High-yield cellulase strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield cellulase strain and application thereof. The preservation number of the high-yield cellulase strain is CCTCC NO: M 2025895, the high-yield cellulase strain is used for producing cellulase, cellulose, cellulose derivatives and substances containing the cellulose or the cellulose derivatives can be decomposed into oligosaccharide, or monosaccharide, or ketone, or furan, or cellulosic ethanol, or cellulosic acid and other substances, the decomposition efficiency is high, and the high-yield cellulase strain is especially suitable for low-temperature and pressurized environments. And a novel high-activity biological method is provided for degradation of cellulose and / or cellulose derivatives.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a high-yield cellulase strain and application thereof. Background Art

[0002] As one of the most important biocatalysts in nature, cellulase plays an irreplaceable role in fields such as biomass conversion, textile and food industries. This type of enzyme system is usually composed of endoglucanases, exoglucanases and β-glucosidases, which can efficiently degrade cellulose into fermentable sugars. However, traditional industrial cellulases have defects such as insufficient low-temperature activity and poor environmental adaptability, and their application is severely limited, especially in extreme environments. It is worth noting that microorganisms bred in extreme deep-sea environments have evolved over a long period of time and often produce cellulase systems with special adaptability, which provides a valuable resource for the development of a new generation of industrial enzymes.

[0003] Chaetomium fungi are an important group of cellulose-degrading microorganisms, renowned for their powerful ability to decompose lignocellulose. Strains of this genus are capable of producing a rich system of cellulases and secondary metabolites, demonstrating great potential in areas such as agricultural waste treatment and biofuel production. While research on cellulase-producing fungi has yielded some promising results, most studies have focused on continental and offshore areas. These relatively accessible and mild environmental conditions provide researchers with convenient research opportunities. However, in the hadal trenches, characterized by extremely high hydrostatic pressure, weak light levels, and unique ecosystems, the distribution and survival of cellulase-producing fungi have rarely been thoroughly investigated. Summary of the Invention

[0004] The present invention discovered that a Chaetomium strain existing in the marine environment, especially the abyssal ecosystem, has evolved a special low-temperature and pressure-resistant catalytic mechanism due to its unique living environment pressure, providing a new possibility for solving the technical bottleneck that limits the application of cellulase in extreme environments such as low temperature and high pressure.

[0005] The invention provides a strain with high cellulase production.

[0006] The present invention provides application of a high-yield cellulase strain in cellulase preparation.

[0007] The present invention provides an application of a strain with high cellulase production in the degradation of cellulose and / or cellulose derivatives.

[0008] The present invention provides an application of a high-yield cellulase strain in the preparation of oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or fiber alcohols, and / or cellulose acid substances.

[0009] The technical solution of the present invention is a high-yield cellulase strain, comprising a strain with a preservation number of CCTCC NO: M2025895, and / or a subculture strain of the strain with a preservation number of CCTCC NO: M 2025895.

[0010] The high-yield cellulase strain provided above by the present invention is used to prepare cellulase, and is used to prepare products containing cellulase, and the products include but are not limited to the high-yield cellulase strain provided above by the present invention, and / or the culture or fermentation of the high-yield cellulase strain, and / or the extract of the culture or fermentation of the high-yield cellulase strain.

[0011] The present invention provides a cellulase-containing product comprising at least one of the following:

[0012] a. A high-yielding cellulase strain provided above in the present invention;

[0013] b. A culture or fermentation product of a high-yielding cellulase strain provided above in the present invention;

[0014] c. An extract of the culture or fermentation product of the high-yielding cellulase strain provided above.

[0015] The high-yield cellulase strain provided herein secretes or produces cellulases, particularly endoglucanases, which act on cellulose and cellulose derivatives to decompose them into oligosaccharides, monosaccharides, ketones, furans, cellools, and / or cellulose acids. The high-yield cellulase strain provided herein, and / or the cellulase-containing product provided herein, are used for degrading cellulose and / or cellulose derivatives, preparing products for degrading cellulose and / or cellulose derivatives; and for preparing oligosaccharides, monosaccharides, ketones, furans, cellools, and / or cellulose acids, preparing products for preparing oligosaccharides, monosaccharides, ketones, furans, cellools, and / or cellulose acids. The oligosaccharides include, but are not limited to, cellooligosaccharides and xylo-oligosaccharides. 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 furfural and 5-hydroxymethylfurfural. The cellulosic alcohols include but are not limited to cellulosic ethanol, methanol, ethylene glycol, etc. The cellulosic acids are substances containing carboxyl groups, including but not limited to cellulosic acid, gluconic acid, lactic acid, acetic acid, etc.

[0016] The present invention provides a product for degrading cellulose and / or cellulose derivatives, comprising at least one of the following:

[0017] a. A high-yielding cellulase strain provided above in the present invention;

[0018] b. A culture or fermentation product of a high-yielding cellulase strain provided above in the present invention;

[0019] c. An extract of the culture or fermentation product of the high-yielding cellulase strain provided above.

[0020] The present invention provides a product for preparing oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or inositols, and / or cellulose acids, comprising at least one of the following:

[0021] a. A high-yielding cellulase strain provided above in the present invention;

[0022] b. A culture or fermentation product of a high-yielding cellulase strain provided above in the present invention;

[0023] c. An extract of the culture or fermentation product of the high-yielding cellulase strain provided above.

[0024] The present invention provides a method for preparing cellulase, which comprises the following steps: placing the high-yield cellulase strain provided by the present invention in a culture medium for culturing or fermenting.

[0025] Furthermore, the temperature and / or pressure of the culture or fermentation can be regulated to regulate the production and yield of the cellulase. The temperature is higher than 10°C, preferably 15°C or higher, preferably 20°C or higher, and preferably 25°C or higher. In one embodiment, the temperature is preferably 15°C-28°C, preferably 20°C-28°C, and preferably 25°C-28°C. The pressure is controlled at 0.1 MPa or higher, preferably 20 MPa or higher, preferably 40 MPa or higher, and preferably 60 MPa or higher. In one embodiment, the pressure is controlled at 0.1-60 MPa, preferably 20-60 MPa, preferably 40-60 MPa, and preferably 60 MPa.

[0026] The present invention provides a method for degrading cellulose and / or cellulose derivatives, comprising the steps of adding the high-yield cellulase strain provided by the present invention and / or cellulase produced by the high-yield cellulase strain 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 the present invention, and can be a culture or fermentation product of the high-yield cellulase strain provided above in the present invention, or an extract of the culture or fermentation product of the high-yield cellulase strain provided above in the present invention. Furthermore, the temperature and / or pressure are regulated during the culture or fermentation process to regulate the production and yield of the cellulase. The temperature is higher than 10°C, preferably the temperature is controlled at above 15°C, preferably the temperature is controlled at above 20°C, preferably the temperature is controlled at above 25°C; as an embodiment, the temperature is preferably 15°C-28°C, preferably the temperature is 20°C-28°C, preferably the temperature is 25°C-28°C; the pressure is controlled at above 0.1Mpa, preferably the pressure is controlled at above 20Mpa, preferably the pressure is controlled at above 40Mpa, preferably the pressure is controlled at above 60Mpa; as an embodiment, the pressure is controlled at 0.1-60Mpa, preferably the pressure is controlled at 20-60Mpa, preferably the pressure is controlled at 40-60Mpa, preferably the pressure is controlled at 60Mpa.

[0028] The present invention provides a method for preparing oligosaccharides, and / or monosaccharides, and / or fiber alcohols, and / or cellulose acids, which comprises the following steps: adding the high-yield cellulase strain provided by the present invention and / or the cellulase prepared by the high-yield cellulase strain provided by the present invention to a substrate containing cellulose and / or a cellulose derivative, and performing an enzymatic hydrolysis reaction.

[0029] The cellulase is obtained by culturing or fermenting the high-yield cellulase strain provided above in the present invention, and can be a culture or fermentation product of the high-yield cellulase strain provided above in the present invention, or an extract of the culture or fermentation product of the high-yield cellulase strain provided above in the present invention. Furthermore, the temperature and / or pressure are regulated during the culture or fermentation process to regulate the production and yield of the cellulase. The temperature is higher than 10°C, preferably the temperature is controlled at above 15°C, preferably the temperature is controlled at above 20°C, preferably the temperature is controlled at above 25°C; as an embodiment, the temperature is preferably 15°C-28°C, preferably the temperature is 20°C-28°C, preferably the temperature is 25°C-28°C; the pressure is controlled at above 0.1Mpa, preferably the pressure is controlled at above 20Mpa, preferably the pressure is controlled at above 40Mpa, preferably the pressure is controlled at above 60Mpa; as an embodiment, the pressure is controlled at 0.1-60Mpa, preferably the pressure is controlled at 20-60Mpa, preferably the pressure is controlled at 40-60Mpa, preferably the pressure is controlled at 60Mpa.

[0030] To address the issue of insufficient activity of traditional cellulases in extreme environments, the present invention developed a deep-sea C. madrasense strain that produces 25% more endoglucanase at low temperatures than a terrestrial model strain. Using a stepped pressure culture technique that simulates a deep-sea environment, and by precisely controlling the pressure gradient of the culture system, the yield of marine C. madrasense was increased by 2.3 times. Furthermore, by adjusting the ratio of the components in the endoglucanase activity assay, experimental costs and workload were reduced, offering a new approach for the industrial application of deep-sea biotechnology.

[0031] The advantages of the present invention are:

[0032] 1. Obtaining symbiotic fungi with unique cellulose degradation potential. Compared to the cellulose-degrading fungi commonly found in soil, decaying wood, or deep-sea sediments, the cellulase-producing bacteria in this scheme originate from the intestinal symbiosis of organisms in the extreme deep-sea environment. Their living environment (high pressure, low temperature, and oligotrophic conditions) allows them to evolve a unique cellulase system with higher catalytic efficiency.

[0033] 2. By adjusting the ratio of each component in the endoglucanase activity assay, experimental costs and workload were reduced. By optimizing the colorimetric reaction conditions and micro-quantification detection technology, the amount of DNS used was reduced by 50% while ensuring data accuracy, thereby reducing experimental costs.

[0034] 3. The marine-derived Chaetomium madrasense strain provided by the present invention, when cultured at 15°C for 22 days, produced an endoglucanase yield of 85-88 U / mL, approximately 25% higher than the terrestrial-derived model strain of Chaetomium madrasense (69-72 U / mL). Compared to traditional terrestrial cellulose-degrading strains, the marine-derived C. madrasense strain of the present invention, through long-term adaptive evolution in deep-sea environments, is suitable for low-temperature biorefining processes and energy-efficient cellulosic ethanol production.

[0035] 4. After 24 hours of high-pressure culture at 60 MPa, the endoglucanase activity of the fermentation broth of the C. madrasense strain provided by the present invention reached 4.91 U / mL, a 2.3-fold increase compared to that under normal pressure (0.1 MPa, 2.12 U / mL), and exhibited a significant pressure-dependent increasing trend (20 MPa: 2.42 U / mL, 40 MPa: 3.60 U / mL). Compared to cellulose-degrading strains cultured at conventional normal pressure, this solution significantly improves endoglucanase activity by increasing pressure, making it suitable for pressure-assisted lignocellulose pretreatment processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Two parallel experiments were set up for C. madrasense HM411 as the hydrolysis zone of cellulase-producing strain.

[0037] Figure 2 These are the results of cellulase activity detection in the fermentation broth of the Haiyuan Madrid Chaete (C. madrasense HM411) strain.

[0038] Figure 3 This is the minimum enzyme production temperature of C. madrasense HM411.

[0039] Figure 4 This is a comparison of the growth of marine-origin C. madrasense HM411 and terrestrial-origin C. madrasense HM412 strains under low temperature.

[0040] Figure 5 This is a comparison of the endoglucanase activities of marine C. madrasense HM411 and terrestrial C. madrasense HM412 strains under low temperature.

[0041] Figure 6 Comparison of enzyme production of marine C. madrasense HM411 under different pressures. DETAILED DESCRIPTION

[0042] The technical solutions of the present invention are described below with reference to specific embodiments, which are not intended to limit the scope of protection of the present invention.

[0043] 1. Strain Activation and Enrichment Fluid Culture

[0044] Chaetomium madrasense, isolated from a deep-sea biological sample, was inoculated onto PDA solid medium and cultured at 28°C for 3 days to activate the strain. The activated strain was then inoculated into enrichment medium and cultured at 28°C in a shaker at 180 rpm for 2 days to obtain an enrichment solution.

[0045] The marine-derived Chaetomium madrasense HM411 was deposited in the China Center for Type Culture Collection (Wuhan University, Wuhan, China, Postal Code 430072) on April 25, 2025, with the deposit number CCTCC NO: M 2025895, the culture name Chaetomium madrasense HM411, and the preservation result was survival.

[0046] 2. Preliminary screening of cellulase-producing bacteria using Congo red staining

[0047] Use a sterile toothpick to pick mycelium and inoculate it into a cellulose-only carbon source medium. Incubate at 28°C for 5 days. After removing the mycelium, add 2 mL of 1 mol / L Congo red solution to the plate and let it stand for 15 minutes. After staining, discard the dye and add an appropriate amount of 1 mol / L sodium chloride solution. Let it stand for 15 minutes before eluting and discarding the waste solution. Identify strains that produce hydrolysis circles.

[0048] Through the initial screening, it was preliminarily determined that the two fungi C. madrasense HM411 had the potential to degrade cellulose. Figure 1 Compared with traditional cellulose-degrading fungi, this fungus is a unique source, being a symbiotic bacterium in the gut of abyssal organisms.

[0049] 3. Improvement of the endoglucanase activity assay and rescreening of strains

[0050] C. madrasense HM411 was inoculated into the enrichment medium and cultured at 28°C for 2 days. The inoculation amount was 1% and inoculated into 100 mL of fermentation medium. The culture was continued for 3 days in a constant temperature shaker at 28°C and 180 rpm. The fermentation broth was centrifuged at 12000 rpm and 4°C for 10 minutes to obtain the crude enzyme solution. The fermentation broth was treated at 100°C for 10 minutes as the control solution to determine the endoglucanase activity of the fermentation broth. 500uL of the crude enzyme solution was added with 1mL of 1% CMCNa substrate buffer and shaken to mix. The enzymatic hydrolysis reaction was carried out at 50°C for 30 minutes (preheated for 5 minutes), and then 1.5mL of DNS color developer was added. The color was developed in a boiling water bath for 10 minutes. After removal, the reaction was terminated by placing it in a cold water bath for 10 minutes. 200uL of the supernatant was taken to measure the absorbance at 540nm and calculate the cellulase activity ( Figure 2 The ratio of crude enzyme solution, 1% CMCNa, and DNS in the reaction system was crude enzyme solution: 1% CMCNa: DNS = 0.1:0.2:0.3. Enzyme activity was defined as the amount of glucose produced per 1 μg of cellulose catalyzed by 1 mL of crude enzyme solution at 50°C and pH 4.8 using 1% CMCNa solution as the substrate. One unit of enzyme activity (U) was defined as the amount of glucose produced per 1 μg of cellulose catalyzed by 1 mL of crude enzyme solution in the hydrolysis reaction.

[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: volume of crude enzyme solution.

[0057] 4. Determination of the minimum enzyme production temperature

[0058] C. madrasense HM411 was inoculated into 100 mL of enrichment medium and cultured in a shaker at 28°C and 180 rpm for 2 days. After the culture was complete, the enrichment culture was inoculated into 100 mL of fermentation medium at a 1% inoculum rate. The culture was continuously incubated at 10, 15, 20, 25, and 28°C in a shaker at 180 rpm. An appropriate amount of the fermentation broth was collected every two days, balanced, and centrifuged to obtain the supernatant. Endoglucanase activity was determined according to the above method.

[0059] The test results at different temperatures showed that 25℃ to 28℃ was the optimal temperature for the production of endoglucanase by C. madrasense HM411. When the temperature was lowered to 15℃, C. madrasense HM411 was still able to grow and produce enzymes, and entered the stable phase on the 16th day. 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] The enriched culture fluid of the marine Madrid Chaete (C. madrasense HM411) and the terrestrial Madrid Chaete (C. madrasense HM412, purchased from the China General Microorganism Culture Collection Center, CGMCC number 3.17112, original number CBS113.83) was inoculated into the induced enzyme production culture medium at an inoculum rate of 1%, and cultured continuously at 15, 20, and 28°C low-temperature shaker at 180 rpm, and the growth diameter of the strain was recorded at 24h, 48h, and 72h.

[0062] Figure 4 The results showed that the growth diameter of terrestrial C. madrasense HM412 was 8.2% higher than that of marine C. madrasense HM411 when cultured at 28℃ for 72h, and that of marine C. madrasense HM411 was 15.3% higher than that of terrestrial C. madrasense HM412 when cultured at 15℃ for 72h.

[0063] Note: Due to different growth environments of strains, C. madrasense from different sources have different tolerance to low temperatures. The culture temperature can be 10-50°C, and reaction temperatures outside the range of 10-50°C can be provided.

[0064] 6. Comparison of enzyme production with terrestrial model strains

[0065] The enriched culture fluids of marine C. madrasense HM411 and terrestrial C. madrasense HM412 were inoculated into the induced enzyme production medium at a 1% inoculation rate, and cultured continuously at 15°C in a low-temperature shaker at 180 rpm. Starting from the 6th day, a certain amount of fermentation fluid was taken every 48 hours to determine the endoglucanase activity, and the cellulase production curves of the two strains at low temperature were drawn.

[0066] Compared with the terrestrial C. madrasense HM412 model strain, the marine C. madrasense HM411 had a stronger enzyme production ability under low temperature conditions. In addition, the terrestrial C. madrasense HM412 entered the stable enzyme production period at 22 days, with an endoglucanase activity of 69-72 U / mL, while the marine C. madrasense HM411 enzyme activity at the same period was 85-88 U / mL ( Figure 5 ).

[0067] 7. Determination of enzyme production of C. madrasense HM411 under different pressures

[0068] After 3 days of culture in PDA medium, a uniformly sized bacterial cake was removed using a 200 µL pipette tip and inoculated into 100 mL of enrichment medium. The culture was continued in a shaker at 28°C and 180 rpm for 2 days. Subsequently, the target strain enrichment solution was inoculated into 100 mL of enzyme medium in a conical flask at a 1% inoculum and continued incubation at 28°C and 180 rpm for another 3 days. The bacterial solution was aliquoted into UV-sterilized autoclave bags, air was removed as much as possible, and the bags were sealed with a sterile sealer. Three replicates were prepared for each sample and labeled with 0.1, 20, and 40 MPa. The labeled autoclave samples were placed in two pressure culture vessels filled with pure water and slowly pressurized to 20 and 40 MPa, respectively, before incubation at room temperature. The cultures were incubated in the pressure culture vessels for 24 hours. Simultaneously, a sample from the autoclave bag was incubated at 0.1 MPa at room temperature as a control. After 24 h of high pressure incubation, the pressure was slowly released, and the samples were immediately placed on ice. The endoglucanase activity was determined according to the above method.

[0069] The experimental results show that ( Figure 6 After 24 hours of high hydrostatic pressure cultivation of C. madrasense HM411, the endoglucanase activity in the fermentation broth increased. At 0.1, 20, 40, and 60 MPa, the endoglucanase activity reached 2.12, 2.42, 3.60, and 4.91 U / mL, respectively. At 60 MPa, the endoglucanase activity increased 2.3-fold compared to 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 this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A high-yield cellulase strain, characterized in that: The invention relates to a strain having a deposit number of CCTCC NO: M 2025895 and / or a subculture strain of the strain having a deposit number of CCTCC NO: M 2025895.

2. The strain according to claim 1 is used for preparing cellulase and / or products containing cellulase.

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 according to claim 1; c. An extract of a culture or fermentation product of the strain according to claim 1.

4. The strain according to claim 1 and / or the product according to claim 3 are used for the degradation of cellulose and / or cellulose derivatives, and / or for the preparation of products for the degradation of cellulose and / or cellulose derivatives.

5. A product for the degradation of cellulose and / or cellulose derivatives, 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 according to claim 1; c. An extract of a culture or fermentation product of the strain according to claim 1.

6. The strain according to claim 1 and / or the product according to claim 3 is used for the preparation of oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or fiber alcohols, and / or cellulose acid substances, and / or products for the preparation of oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or fiber alcohols, and / or cellulose acid substances.

7. A product for the preparation of oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or inositols, and / or cellulose acids, 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 according to claim 1; c. An extract of a culture or fermentation product of the strain according to claim 1.

8. A method for preparing cellulase, characterized in that: The steps include: The strain according to claim 1 is placed in a culture medium for cultivation or fermentation.

9. A method for degrading cellulose and / or cellulose derivatives, characterized in that: The steps include: The strain according to claim 1, the product according to claim 3, or the degradation product according to claim 5 is added to a substrate containing cellulose and / or a cellulose derivative to carry out an enzymatic hydrolysis reaction.

10. A method for preparing oligosaccharides, and / or monosaccharides, and / or ketones, and / or furans, and / or inositols, and / or cellulose acids, characterized in that: The steps include: The strain according to claim 1, the product according to claim 3, or the preparation product according to claim 7 is added to a substrate containing cellulose and / or a cellulose derivative to carry out an enzymatic hydrolysis reaction.

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