is treated at 121 DEG C for 20min, and then cooled to 55 DEG C after sterilization.
[0023] S3, particle addition: no additional nanoparticles are added to the fermentation tank in this embodiment.
[0024] S4, inoculation: 300 mL of Thermus thermophilus seed liquid was inoculated into 3 L of medium in the fermenter at 10% (v / v) for the purpose of increasing the culture, and the OD of the fermentation broth at the initial time was measured 600 ≈0.25.
[0025] The Thermus thermophilus seed liquid was obtained by culturing Thermus thermophilus in a shake flask. Thermus thermophilus was inoculated into a 500 mL high-temperature borosilicate glass shake flask with the same medium formula as the fermenter, and the liquid volume was 300 mL. The flask was incubated in a constant-temperature shaker at 75°C and 150 rpm for 48-72 h. The whole process was at natural pH without light supplementation. When the OD reached a certain concentration, the Thermus thermophilus seed liquid was obtained and used for inoculation in the fermenter.
[0026] The Thermus thermophilus (Thermus thermophilus) ZHH-88 was used, and the strain was deposited at the China Center for Type Culture Collection (CCTCC) on December 12, 2025, with the accession number CCTCC M 20252862.
[0027] S5, fermenter parameter control: 0-6h: control the temperature at 55°C, the dissolved oxygen at 80%, stir at 300rpm, input 1.5vvm of air, and adjust the pH to 7.2±0.05; 6-6.5h: linearly increase the temperature to 75°C, and simultaneously decrease the dissolved oxygen to 30%, and increase the stirring speed to 350rpm; 6.5-12h: control the temperature at 75°C, the dissolved oxygen at 30%, and the stirring speed remains at 350rpm, input 1vvm of air and 0.3vvm of pure oxygen. During this period, a blue light LED irradiation system with a light intensity of 40W·m -2 , and a wavelength of 405nm was used. At 12 hours, the blue light irradiation was stopped; 12-12.5h: cool down to 30°C for 5min, stop stirring and aeration, and obtain the final fermentation broth.
[0028] S6, harvest: centrifuge the final fermentation broth at 8000xg at 4°C for 10min, and collect the concentrated bacteria after centrifugation. Resuspend the bacteria in 3L of PBS solution, then heat sink at 55°C for 15min, then ice bath for 15min, then centrifuge at 12000xg for 20min after ice bath. The supernatant after centrifugation was filtered through a 0.22µm filter membrane to remove cell wall impurities, and a clear enzyme solution was obtained.
[0029] Results: The dry weight DCW of the bacteria in the clear enzyme solution was 4.9g / L, and the CPD photolyase was 1.3mg / L.
[0030] Example 2: A method for optimizing Thermus thermophilus fermentation to produce high-yield photosynthetic bacteria, this embodiment is basically the same as embodiment 1, the difference is that: S3, particle addition: add nano-TiO2 (anatase type, 20 nm, pre-550℃ calcination for 4h) to the fermenter, the addition weight of nano-TiO2 is 0.08% (w / v) of the fermentation liquid volume, that is, 0.8 grams of nano-TiO2 is added per liter of fermentation liquid, and is dispersed by sterile magnetic stirring at a speed of 200 rpm for 5 min.
[0031] Results: The dry weight DCW of the bacteria is 5.8g / L, and the CPD photosynthetic enzyme yield is 12.4mg / L.
[0032] As can be seen from the above, the addition of anatase nano-TiO2 makes the dry weight DCW of the bacteria increase by 18% compared with embodiment 1, and the CPD photosynthetic enzyme yield is 9.5 times that of embodiment 1, which is far more than the increase in bacteria.
[0033] Anatase nano-TiO2 will produce weak electron-hole pairs under 405nm blue light, promote FAD reduction-oxidation cycle, directly amplify enzyme specific activity, make enzyme specific activity ≥1300U / g; At the same time, its high hydrophilic surface significantly reduces the bubble diameter, and the mass transfer coefficient kLa is improved to 164h - ¹, so that the bacteria can obtain sufficient oxygen supply at a lower density, so that more carbon source and energy are used for enzyme synthesis rather than biomass accumulation. The results suggest that the addition of TiO2 is more suitable for the preparation route of "high enzyme / low bacteria", which can significantly reduce the subsequent purification load.
[0034] Example 3: A method for optimizing Thermus thermophilus fermentation to produce high-yield photosynthetic bacteria, this embodiment is basically the same as embodiment 1, the difference is that: S3, particle addition: add nano-SiO2 (gas phase method, particle size is 12nm, specific surface area measured by BET method is 200m 2 / g, 550℃ calcination for 4h) to the fermenter, the addition weight of nano-TiO2 is 0.05% (w / v) of the fermentation liquid volume, that is, 0.5 grams of nano-TiO2 is added per liter of fermentation liquid, and is dispersed by sterile stirring at a speed of 200 rpm for 5 min.
[0035] Results: The dry weight DCW of the bacteria is 6.5g / L, and the CPD photosynthetic enzyme yield is 6.7mg / L.
[0036] As can be seen from the above, the addition of nano-SiO2 makes the dry weight DCW of the bacteria reach 6.5g / L, which is 33% higher than that of embodiment 1, and the CPD photosynthetic enzyme yield is 6.7mg / L, which is 5.2 times that of embodiment 1.
[0037] The three-dimensional network structure of gas-phase nano-SiO2 can effectively reduce the apparent viscosity of the liquid and increase the bubble residence time, thereby increasing the mass transfer coefficient kLa to 210 h - ¹, so as to make the biomass accumulation of the bacteria most sufficient. This mode is suitable for "bacteria-enzyme double high" type production, and the enzyme potential can be further released by subsequent feeding or reducing the induction temperature.
[0038] Example 4: A method for optimizing Thermus thermophilus fermentation to produce high-yield photosynthetic enzyme, which is basically the same as example 1, the difference is: S3, particle addition: add nano MCM-41 molecular sieve (mesoporous, pore size 6 nm, particle size 2 µm, calcined at 550°C for 4 h) to the fermentation tank, the total amount of nano MCM-41 molecular sieve added is 0.10% (w / v) of the fermentation liquid volume, that is, 1 gram of nano MCM-41 molecular sieve is added per liter of fermentation liquid, and it is dispersed for 5 min at a sterile stirring speed of 200 rpm.
[0039] Results: The dry cell weight DCW is 5.5 g / L, and the CPD photosynthetic enzyme yield is 5.2 mg / L.
[0040] As can be seen from the above, the addition of MCM-41 molecular sieve in example 4 makes the dry cell weight DCW 5.5 g / L, which is 12% higher than example 1, and the CPD photosynthetic enzyme yield is 5.2 mg / L, which is 4 times that of example 1.
[0041] The mesoporous structure of MCM-41 molecular sieve 1-2 µm provides a large number of bubble nucleation sites, and the mass transfer coefficient kLa reaches 172 h - ¹, thereby increasing the dry cell weight DCW and the CPD photosynthetic enzyme yield. Moreover, MCM-41 is chemically inert at high temperatures, easy to centrifuge and recover, and can be reused, thereby reducing costs.
[0042] It should be understood that the above examples are only used to illustrate the technical solutions of the present application, but not to limit them. For those skilled in the art, the technical solutions described in the above examples can be modified, or some technical features can be replaced by equivalents; all these modifications and replacements shall fall within the protection scope of the appended claims of the present application.
Claims
1. A method for optimizing the fermentation of thermophilic bacteria to produce high yields of photolyase, characterized in that: Includes the following steps: S1. Prepare the container; S2, Preparation of culture medium; S3. Inoculation: The thermophilic thermophilic bacteria seed liquid is aseptically inoculated into the culture medium of the fermenter for enrichment culture; S4. Fermenter parameter control: 0-6h: Control the temperature at 50-60℃, dissolved oxygen at 75-85%, and introduce air; 6-6.5h: Linearly increase temperature to 70-80℃, dissolved oxygen simultaneously decreases to 25-35%; 6.5-12h: Maintain a constant temperature of 70-80℃, dissolved oxygen of 25-35%, introduce air and pure oxygen, and simultaneously irradiate with blue light at a wavelength of 405nm for 12 hours and then stop. 12-12.5h: Rapidly cool to 25-35℃, stop aeration, and obtain the final fermentation broth; S5. Harvesting: Centrifuge the final fermentation broth, collect the bacterial cells, resuspend the bacterial cells in PBS solution, and then perform heat precipitation, ice bath, second centrifugation, and filtration to obtain a clear enzyme solution.
2. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 1, characterized in that: In step S2, the culture medium comprises the following components: yeast extract 10-15 g / L, peptone 5-10 g / L, glycerol 2-8 g / L, NaCl 1-6 g / L, KH2PO4 0.5-2 g / L, MgSO4·7H2O 0.2-1 g / L, FeCl3·6H2O 0.005-0.02 g / L, and the pH of the culture medium is 7.0-7.
4.
3. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 1, characterized in that: The thermophilic thermophilic bacteria seed culture in step S3 was obtained by culturing thermophilic thermophilic bacteria in a shake flask. The thermophilic thermophilic bacteria used was strain with preservation number CCTCC M 20252862.
4. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 1, characterized in that: OD of fermentation broth at the initial moment in step S3 600 It is 0.2-0.
3.
5. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 1, characterized in that: It also includes adding nano-TiO2, nano-SiO2, or nanoscale molecular sieves into the fermenter between culture medium preparation and inoculation.
6. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 5, characterized in that: The weight of nano-TiO2 added is 0.06-0.10% of the fermentation liquid volume, and the particle size of nano-TiO2 is 10-30nm.
7. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 5, characterized in that: The weight of nano-SiO2 added is 0.04-0.06% of the fermentation liquid volume, and the particle size of nano-SiO2 is 5-20nm.
8. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 5, characterized in that: The weight of the added nanoscale molecular sieve is 0.08-0.12% of the fermentation broth volume. The particle size of the nanoscale molecular sieve is 1-5µm and the pore size is 2-8nm.
9. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 1, characterized in that: In step S4, from 0 to 6 hours, stirring is performed simultaneously at a speed of 250-350 rpm, with an air flow rate of 1.2-1.8 vvm; from 6 to 6.5 hours, the stirring speed is increased to 300-400 rpm; from 6.5 to 12 hours, the air flow rate is 0.8-1.2 vvm, the pure oxygen flow rate is 0.1-0.5 vvm, and the stirring speed is 300-400 rpm; the blue light intensity is 40 W·m. -2 ; 12-12.5h, cooling time is 2-8min.
10. The method for optimizing the fermentation of thermophilic bacteria to produce high-yield photolyase according to claim 1, characterized in that: In step S5, the centrifugation temperature is 2-6℃, the centrifugation force is 6800×g-9200×g, and the centrifugation time is 8-12 min; the heat sink temperature is 47-64℃, and the heat sink time is 12-18 min; the ice bath time is 12-18 min; the centrifugation force for the second centrifugation is 10000×g-14000×g, and the second centrifugation time is 17-23 min.