A high-yield cellulase-producing strain and application thereof
By using the abyssal strain C. madrasense, the production method of cellulase was optimized, which solved the problem of insufficient activity of traditional cellulase under extreme environments, and realized the application of highly efficient cellulase, which is suitable for biorefining processes under low temperature and high pressure conditions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI OCEAN UNIV
- Filing Date
- 2025-05-29
- Publication Date
- 2026-08-04
AI Technical Summary
Traditional cellulases have insufficient activity under extreme conditions such as low temperature and high pressure, which limits their application range, especially in extreme environments such as the deep sea where they have not been fully studied and utilized.
We developed a strain of the genus C. madrasense derived from the abyssal environment. By using a stepped pressure culture technique that simulates the abyssal environment and optimizing enzyme activity methods, we improved the yield and activity of cellulase and adapted it to low temperature and high pressure conditions.
The yield of endoglucanase was increased by 25% at low temperatures and the enzyme activity was increased by 2.3 times under high pressure, making it suitable for biorefining processes under low temperature and high pressure conditions, thus reducing experimental costs and workload.
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Figure CN120682940B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, specifically relating to a strain that produces high levels of cellulase and its applications. Background Technology
[0002] Cellulases, as one of the most important biocatalysts in nature, play an irreplaceable role in biomass conversion, textiles, and the food industry. These enzyme systems typically consist of endoglucanases, exoglucanases, and β-glucosidases working synergistically to efficiently degrade cellulose into fermentable sugars. However, traditional industrial cellulases suffer from insufficient activity at low temperatures and poor environmental adaptability, severely limiting their application, particularly in extreme environments. Of particular interest are the cellulase systems with unique adaptability that microorganisms nurtured in the extreme environments of the deep sea have evolved over long periods, providing a valuable resource for developing next-generation industrial enzymes.
[0003] *Chaetomium* fungi are an important group of cellulose-degrading microorganisms, renowned for their powerful ability to break down lignocellulose. Strains of this genus can produce abundant cellulase systems and secondary metabolites, showing great potential in agricultural waste treatment and biofuel production. Although some progress has been made in the study of cellulase-producing fungi, most related research has focused on continental and near-shore areas. These relatively accessible areas with relatively mild environmental conditions provide researchers with convenient research conditions. However, the distribution and survival status of cellulase-producing fungi in the deep-sea trenches with extremely high hydrostatic pressure, low light, and unique ecosystems have rarely been investigated in depth. Summary of the Invention
[0004] This invention has discovered a strain of the genus *Ceratophyllum* that exists in marine environments, especially deep-sea ecosystems. Due to the unique environmental pressures it faces, it has evolved a special low-temperature and high-pressure catalytic mechanism, providing a new possibility for overcoming the technical bottleneck that limits the application of cellulase in extreme environments such as low temperature and high pressure.
[0005] This invention provides a strain that produces high levels of cellulase.
[0006] This invention provides the application of a high-yield cellulase strain in cellulase preparation.
[0007] This invention provides the application of a high-producing cellulase strain in the degradation of cellulose and / or cellulose derivatives.
[0008] This invention provides the application of a high-cellulase-producing strain in the preparation of oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or cellulose alcohols, and / or cellulic acids.
[0009] The technical solution of the present invention is a strain that produces high levels of cellulase, comprising a strain with accession number CCTCC NO: M2025895, and / or a passaged strain of the strain with accession number CCTCC NO: M 2025895.
[0010] The present invention provides a high-cellulase-producing strain for preparing cellulase and for preparing cellulase-containing products, including but not limited to the high-cellulase-producing strain provided above, and / or cultures or ferments of high-cellulase-producing strains, and / or extracts of cultures or ferments of high-cellulase-producing strains.
[0011] This invention provides a product containing cellulase, comprising at least one of the following:
[0012] a. The cellulase-producing strain provided above in this invention;
[0013] b. A culture or fermentation product of a high-cellulase-producing strain provided above in this invention;
[0014] c. An extract of a culture or fermentation product of a high-cellulase-producing strain provided above in this invention.
[0015] The present invention provides a high-cellulase-producing strain that secretes or produces cellulase, particularly endoglucanase, which acts on cellulose and cellulose derivatives to decompose them into oligosaccharides and / or monosaccharides, and / or ketones, and / or furans, and / or cellulose alcohols, and / or cellulic acids. The high-cellulase-producing strain and / or cellulase-containing product provided by the present invention are used for the degradation of cellulose and / or cellulose derivatives to prepare products for the degradation of cellulose and / or cellulose derivatives; and for the preparation of oligosaccharides and / or monosaccharides, and / or ketones, and / or furans, and / or cellulose alcohols, and / or cellulic acids, to prepare products for the preparation of oligosaccharides and / or monosaccharides, and / or ketones, and / or furans, and / or cellulose alcohols, and / or cellulic acids. The oligosaccharides include, but are not limited to, cellulose oligosaccharides and xylooligosaccharides. The monosaccharides include, but are not limited to, hexoses and pentoses. The ketones include, but are not limited to, acetone and 2,3-butanedione. The furans include, but are not limited to, uronic acid and 5-hydroxymethylfurfural. The cellulosic alcohols include, but are not limited to, cellulose ethanol, methanol, ethylene glycol, etc., and the cellulosic acids are substances containing a carboxyl group, including, but not limited to, cellulose acid, gluconic acid, lactic acid, acetic acid, etc.
[0016] This invention provides a product for the degradation of cellulose and / or cellulose derivatives, comprising at least one of the following:
[0017] a. The cellulase-producing strain provided above in this invention;
[0018] b. A culture or fermentation product of a high-cellulase-producing strain provided above in this invention;
[0019] c. An extract of a culture or fermentation product of a high-cellulase-producing strain provided above in this invention.
[0020] This invention provides a product for preparing oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or cellulose alcohols, and / or cellulosic acids, comprising at least one of the following:
[0021] a. The cellulase-producing strain provided above in this invention;
[0022] b. A culture or fermentation product of a high-cellulase-producing strain provided above in this invention;
[0023] c. An extract of a culture or fermentation product of a high-cellulase-producing strain provided above in this invention.
[0024] The present invention provides a method for preparing cellulase, comprising the steps of: placing the high-yield cellulase strain provided by the present invention in a culture medium for culture or fermentation.
[0025] Furthermore, the temperature and / or pressure of the culture or fermentation are adjusted to regulate the yield and productivity of cellulase. The temperature is above 10°C, preferably above 15°C, preferably above 20°C, and preferably above 25°C; as one embodiment, the preferred temperature is 15°C-28°C, preferably 20°C-28°C, and preferably 25°C-28°C; the pressure is controlled above 0.1 MPa, preferably above 20 MPa, preferably above 40 MPa, and preferably above 60 MPa; as one embodiment, the pressure is controlled between 0.1-60 MPa, preferably between 20-60 MPa, preferably between 40-60 MPa, and preferably above 60 MPa.
[0026] The present invention provides a method for the degradation of cellulose and / or cellulose derivatives, comprising the steps of: adding the strain of high cellulase production provided by the present invention and / or cellulase prepared from the strain of high cellulase production provided by the present invention to a substrate containing cellulose and / or cellulose derivatives, and performing an enzymatic hydrolysis reaction.
[0027] The cellulase is obtained by culturing or fermenting the high-yield cellulase strain provided above in this invention. It can be a culture or fermentation product of the high-yield cellulase strain provided above in this invention, or an extract of the culture or fermentation product of the high-yield cellulase strain provided above in this invention. Further, the temperature and / or pressure are controlled during the culturing or fermentation process to regulate the yield and productivity of the cellulase. The temperature is above 10°C, preferably above 15°C, preferably above 20°C, and preferably above 25°C; as one embodiment, the preferred temperature is 15°C-28°C, preferably 20°C-28°C, and preferably 25°C-28°C; the pressure is controlled above 0.1 MPa, preferably above 20 MPa, preferably above 40 MPa, and preferably above 60 MPa; as one embodiment, the pressure is controlled between 0.1-60 MPa, preferably between 20-60 MPa, preferably between 40-60 MPa, and preferably above 60 MPa.
[0028] This invention provides a method for preparing oligosaccharides, and / or monosaccharides, and / or cellulose alcohols, and / or cellulose acids, comprising the steps of: adding the high-cellulase-producing strain provided by the present invention and / or cellulase prepared from the high-cellulase-producing strain provided by the present invention to a substrate containing cellulose and / or cellulose derivatives, and performing an enzymatic hydrolysis reaction.
[0029] The cellulase is obtained by culturing or fermenting the high-yield cellulase strain provided above in this invention. It can be a culture or fermentation product of the high-yield cellulase strain provided above in this invention, or an extract of the culture or fermentation product of the high-yield cellulase strain provided above in this invention. Further, the temperature and / or pressure are controlled during the culturing or fermentation process to regulate the yield and productivity of the cellulase. The temperature is above 10°C, preferably above 15°C, preferably above 20°C, and preferably above 25°C; as one embodiment, the preferred temperature is 15°C-28°C, preferably 20°C-28°C, and preferably 25°C-28°C; the pressure is controlled above 0.1 MPa, preferably above 20 MPa, preferably above 40 MPa, and preferably above 60 MPa; as one embodiment, the pressure is controlled between 0.1-60 MPa, preferably between 20-60 MPa, preferably between 40-60 MPa, and preferably above 60 MPa.
[0030] To address the issue of insufficient activity of traditional cellulases under extreme environments, this invention developed a deep-sea-derived *C. madrasense* strain. This strain exhibits a 25% higher endoglucanase production rate at low temperatures compared to terrestrial model strains. Based on a stepped pressure culture technique simulating the deep-sea environment, the yield of marine-derived *C. madrasense* was increased by 2.3 times by precisely controlling the pressure gradient of the culture system. Furthermore, by adjusting the proportions of components in the method for determining endoglucanase activity, experimental costs and workload were reduced, providing a new solution for the industrial application of deep-sea biotechnology.
[0031] The advantages of this invention are:
[0032] 1. Obtaining symbiotic fungi with a unique origin and cellulose degradation potential. The key difference lies in the fact that, compared to cellulose-degrading fungi commonly found in existing technologies from soil, decaying wood, or deep-sea sediments, the cellulase-producing bacteria in this scheme originate from a biological gut symbiotic system in an extreme deep-sea environment. Their living environment (high pressure, low temperature, oligotrophic conditions) has enabled them to evolve a unique cellulase system with higher catalytic efficiency.
[0033] 2. By adjusting the proportions of each component in the method for determining endoglucanase activity, experimental costs and workload were reduced. This is achieved by optimizing the colorimetric reaction conditions and using micro-quantitation detection techniques, reducing the amount of DNS used by 50% while ensuring data accuracy, thereby lowering experimental costs.
[0034] 3. The marine-derived *Chaetomium madrasense* strain provided by this invention, when cultured at 15°C for 22 days, achieved an endoglucanase yield of 85-88 U / mL, which is approximately 25% higher than the terrestrial-derived *Chaetomium madrasense* model strain (69-72 U / mL). This is because, compared to traditional terrestrial-derived cellulose-degrading strains, the marine-derived *Chaetomium madrasense* strain of this invention has undergone long-term adaptation and evolution in the deep-sea environment, making it suitable for low-temperature biorefining processes and energy-efficient cellulosic ethanol production.
[0035] 4. The *C. madrasense* strain provided by this invention exhibits an endoglucanase activity of 4.91 U / mL in its fermentation broth after 24 hours of high-pressure culture at 60 MPa, a 2.3-fold increase compared to the activity under normal pressure (0.1 MPa, 2.12 U / mL), and shows a significant pressure-dependent increasing trend (20 MPa: 2.42 U / mL, 40 MPa: 3.60 U / mL). This is significant because, compared to conventionally cultured cellulose-degrading strains under normal pressure, this method substantially increases endoglucanase activity by increasing pressure, making it suitable for pressure-assisted lignocellulose pretreatment processes. Attached Figure Description
[0036] Figure 1 Two parallel experiments were set up to use C. madrasense HM411 as the hydrolysis zone for cellulase-producing strains.
[0037] Figure 2 The results show the cellulase activity of the fermentation broth of the Haiyuan Madrid hairy shell (C. madrasense HM411) strain.
[0038] Figure 3 This is the lowest enzyme production temperature for Haiyuan Madrid scab (C. madrasense HM411).
[0039] Figure 4 Comparison of the growth of marine C. madrasense HM411 and terrestrial C. madrasense HM412 strains under low temperature.
[0040] Figure 5 Comparison of endoglucanase activities of marine C. madrasense HM411 and terrestrial C. madrasense HM412 strains at low temperatures.
[0041] Figure 6 Comparison of enzyme production by C. madrasense HM411 under different pressures. Detailed Implementation
[0042] The technical solution of the present invention will be described below with specific embodiments, but these are not intended to limit the scope of protection of the present invention.
[0043] 1. Strain activation and enrichment broth culture
[0044] Marine Chaetomium madrasense isolated from abyssal organisms was inoculated onto PDA solid medium and cultured at 28°C for 3 days to activate the strain. The activated strain was then picked and inoculated into enrichment medium, and cultured at 28°C and 180 rpm for 2 days to obtain the enriched solution.
[0045] The marine-derived *C. madrasense* HM411 was deposited on April 25, 2025, at the China Center for Type Culture Collection (Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO: M 2025895 and culture name *Chaetomium madrasense* HM411. The culture was found to be viable.
[0046] 2. Initial screening of cellulase-producing bacteria using Congo red staining method.
[0047] Mycelia were picked up with a sterile toothpick and inoculated into a cellulose-only carbon source medium, and incubated statically at 28°C for 5 days. After incubation, 2 mL of 1 mol / L Congo red solution was added to the plate, and the plate was incubated for 15 min to stain. After staining, the staining solution was discarded, and an appropriate amount of 1 mol / L sodium chloride solution was added. After elution for 15 min, the waste liquid was discarded to identify the strain capable of producing a hydrolysis zone.
[0048] Preliminary screening has identified two fungal strains, *C. madrasense* HM411, as having potential for cellulose degradation. Figure 1 As shown. Compared to traditional sources of cellulose-degrading fungi, this fungus has a more unique origin, being a symbiotic bacterium in the gut of deep-sea organisms.
[0049] 3. Improvement of the method for measuring endoglucanase activity and secondary screening of strains
[0050] C. madrasense HM411 was inoculated into enrichment medium and cultured at 28℃ for 2 days. Then, 1% of the inoculum was added to 100 mL of fermentation medium and cultured continuously for 3 days at 28℃ and 180 rpm in a constant temperature shaker. The fermentation broth was centrifuged at 12000 rpm and 4℃ for 10 min to obtain crude enzyme solution. The fermentation broth was treated at 100℃ for 10 min as a control solution, and the activity of endoglucanase in the fermentation broth was measured. 500 μL of the crude enzyme solution was taken, and 1 mL of 1% CMCNa substrate buffer was added and vortexed to mix. Enzymatic hydrolysis was performed in a 50℃ water bath for 30 min (preheated for 5 min). Then, 1.5 mL of DNS chromogenic reagent was added, and the mixture was incubated in a boiling water bath for 10 min. The reaction was terminated by placing the mixture in a cold water bath for 10 min. 200 μL of the supernatant was taken and the absorbance was measured at 540 nm, and the cellulase activity was calculated. Figure 2 In this reaction system, the ratio of crude enzyme solution, 1% CMCNa, and DNS is crude enzyme solution: 1% CMCNa: DNS = 0.1:0.2:0.3. Enzyme activity is defined as the amount of enzyme produced (U) by catalyzing 1 μg of cellulose to produce glucose in 1 mL of crude enzyme solution during hydrolysis at 50°C and pH 4.8, using 1% CMCNa solution as the substrate.
[0051] Enzyme activity (U / mL) = (a×n+1000) / (t×v)
[0052] a: Glucose concentration obtained from the glucose standard curve
[0053] n: Dilution factor of enzyme solution
[0054] 1000: Conversion factor between ug and mg
[0055] t: reaction time
[0056] v: Crude enzyme solution volume.
[0057] 4. Determination of the minimum enzyme production temperature
[0058] C. madrasense HM411 was inoculated into 100 mL of enrichment medium and cultured on a shaker at 28 °C and 180 rpm for 2 days. After the culture was completed, the enrichment broth was inoculated into 100 mL of fermentation medium at an inoculation rate of 1%, and cultured continuously on a shaker at 180 rpm for 10, 15, 20, 25, and 28 °C. Every 2 days, an appropriate amount of fermentation broth was taken, balanced, centrifuged to obtain the supernatant, and the endoglucanase activity was determined according to the above method.
[0059] Test results at different temperatures showed that 25℃ to 28℃ was the optimal temperature for endoglucanase production by *C. madrasense* HM411. When the temperature was lowered to 15℃, *C. madrasense* HM411 could still grow and produce enzyme, entering a stable phase on day 16, and the enzyme activity was the same as that produced at the optimal temperature. Figure 3 ).
[0060] 5. Comparison of growth with terrestrial model strains
[0061] Marine-sourced *C. madrasense* HM411 and terrestrial-sourced *C. madrasense* HM412 (purchased from China General Microbiological Culture Collection Center, CGMCC number 3.17112, original number CBS113.83) enrichment cultures were inoculated into enzyme-inducing medium at an inoculum rate of 1% and continuously cultured at 15, 20, and 28°C on a low-temperature shaker at 180 rpm. The growth diameter of the strains was recorded at 24 h, 48 h, and 72 h.
[0062] Figure 4 The results showed that when cultured at 28℃ for 72 h, the growth diameter of terrestrial C. madrasense HM412 was 8.2% higher than that of marine C. madrasense HM411, while when cultured at 15℃ for 72 h, the marine C. madrasense HM411 was 15.3% higher than that of terrestrial C. madrasense HM412.
[0063] Note: Due to different growth environments, C. madrasense from different sources has different tolerance to low temperatures. The culture temperature can be 10-50℃, and reaction temperatures outside the 10-50℃ range can be provided.
[0064] 6. Comparison of enzyme production with terrestrial model strains
[0065] The enriched cultures of *C. madrasense* HM411 (Haiyuan) and *C. madrasense* HM412 (Luyuan) were inoculated into enzyme-inducing medium at an inoculation rate of 1%, and continuously cultured at 180 rpm in a shaker at 15°C. Starting from day 6, a certain amount of fermentation broth was taken every 48 hours to determine the activity of endoglucanase, and cellulase production curves of the two strains at low temperature were plotted.
[0066] Compared to the terrestrial C. madrasense HM412 model strain, the marine C. madrasense HM411 exhibits stronger enzyme production capacity under low-temperature conditions. Furthermore, the terrestrial C. madrasense HM412 enters a stable enzyme production phase at 22 days, with an endoglucanase activity of 69-72 U / mL, while the marine C. madrasense HM411 at the same stage exhibits an enzyme activity of 85-88 U / mL. Figure 5 ).
[0067] 7. Determination of enzyme production by *C. madrasense* HM411 under different pressures
[0068] The bacterial strain cultured on PDA medium for 3 days was used to collect uniformly sized bacterial discs using a 200 μL pipette tip and inoculated into 100 mL of enrichment medium. The culture was then continuously incubated for 2 days at 28°C and 180 rpm in a shaker. Subsequently, at an inoculation rate of 1%, the target strain enrichment was inoculated into 100 mL of enzyme medium in an Erlenmeyer flask and continuously incubated for 3 days at 28°C and 180 rpm in a shaker. The bacterial solutions were then aliquoted into UV-sterilized high-pressure sealed bags, ensuring minimal air was removed, and sealed with a sterile sealer. Three replicates were set for each sample, labeled 0.1, 20, and 40 MPa, respectively. The labeled high-pressure samples were placed in two pressure incubation containers filled with pure water, and the pressure was slowly increased to 20 and 40 MPa, respectively, and incubated at room temperature. The samples were continuously incubated in the pressure incubation containers for 24 hours. Simultaneously, samples from high-pressure sealed bags were incubated at room temperature under the same conditions at 0.1 MPa as a control. After high-pressure culture for 24 hours, the pressure was slowly released, and the sample was immediately placed on ice. The activity of endoglucanase was then determined according to the above method.
[0069] Experimental results show that ( Figure 6 After culturing *C. madrasense* HM411 under high hydrostatic pressure for 24 h, the activity of endoglucanase in the fermentation solution showed an increasing trend. At pressures of 0.1, 20, 40, and 60 MPa, the endoglucanase activities were 2.12, 2.42, 3.60, and 4.91 U / mL, respectively. The endoglucanase activity at 60 MPa was 2.3 times higher than that at 0.1 MPa.
[0070] Note: The above examples are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.
Claims
1. A strain that produces high levels of cellulase under low temperature and high pressure, characterized in that, The strain is classified as Chaetomium madrasense HM411, with accession number CCTCC NO: M 2025895.
2. The application of the strain described in claim 1 in the preparation of cellulase and / or cellulase-containing products under low temperature and high pressure.
3. A product containing cellulase, characterized in that, Contains at least one of the following: a. The strain according to claim 1; b. A culture or fermentation product of the strain of claim 1.
4. A method for preparing cellulase, characterized in that, The steps include: The strain described in claim 1 is placed in a culture medium for cultivation or fermentation.