Cellulomonas iranica strain lg2020 and uses thereof

Abstract

The present application relates to the technical field of microorganism, and in particular to a Cellulomonas iranensis LG2020 and application thereof.The Cellulomonas iranensis LG2020 with the functions of degrading cellulose and lignocellulose has a preservation number of GDMCC No.62533.Through filter paper degradation test, the residual rate of filter paper is 1.72% on the 7th day; through straw degradation capacity test, after the 25th day, the Cellulomonas iranensis has decomposed the straw into very thin fragments, which indicates that the Cellulomonas iranensis has good straw degradation capacity, and therefore, the application also provides application of the strain LG2020 in preparation of cellulase and / or lignin degrading enzyme.

Description

[Technical Field]

[0001] This invention relates to the field of microbial technology, specifically to the Iranian cellulomonas strain LG2020 and its applications. [Background Technology]

[0002] Cellulose is a long-chain, high-molecular-weight heteropolymer composed of D-glucose linked by β-1,4 glycosidic bonds. It is the most abundant component (30-50%) in plant cell walls and is one of the most widely distributed and structurally complex polysaccharides in nature, typically composed of 150-15,000 glucose units. Under normal temperature conditions, the hydrogen bonds and van der Waals forces between cellulose molecules in plant cell walls polymerize to form a secondary structure of cellulose sheets. These secondary structures, linked by partial hydrophobic interactions, stack to form a tertiary fibrous structure—microfibrils. This structure resists free diffusion transport, meaning it resists the transport of many small molecules within the plant, such as water molecules, enzyme molecules, and even hydrogen ions, further preventing hydrolysis by enzymes or damage from organic reagents. Further polymerization of the tertiary fibrous structure forms filamentous crystalline fibers, maintaining the cellulose structure in a relatively stable state. This makes the cellulose macromolecule insoluble in water and most organic solvents, making cellulose relatively difficult to degrade and utilize in nature. Lignocellulose mainly exists in this crystalline form. However, cellulose molecules that are easily hydrolyzed generally form amorphous cellulose chains, creating amorphous cellulose. The hydrolysis process begins with the long cellulose chains breaking down into shorter chains via oxygen bridges. Then, the oxygen bridges completely break, leading to the complete hydrolysis of cellulose molecules into individual reducing sugar molecules, glucose. These glucose molecules can then be further utilized by microorganisms through glycolysis to produce pyruvate, which is then converted into alcohols, lactic acid, or enters the tricarboxylic acid cycle to generate other substances. Currently, researchers mainly use physical, chemical, and biological methods to degrade cellulose. Physical and chemical methods are not only costly but also pollute the environment and damage the ecosystem. Microbial degradation of lignocellulose has advantages such as low energy consumption and being environmentally friendly, but it has disadvantages such as a long degradation cycle and demanding degradation conditions. Therefore, we have found that highly efficient degrading microorganisms are key to enabling the large-scale application of biological cellulose degradation. To discover more efficient cellulose-degrading microbial resources, we collected samples from different environments to screen for microorganisms with cellulose-degrading capabilities. Meanwhile, we conducted some research on the degradation ability and biochemical characteristics of the screened cellulose-degrading bacteria, providing some theoretical basis for the further development and utilization of cellulose-degrading bacteria. [Summary of the Invention]

[0003] In view of this, the purpose of this invention is to provide a Cellulomonasiranensis strain LG2020 and its applications.

[0004] To achieve the above objectives, this invention screened and obtained a Cellulomonasiranensis strain LG2020, with accession number GDMCC No. 62533, accession date: June 13, 2022, accession address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC).

[0005] To further illustrate, the Cellulomonas iranensis strain LG2020 grows well on LB liquid medium; the colonies are golden yellow, about 1 mm in diameter, round, convex, with neat edges, opaque, smooth, glossy, and moist.

[0006] The present invention also provides the application of Cellulomonas iranensis strain LG2020 in the preparation of cellulase and / or lignin-degrading enzyme.

[0007] To further explain, the enzyme production conditions for preparing cellulase and / or lignin-degrading enzyme are: a rotation speed of 200 r / min, a culture medium volume of 200 mL / 500 mL, an initial pH of 6.4, an inoculum size of 15%, a fermentation time of 3 days, and a culture temperature of 37℃.

[0008] To further clarify, the enzyme-producing culture medium conditions for preparing cellulase and / or lignin-degrading enzymes are as follows: optimal CMC-Na concentration of 2%, peptone concentration of 2%, DMF concentration of 0.5%, bovine serum albumin concentration of 1%, Tween-80 concentration of 0.5%, PEG6000 concentration of 0.5%, and 10 mM zinc chloride.

[0009] The present invention also provides the application of the aforementioned Cellulomonas iranensis strain LG2020 in the degradation of lignocellulose.

[0010] This invention also provides the application of the aforementioned Cellulomonas iranensis strain LG2020 in the degradation of plant straw.

[0011] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0012] The *Cellulomonas iranensis* strain LG2020 of this invention exhibits excellent ability to degrade cellulose and lignocellulose. In a filter paper strip degradation test, the filter paper residue rate was 1.72% on day 7. In a straw degradation test, after day 25, *Cellulomonas iranensis* had decomposed the straw into very thin fragments, demonstrating its excellent straw degradation ability. In contrast, commercially available lignocellulose-degrading bacteria did not show significant changes in the overall morphology of straw after 25 days of degradation. Furthermore, by considering enzyme production conditions and culture medium components, combined with response surface methodology optimization results, the optimal conditions for enzyme production by Cellulomonas iranensis strain LG2020 were determined as follows: culture medium volume 200 mL / 500 mL, initial pH 6.44, inoculum size 15%, fermentation time 3 days, culture temperature 37℃, rotation speed 200 r / min, optimal carbon source CMC-Na with an optimal concentration of 2%, optimal nitrogen source peptone with an optimal concentration of 2.14%, optimal bovine serum albumin concentration of 1.17%, optimal concentrations of DMF, Tween-80, and PEG6000 of 0.5%, and optimal ZnCl2 concentration of 10 mM. Under the optimal fermentation conditions described above, the CMC, FPA, and xylanase activities of Cellulomonas iranensis strain LG2020 were 49.8977 U / mL, 22.9328 U / mL, and 90.777 U / mL, respectively, representing increases of 9.712 times, 6.602 times, and 9.634 times compared to the unoptimized values. The relative errors between the measured and predicted values ​​were 1.76%, 1.38%, and 2.00%, respectively. In conclusion, strain LG2020 of this invention has broad application prospects in the field of cellulose and lignocellulose degradation. [Attached Image Description]

[0013] Figure 1 Morphological observation of strain LG2020: A: Gram staining observation; B: Colony morphology observation; C: Scanning electron microscopy observation.

[0014] Figure 2 Results of 16S rDNA PCR amplification of strain LG2020.

[0015] Figure 3 Phylogenetic tree based on 16S rDNA gene sequence homology of strain LG2020.

[0016] Figure 4 : Figure 4 (A) shows the glucose standard curve. Figure 4 (B) shows the xylose standard curve.

[0017] Figure 5 Effects of pH on the activity and stability of CMC, FPA, and xylanase.

[0018] Figure 6 Effects of temperature on the activity and stability of CMC, FPA, and xylanase.

[0019] Figure 7 : Graph showing the effect of filter paper strip degradation on filter paper residue rate.

[0020] Figure 8 : Graph showing the effect of filter paper strip degradation on filter paper. Figure 8 (A) is the blank group. Figure 8 (B) is the treatment group.

[0021] Figure 9 Comparison of straw degradation effects. A0: Degradation by *Cellulosum iridis* in 0 days; A25: Degradation by *Cellulosum iridis* in 25 days; B0: Degradation by commercially available lignocellulosic degrading bacteria in 0 days; B25: Degradation by commercially available lignocellulosic degrading bacteria in 25 days.

[0022] Figure 10 : Effect of shaker speed on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0023] Figure 11 Effects of liquid volume on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0024] Figure 12 The effect of culture medium pH on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0025] Figure 13 Effects of inoculum size on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0026] Figure 14 Effects of culture time on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0027] Figure 15 Effects of culture temperature on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0028] Figure 16 Effects of different carbon sources (A) and optimal carbon source concentration (B) on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0029] Figure 17 Effects of different nitrogen sources (A) and optimal nitrogen source concentration (B) on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0030] Figure 18 Effects of DMF on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0031] Figure 19 Effects of bovine serum albumin on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0032] Figure 20 Effects of Tween-80 on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0033] Figure 21 Effects of PEG6000 on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0034] Figure 22 The effects of different metal ions on CMC enzyme activity, FPA enzyme activity, xylanase activity and growth.

[0035] Figure 23 Contour lines and response surface plots of the effect of peptone concentration and bovine serum albumin concentration on CMC enzyme activity.

[0036] Figure 24 Contour lines and response surface plots of the effect of peptone concentration and culture medium pH on FPA enzyme activity.

[0037] Figure 25 Contour lines and response surface plots of the effect of peptone concentration and bovine serum albumin concentration on xylanase activity.

Detailed Implementation Methods

[0038] All features disclosed in this specification, or steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps. Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0039] Example 1: Isolation and Identification of Cellulomonas iridis

[0040] 1.1 Experimental Materials

[0041] 1.1.1 Sample Collection The samples were collected from mud and soil samples from the snail breeding base in Ligao Town, Liuzhou City, Guangxi Province.

[0042] 1.1.2 Culture medium

[0043] Initial screening liquid culture medium: CMC-Na 15g, K2HPO4 1g, NH4NO3 1g, MgSO4·7H2O 0.5g, NaCl 0.5g, pH natural, ddH2O to 1L, sterilize at 121℃ for 20min.

[0044] Primary screening solid culture medium: Add 2% agar to the primary screening liquid culture medium.

[0045] Secondary screening medium: The formula is the same as that for the primary screening medium.

[0046] LB liquid medium: 10g peptone, 5g yeast extract, 10g NaCl, pH at rest, bring to 1L with ddH2O, and sterilize at 121℃ for 20min.

[0047] LB solid medium: 2% agar is added to LB liquid medium.

[0048] Filter paper disintegration medium: K2HPO4 1 g, NH4NO3 1 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, 3 g filter paper strips (1 cm × 6 cm), pH at rest, add ddH2O to a final volume of 1 L, sterilize at 121℃ for 20 min.

[0049] Enzyme-producing medium: CMC-Na 10g, peptone 20g, MgSO4·7H2O 0.3g, K2HPO4·3H2O 1.5g, NaCl 5g, CaCl2·2H2O 0.1g, ddH2O to a final volume of 1L, pH 6.4, sterilized at 121℃ for 20min.

[0050] 1.1.3 Main Solution

[0051] 1 mg / mL glucose standard solution: Take 100 mg of glucose (dried at 80℃ to constant weight), dissolve it in ddH2O, make up to 100 mL, and store at 4℃ for later use.

[0052] 1 mg / mL xylose standard solution: Take 100 mg xylose (dried at 80℃ to constant weight), dissolve in ddH2O, make up to 100 mL, and store at 4℃ for later use.

[0053] 0.1 mol / L citrate buffer: Dissolve 21.014 g of citric acid (molecular weight 210.14) in ddH2O, bring the volume up to 1000 mL, and store at 4 °C for later use.

[0054] 0.1 mol / L sodium citrate buffer: Dissolve 29.412 g of sodium citrate (molecular weight 294.12) in ddH2O, bring the volume up to 1000 mL, and store at 4 °C for later use.

[0055] pH 4.5, 0.05 mol / L citrate-sodium citrate buffer: Accurately measure 27.12 mL of citric acid solution and 22.88 mL of sodium citrate solution, then bring the volume to 100 mL. Mix well to obtain pH 4.5, 0.05 mol / L citrate-sodium citrate buffer. Store at 4°C for later use.

[0056] 1% Sodium Carboxymethyl Cellulose Solution: Weigh 1g of sodium carboxymethyl cellulose (CMC-Na), add an appropriate amount of 0.05mol / L citrate-sodium citrate buffer solution at pH 4.5, heat to dissolve until CMC-Na is completely dissolved, cool, and then bring the volume to 100mL with buffer solution. Mix well and store at 4℃ for later use.

[0057] 1% xylan solution: Weigh 1.0g of xylan and dissolve it in a prepared pH 4.5 0.05mol / L citrate-sodium citrate buffer solution. Dilute to 100mL and store at 4℃ for later use.

[0058] 3,5-Dinitrosalicylic acid reagent (DNS): Weigh 6.3 g of 3,5-dinitrosalicylic acid and add 262 mL of 2 mol / L NaOH to a hot solution of potassium sodium tartrate (182 g of potassium sodium tartrate dissolved in 500 mL of ddH2O). Add 5 g of phenol and 5 g of sodium sulfite to the solution, stir to dissolve, cool, and then bring the volume to 1000 mL with ddH2O. Store in a brown bottle. Let stand at room temperature for one week before use.

[0059] 1.2.1 Screening of cellulose-degrading bacteria

[0060] (1) Initial screening of strains

[0061] A certain amount of soil sample was added to 250 mL of initial screening liquid culture medium and cultured in a shaker at 37℃ and 150 r / min for 3-7 days. The color change of the solution in the culture medium was observed every 12 hours until the culture medium became turbid. The enrichment solution was sampled and diluted. 900 μL of culture medium was transferred to a 1.5 mL centrifuge tube, and 100 μL of the enrichment suspension was diluted in a 1.5 mL centrifuge tube. The dilution gradient was 10⁻⁵-10⁻⁸. 10 μL of each solution was spread onto the enrichment medium to form a blank control. The samples were cultured in a biochemical incubator at 37℃, and the growth of bacteria was observed every 12 hours. Bacteria were cultured after 72-120 hours. Vigorous and healthy strains were selected and transferred to a secondary screening medium for further screening.

[0062] (2) Secondary screening of strains

[0063] The strains obtained from the primary screening medium were transferred to the secondary screening medium and cultured in a constant temperature incubator at 37°C for 3-5 days. Then, the strains with better growth were selected and inoculated into filter paper disintegration medium to test the filter paper residue rate. At the same time, the CMC enzyme activity, FPA enzyme activity, and xylanase activity were determined by the DNS method. Taking into account the filter paper residue rate and the results of the three enzyme activities, strains with high cellulose degradation ability were finally screened.

[0064] 1.2.2 Preparation of crude enzyme solution from cellulose-degrading bacteria

[0065] The selected strains were inoculated onto LB liquid medium and cultured at 37°C and 150 r / min to prepare a 106 CFU bacterial suspension. This suspension was then inoculated into the enzyme-producing fermentation medium at a 10% inoculation rate and cultured at 37°C and 150 r / min for 3 days to obtain the fermentation broth. The fermentation broth was centrifuged at 10000 r / min for 10 min, and the supernatant was taken as the crude enzyme solution.

[0066] 1.2.3 Identification and Preservation of Cellulose-Degrading Bacteria

[0067] (1) Morphological observation of the strain

[0068] The final selected strains were streaked onto LB agar plates, inverted, and incubated at 37°C for 3 days. Afterward, the colony morphology, color, and size were observed on the plates, and single colonies were selected for Gram staining and microscopic examination. Simultaneously, following Zeng Lingjie's method, morphological changes in the strains were observed using scanning electron microscopy.

[0069] (2) Molecular biological identification of the strain

[0070] Using LB bacterial culture as a DNA template, the 16S rDNA sequence was amplified by PCR using a pair of universal primers for bacterial 16S rRNA: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′). The PCR reaction system is shown in Table 1.

[0071] Table 1. PCR reaction system (25 μL)

[0072]

[0073] The amplification conditions are as follows:

[0074]

[0075] The reaction system was mixed and amplified according to the amplification conditions.

[0076] After PCR amplification, electrophoresis was performed. After electrophoresis, the gel was placed in a UV spectrometer to observe the bands. Then, the PCR amplification products were recovered and purified using a standard agarose gel DNA recovery kit (Beijing Tiangen Biotech Co., Ltd.) (see the kit's instruction manual for specific procedures). The recovered DNA fragments were then sent to Guangzhou BGI Genomics Co., Ltd. to obtain the strain's 16S rDNA identification results. The results were then accessed through the NCBI website, the BLAST tool was selected, the obtained sequence was entered, and BLAST was run. The results were analyzed based on the highest score, overall score, base coverage, and E-value (expected value) consistency. Finally, a phylogenetic tree was constructed using MEGA 5.0 software to determine the strain's taxonomic position.

[0077] (3) Preservation of strains

[0078] Inoculate a single colony into LB liquid medium and incubate at 37°C and 150 rpm for 1-3 days, then mix with 50%...

[0079] Glycerol was mixed in a 1:1 ratio and placed into sterile 1.5 mL centrifuge tubes for freezing at -80°C.

[0080] 1.2.4 Study on the enzymatic properties of cellulose-degrading bacteria

[0081] (1) Construction of standard curves for glucose and xylose

[0082] Following Zhao Yu's method, 0-2.0 mL of a 1 mg / mL glucose / xylose standard solution was placed in a colorimetric tube, and ddH2O was added to bring the volume to 2 mL. Then, 3 mL of DNS (as shown in Table 2) was added and mixed well. The tube was then boiled in a water bath for 5 min, cooled to room temperature, and brought to a final volume of 25 mL with ddH2O. The mixture was inverted and mixed thoroughly, and the OD540 nm was measured using a spectrophotometer. Standard curves for glucose and xylose were plotted with the optical density value on the ordinate and the glucose / xylose content (mg) on ​​the abscissa.

[0083] Table 2 Preparation of Standard Glucose / Xylose Solution

[0084] serial number 0 1 2 3 4 5 6 7 8 9 10 Glucose / xylose standard solution 0 0 0 0 0 1 1 1 1 1 2.0 <![CDATA[ddH2O / mL]]> 2 1 1 1 1 1 0 0 0 0 0 DNS solution / mL 3 3 3 3 3 3 3 3 3 3 3

[0085] (2) Determination of endo-β-glucanase (CMC) activity

[0086] Take 1 mL of 1% CMC-Na solution, add 0.5 mL of citrate buffer (pH 4.5) and 0.5 mL of crude enzyme solution, react in a 50℃ water bath for 30 min, add 3 mL of DNS reagent, shake thoroughly, boil in a water bath for 10 min, remove and bring to a final volume of 25 mL, invert and mix well, and measure its OD540 nm. Separately, use the enzyme solution boiled at 100℃ for 20 min as a control.

[0087] (3) Determination of filter paper enzyme (FPA enzyme) activity

[0088] Take 50 mg (1 cm × 6 cm) of Xinhua filter paper as substrate, add 1.5 mL of citrate buffer at pH 4.5, change the reaction time to 60 min, and follow the same steps as for determining CMC enzyme activity.

[0089] (4) Determination of xylanase activity

[0090] The assay procedure is the same as that for CMC enzyme activity assay; only the substrate needs to be changed to a 1% xylan solution.

[0091] (5) Optimal pH of enzyme reaction

[0092] According to the preparation method of crude enzyme solution in 2.2.3, the effect of buffer solutions with different pH values ​​on enzyme activity at 50℃ was determined. Citrate-sodium citrate buffer solutions with pH ranges of 2.5-8 were used to measure CMC enzyme activity, FPA enzyme activity, and xylanase activity. The relative enzyme activity was calculated with the enzyme activity at the optimal pH of the enzyme reaction as 100%.

[0093] (6) Optimal temperature of enzyme reaction

[0094] According to the preparation method of crude enzyme solution in 2.2.3, the effect of different temperatures on enzyme activity was determined at the optimal pH value. The temperature in the enzymatic reaction was adjusted to 30-80℃, and the CMC enzyme activity, FPA enzyme activity, and xylanase activity were measured. The relative enzyme activity was calculated with the enzyme activity at the optimal temperature of the enzyme reaction as 100%.

[0095] (7) Definition of enzyme activity

[0096] Enzyme activity is defined as the amount of enzyme required to produce 1 microgram of reducing sugar from a substrate solution in 1 minute at 50°C and pH 4.5. It is abbreviated as U / mL. The enzyme activity formula is:

[0097]

[0098] Where: U - cellulase activity, U / mL; reducing sugar content, mg; 1000 - conversion factor; n - dilution factor; T - reaction time, min; V - enzyme volume, mL.

[0099] 1.3.1 Screening results of cellulose-degrading bacteria

[0100] Initial screening was conducted on soil samples from a snail farming base in Ligao Town, Liuzhou City, Guangxi Province, using a medium with CMC-Na as the sole carbon source. The results were then spread onto a solid medium plate for secondary screening, yielding a strain with good growth. This strain was named HF, and the CMC, FPA, and xylanase activities of the HF strain were 5.1378 U / mL, 3.4737 U / mL, and 9.4223 U / mL, respectively.

[0101] 1.3.2 Morphological observation results of the strain

[0102] like Figure 1 As shown, under a microscope, Gram staining is positive, but the staining is easily destained. Colony morphology reveals golden-yellow colonies, approximately 1 mm in diameter, round, convex, with neat edges, opaque, smooth, glossy, and moist. Scanning electron microscopy shows that HF ​​bacteria are irregular, straight, or slightly curved rod-shaped, 0.4 × 2.5–4.0 μm, occasionally exhibiting typical rod-like branching.

[0103] 1.3.3 Molecular biological identification results of the strain

[0104] The 16S rDNA sequence of HF strain was amplified using colony PCR technology, with HF strain as the DNA template. The PCR amplification results of the strain are as follows: Figure 2 As shown in the figure, channel 1 is a blank group with no bands, while the target bands amplified in channels 2 and 3 are approximately 1500 bp in size, which is consistent with the theoretical value.

[0105] Sequencing results of the HF strain were compared with BLAST sequences in the NCBI GenBank database for homology analysis. A phylogenetic tree was then constructed using MEGA 5.0 software, and the results are as follows: Figure 3 As shown. After sequence alignment, HF bacteria showed 100% sequence similarity to Cellulomonas iranensis of the genus Cellulomonas sp.

[0106] 1.3.4 Study on the enzymatic properties of the strain

[0107] (1) Construction of standard curves for glucose and xylose

[0108] Based on the experimental results, the glucose / xylose content (mg) is plotted on the x-axis, and the corresponding absorbance OD540nm value is plotted on the y-axis. For example... Figure 4 (A) shows the standard curve for glucose, and its corresponding regression equation is: y = 0.6407x + 0.0089, (R²) 2 =0.9992. ) For example Figure 4(B) shows the standard curve for xylose, and its corresponding regression equation is: y = 0.7033x - 0.001, (R² - π / 2)² = 0.7033x - 0.001. 2 =0.9992. Both meet the requirements of the standard curve.

[0109] (2) Optimal pH of enzyme reaction

[0110] To determine the optimal pH for the three enzymes, the pH of the reaction system was first adjusted to 2.5-8.0 using a citrate-sodium citrate buffer solution. The results are as follows: Figure 5 As shown, at pH 2.5, the relative enzyme activities of CMC, FPA, and xylanase were 28.85%, 22.03%, and 25.84%, respectively. Between pH 2.5 and 4.5, the activities of the three enzymes showed a significant increasing trend, reaching their maximum at pH 4.5. When the pH of the reaction system was greater than 4.5, the enzyme activities decreased, showing a downward trend. When the pH reached 8.0, the relative enzyme activities of CMC, FPA, and xylanase were 13.74%, 10.78%, and 12.29%, respectively. Therefore, the optimal pH for the enzyme reaction of these three enzymes was 4.5.

[0111] (3) Optimal temperature of enzyme reaction

[0112] To determine the optimal temperature for the enzyme reaction system of the HF strain, we measured the effect of 30-80℃ on the enzyme, and the results are as follows: Figure 6 As shown, the optimal reaction temperature for CMC, FPA, and xylanase is 50℃. When the reaction system temperature is below 50℃, the activities of the three enzymes increase with increasing temperature, showing a significant upward trend with roughly the same rate of increase. At temperatures of 45-55℃, the relative enzyme activities of the three enzymes remain above 80%. When the temperature is above 50℃, the activities of the three enzymes decrease with increasing temperature, possibly because excessively high temperatures alter the enzyme structure, preventing binding to the substrate and even leading to enzyme inactivation. When the temperature rises to 80℃, the relative enzyme activities of CMC, FPA, and xylanase are 25.35%, 16.21%, and 20.28%, respectively.

[0113] 1.3.5 Results of filter paper strip degradation test

[0114] To test the ability of the HF strain to degrade filter paper, we measured the filter paper residue rate. For example... Figure 7As shown, the degradation rate of the filter paper by the strain was relatively slow in the first 3 days, possibly because the bacteria were adapting to the filter paper disintegration medium or using the medium for growth and reproduction. When the culture reached day 3, the residual rate of the filter paper was 81.56%. After that, the residual rate of the filter paper dropped rapidly, reaching 54.79% on day 4. The period from day 3 to day 6 was the period of fastest degradation. By day 6, the residual rate of the filter paper was 5.25%. After that, the rate of residual rate of the filter paper slowed down significantly, reaching 1.72% on day 7. That is, the HF strain degraded 3 g / L of filter paper.

[0115] In addition, we set up a blank group (without added bacterial cells) and a treatment group (with added bacterial cells), from... Figure 8 (A) We found that the filter paper strips in the blank group were not degraded, the solution was clear, while from... Figure 8 (B) We found that the filter paper strips were missing in the treatment group, and the culture medium turned yellow and turbid, with tiny flocculent precipitates. The filter paper residue rate and experimental phenomena indicate that the HF strain has a strong ability to degrade cellulose.

[0116] As can be seen, this experiment screened a cellulose-degrading bacterium, HF, from mud samples from a snail farming base. The CMC, FPA, and xylanase activities of this strain were 5.1378 U / mL, 3.4737 U / mL, and 9.4223 U / mL, respectively, and it was identified as *Cellulomonas iranensis*. To obtain the highest enzyme activity of the HF strain, we conducted some enzymatic property experiments, finding that the optimal pH and temperature for the three enzyme activities of the HF strain were 4.5 and 50℃, respectively. Finally, to verify the cellulose-degrading ability of the HF strain, we conducted a filter paper strip degradation test. We found that the filter paper strips in the blank group were completely unaffected, while the solution in the treatment group became turbid, and the residual rate of the filter paper was 1.72%, indicating that the HF strain has a strong filter paper degradation ability.

[0117] Straw degradation results: see Figure 9 ,from Figure 9 It can be seen that after 25 days, *Cellulosum iridis* had decomposed the straw into very thin fragments, demonstrating its excellent ability to degrade straw. In contrast, commercially available lignocellulosic degrading bacteria did not significantly alter the overall shape of the straw after 25 days of degradation.

[0118] After sequence alignment, HF bacteria showed 100% sequence similarity to Cellulomonas iranensis of the genus Cellulomonas sp.

[0119] The strain has been identified by microbial taxonomy and has been deposited: named Cellulomonas iranensis strain LG2020, with accession number GDMCC No. 62533, deposit date: June 13, 2022, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC).

[0120] 1.3.6 Degradation of rice straw and sugarcane leaves by strain LG2020

[0121] Naturally dried rice straw and sugarcane leaves were pulverized into filaments approximately 1 cm in length and dried at 80℃ to constant weight. 500 ml of fresh Iranian cellulobacillus LG2020 culture solution was prepared. Under aseptic conditions, 10 grams of the pulverized rice straw and sugarcane leaves were accurately weighed into the culture solution, stirred thoroughly, and incubated at 30℃. Twelve bottles were prepared for each type of straw. Two bottles from each type were collected daily. The rice straw or sugarcane leaves were washed with clean water, dried, and weighed. The average value was used to calculate the degradation rate. The test was conducted for 6 days, and the results are shown in Table 3.

[0122] Table 3

[0123]

[0124] It is evident that LG2020 bacterial solution achieved a degradation rate of 52.3% for rice straw in 4 days and 64.5% in 6 days. LG2020 bacterial solution was even more effective in degrading sugarcane leaves, with a degradation rate of 58.5% in 4 days and 72.2% in 6 days.

[0125] 2.1.1 Preparation of crude enzyme solution from strain

[0126] Preparation of crude enzyme solution from HF strain: The selected strain was inoculated onto LB liquid medium and cultured at 37℃ and 150 r / min to prepare 10... 6 CFU bacterial suspension was inoculated into the enzyme-producing fermentation medium at a 10% inoculum and cultured at 37℃ and 150 rpm for 3 days to obtain the fermentation broth. The fermentation broth was centrifuged at 10000 rpm for 10 min, and the supernatant was collected as the crude enzyme solution. Then, the CMC enzyme activity, FPA enzyme activity, xylanase activity, and OD600 were measured, with 3 replicates per group.

[0127] 2.1.2 Optimization of external conditions for enzyme production by the strain

[0128] (1) Effect of shaking speed on enzyme production

[0129] The enzyme was cultured at different shaking speeds ranging from 75 to 250 r / min. The maximum enzyme activity at each shaking speed was taken as 100%, and the relative enzyme activity was calculated.

[0130] (2) Effect of culture medium volume on enzyme production

[0131] Fill 500mL Erlenmeyer flasks with 100-300mL of culture medium and incubate at the optimized rotation speed determined in the previous step. Calculate the relative enzyme activity, taking the maximum enzyme activity at each culture medium volume as 100%.

[0132] (3) Effect of initial pH of culture medium on enzyme production: Under the conditions optimized in the above two steps, the initial pH of the culture medium was adjusted to 4.0-8.4 for cultivation. The maximum enzyme activity at the initial pH of different culture media was taken as 100%, and the relative enzyme activity was calculated.

[0133] (4) Effect of inoculum size on enzyme production

[0134] Under the optimal conditions described above, bacterial cultures with inoculum concentrations ranging from 2.5% to 20% were inoculated into enzyme-producing media and cultured. The maximum enzyme activity at each inoculum concentration was taken as 100%, and the relative enzyme activity was calculated.

[0135] (5) Effect of culture time on enzyme production

[0136] Under the optimal conditions described above, the culture medium is incubated for 1-7 days. The maximum enzyme activity at different incubation times is taken as 100%, and the relative enzyme activity is calculated.

[0137] (6) Effect of culture temperature on enzyme production

[0138] Under the optimal conditions described above, the culture medium was incubated at 27-46℃. The maximum enzyme activity at each incubation temperature was taken as 100%, and the relative enzyme activity was calculated.

[0139] 2.1.3 Optimization of enzyme production culture medium conditions for the strain

[0140] Under optimal external conditions for enzyme production based on HF strains, the enzyme production medium was further optimized.

[0141] (1) Effects of different carbon sources and carbon source concentrations on enzyme production

[0142] The optimal carbon source for the culture medium was determined using 10 g / L soluble starch, glucose, sucrose, CMC-Na, lactose, fructose, filter paper, and maltose as carbon sources, with no added carbon source as a control. The relative enzyme activity of the blank group was calculated as 100%.

[0143] After determining the optimal carbon source for culture, a carbon source concentration gradient of 0.5-3% was designed. The relative enzyme activity was calculated with the maximum enzyme activity at different nitrogen source concentrations as 100%.

[0144] (2) Effects of different nitrogen sources and nitrogen source concentrations on enzyme production

[0145] The optimal nitrogen source for the culture medium was determined by using 20 g / L of peptone, corn steep liquor, soybean flour, sodium nitrate, ammonium sulfate, ammonium chloride, urea, potassium nitrate, and ammonium nitrate as nitrogen sources, with no nitrogen source added as a control. The relative enzyme activity of the blank group was calculated as 100%.

[0146] After determining the optimal nitrogen source for culture, a nitrogen source concentration gradient of 0.5-3% was designed. The maximum enzyme activity at different nitrogen source concentrations was taken as 100%, and the relative enzyme activity was calculated.

[0147] (3) Effect of DMF on enzyme production

[0148] Add 0-2% DMF to the optimized enzyme-producing medium as described above, with no DMF added as a control. Calculate the relative enzyme activity of the blank group as 100%.

[0149] (4) Effect of bovine serum albumin on enzyme production

[0150] Bovine serum albumin (BSA) at concentrations of 0-2% was added to the optimized enzyme-producing medium, with no BSA added serving as a control. The relative enzyme activity of the blank group was calculated as 100%.

[0151] (5) Effect of Tween-80 on enzyme production

[0152] The optimized enzyme-producing medium was supplemented with 0-2% Tween-80, with no Tween-80 added as a control. The enzyme activity of the blank group was taken as 100%, and its relative enzyme activity was calculated.

[0153] (6) Effect of PEG6000 on enzyme production

[0154] PEG6000 at concentrations of 0-2% was added to the optimized enzyme-producing medium, with no PEG6000 added serving as a control. The relative enzyme activity of the blank group was calculated as 100%.

[0155] (7) Effects of different metal ions on enzyme production

[0156] The optimized enzyme-producing medium was supplemented with 10 mM concentrations of Zn²⁺, Mg²⁺, Al³⁺, Cu²⁺, K⁺, Ag⁺, Ca²⁺, Mn²⁺, Fe²⁺, Hg⁺, and Fe³⁺, respectively, with the unadded metal ions serving as a control. The relative enzyme activity of the blank group was calculated as 100%.

[0157] 2.1.4 Optimization of enzyme production conditions using response surface methodology

[0158] Based on the single-factor experiments, and according to the central composite design principle, four factors—peptone concentration, bovine serum albumin concentration, initial pH of the culture medium, and culture temperature—were selected as independent variables affecting enzyme activity. The relationships between these factors were further investigated. A four-factor, three-level experiment was conducted using DesignExpert 10 and SAS 9.1 software to determine the optimal culture conditions for enzyme activity. The factor level coding table is shown in Table 4.

[0159] Table 4. Factors and Levels in Box-Behnken Experiment Design

[0160]

[0161] 2.1.5 Data Statistics and Analysis

[0162] Each experimental group was biologically replicated three times. Data were plotted using Origin Pro 9.1 and GraphPad Prism 8.0.1 software.

[0163] 2.2 Results and Analysis

[0164] 2.2.1 Effect of shaker speed on enzyme production

[0165] The rotation speed not only affects the rate of cell growth and metabolism, but also the amount of dissolved oxygen during fermentation, directly influencing cell growth and enzyme activity. Experimental results are as follows: Figure 10 As shown, when the shaker speed is less than 200 r / min, the activities of the three enzymes and OD600 of the HF strain gradually increase with increasing shaker speed. This is because the increased speed increases the contact area between the culture medium and air, allowing a large amount of oxygen to dissolve into the medium and replenish the oxygen consumed by the bacteria during growth and reproduction. The production of the three enzymes is highest at a shaker speed of 200 r / min. When the shaker speed exceeds 200 r / min, the OD600 of the HF strain increases, while the enzyme activity shows a rapid decreasing trend. This may be because the excessively high shaker speed leads to excessively rapid bacterial growth and metabolism, causing the bacteria to enter the apoptosis phase prematurely. Therefore, a shaker speed of 200 r / min is most favorable for enzyme production in the HF strain.

[0166] 2.2.2 Effect of culture medium volume on enzyme production

[0167] The volume of liquid directly affects the oxygen concentration in the Erlenmeyer flask. Based on the optimized culture conditions of the strain, HF strain is an aerobic bacterium. In this experiment, nine groups were set up in 500mL Erlenmeyer flasks, with liquid volumes ranging from 100-300mL. The results are as follows: Figure 11As shown, the volume of the culture medium did not significantly affect the OD600 of the HF strain. When the volume was 100 mL, the relative enzyme activities of the three enzymes were 49.17%, 19.18%, and 50.64%, respectively. When the volume was less than 200 mL, the relative enzyme activities of all three enzymes increased with increasing volume, with FPA showing the most significant increase, increasing by about 80%. The relative enzyme activities of xylanase were also higher than those of CMC and FPA. The activities of all three enzymes were at their maximum when the volume was 200 mL. When the volume was between 175 and 225 mL, the relative enzyme activity of xylanase was above 90%. As the volume increased, the activities of all three enzymes decreased, and the rate of decrease of xylanase activity was linear. This may be because a larger volume reduces the surface area of ​​the culture medium in contact with air, depriving the bacteria of oxygen and thus reducing their enzyme activity. Therefore, selecting a liquid volume of 200 mL out of a 500 mL container is most conducive to enzyme production by the HF strain.

[0168] 2.2.3 Effect of initial pH of culture medium on enzyme production

[0169] This experiment determined the enzyme production capacity of the culture medium with an initial pH between 4.0 and 8.4. The results are as follows: Figure 12 As shown in the figure, the OD600 and enzyme activity of the HF strain increased between pH 4.0 and 6.4, with relative enzyme activities generally above 80% between pH 6.0 and 7.2. However, they decreased at pH values ​​above 6.4. Furthermore, the relative enzyme activity of PFA was found to be higher than that of CMC and xylanase. At pH 8.4, the relative enzyme activities of the three enzymes were 52.55%, 56.46%, and 32.15%, respectively. Additionally, the decreasing trends of PFA and CMC enzyme activities were found to be roughly the same. In conclusion, the HF strain is suitable for enzyme production fermentation in weakly acidic and neutral environments. Therefore, a culture medium pH of 6.4 is most favorable for enzyme production by the HF strain.

[0170] 2.2.4 Effect of inoculum size on enzyme production

[0171] The inoculum size affects enzyme activity and the fermentation cycle to varying degrees. The results are as follows... Figure 13As shown, increasing the inoculum size did not significantly affect the growth of the HF strain. When the inoculum size was 2.5%, the relative enzyme activities of the three enzymes were 15.59%, 18.92%, and 22.27%, respectively. When the inoculum size was between 2.5% and 15%, the yield of the three enzymes gradually increased with increasing inoculum size, but the growth rate did not exhibit a stable, regular pattern. The strain exhibited the highest enzyme activity at an inoculum size of 15%. When the inoculum size exceeded 15%, the enzyme yield decreased with increasing inoculum size. This may be because excessive inoculum size leads to excessive bacterial proliferation, excessive absorption of nutrients, and a rapid decrease in dissolved oxygen content, resulting in decreased enzyme activity. Therefore, an inoculum size of 15% is most favorable for enzyme production in the HF strain.

[0172] 2.2.5 Effect of culture time on enzyme production

[0173] To determine the optimal enzyme production time for the HF strain, we measured the enzyme activity from day 1 to 7. The experimental results are as follows: Figure 14 As shown, in the early stage of cultivation, due to the short fermentation time, the cells were still in the logarithmic growth phase and had not yet entered the stationary phase, resulting in an insufficient number of HF strains. Furthermore, most cells had not yet reached the enzyme-producing phase; on day 1, the relative enzyme activities of the three enzymes were all around 50%. As the fermentation time increased, the amount of enzyme produced by the cells accumulated, reaching its maximum on day 3, at which point the cells entered the stationary phase. With further cultivation, after day 3, the cells began to enter the apoptosis phase, slowly aging and undergoing autolysis. In addition, the accumulation of metabolic products in the enzyme-producing medium was detrimental to cell growth and enzyme production, causing the enzyme activity in the fermentation broth to decrease. Therefore, a cultivation time of day 3 was most favorable for enzyme production by the HF strain.

[0174] 2.2.6 Effect of culture temperature on enzyme production

[0175] Temperature affects the growth and reproduction rate of bacteria. At lower temperatures, bacterial growth is slow, making it difficult for them to absorb nutrients for enzyme production, resulting in lower enzyme yields. Conversely, at higher temperatures, bacterial growth and metabolism are faster, causing bacteria to prematurely enter the stationary and apoptosis phases, reducing the bacterial population and consequently decreasing enzyme production capacity. This experiment established seven temperature gradients, and the enzyme production of the HF strain is shown below. Figure 15 As shown, when the temperature is below 37℃, both the OD600 and enzyme activity of the HF strain increase with increasing temperature; when the temperature range is 34-40℃, the enzyme activity of the strain is relatively high, and the relative enzyme activities of CMC enzyme and xylanase are above 80%; when the temperature is above 37℃, both OD600 and enzyme activity decrease; when the temperature is 46℃, the relative enzyme activities of CMC enzyme, FPA enzyme, and xylanase are 13.94% and 13.46%, respectively.

[0176] The percentage was 33.34%. This may be because excessively high temperatures reduce enzyme activity within the strain, thus affecting metabolic processes. We also found that temperature has a greater impact on FPA enzyme activity. Therefore, a culture temperature of 37℃ is most favorable for enzyme production by the HF strain.

[0177] 2.2.7 Effects of different carbon sources and carbon source concentrations on enzyme production

[0178] Carbon sources not only provide nutrients for the growth of HF strains but also induce cellulase activity. The optimization results of carbon sources are as follows: Figure 16 As shown in (A), the results indicate that when soluble starch, glucose, sucrose, CMC-Na, filter paper, maltose, and fructose were used as the sole carbon source, the bacteria could grow and reproduce in large quantities, and all produced CMC enzyme, FPA enzyme, and xylanase, all at higher levels than the control group. Using the relative enzyme activity of the control group as 100%, it was found that all carbon sources could promote enzyme activity production by strain HF. Among these, the strain produced the highest enzyme activity when CMC-Na was used as the carbon source, with relative enzyme activities of CMC enzyme, FPA enzyme, and xylanase at 289.35%, 383.02%, and 285.50%, respectively, significantly higher than the other carbon sources. Furthermore, we found that the relative enzyme activity of FPA enzyme was higher than that of CMC enzyme and xylanase among carbon sources other than maltose. The relative enzyme activities of PFA enzyme in CMC-Na and filter paper were approximately 80% higher than the other two enzymes, suggesting that these two carbon sources are more conducive to PFA enzyme production. Therefore, we chose CMC-Na as the carbon source for further optimization.

[0179] Meanwhile, we also optimized the concentration of CMC-Na, and the results are as follows: Figure 16 As shown in (B), when the CMC-Na concentration is between 0.5% and 1.0%, the activities of the three enzymes continuously increase with increasing concentration. However, when the concentration is between 1% and 2.5%, the relative activities of the three enzymes are not significantly different, generally remaining above 90%. At a CMC-Na concentration of 2.0%, the relative activities of the three enzymes are 100%. When the CMC-Na concentration is higher than 2.0%, the relative activities of the three enzymes slowly decrease, possibly because the excessively high CMC-Na concentration inhibits the growth and reproduction of the bacteria. Therefore, a CMC-Na concentration of 2% is most favorable for enzyme production in the HF strain.

[0180] 2.2.8 Effects of different nitrogen sources and nitrogen source concentrations on enzyme production

[0181] Nitrogen sources are essential components of nucleic acids and enzymes in microbial cells. Nine different nitrogen sources were selected for fermentation and enzyme production culture of HF strains. The relative enzyme activities of the three enzymes measured for each nitrogen source are as follows: Figure 17As shown in (A), the figure shows that, with the relative enzyme activity of the blank group as 100%, the enzyme activity measured using peptone as the sole nitrogen source was the highest and significantly higher than other nitrogen sources. The relative enzyme activities of CMC enzyme, FPA enzyme, and xylanase were 323.34%, 410.12%, and 324.29%, respectively. Corn syrup powder also significantly affected enzyme activity, with the three enzyme activities being approximately 1.73, 1.49, and 1.93 times that of the blank group, respectively. Soybean flour promoted xylan activity, with a relative enzyme activity of 192.44%, approximately twice that of the blank group; however, it inhibited CMC and FPA enzyme activities. Therefore, in the single-factor optimization of the effect of different nitrogen sources on enzyme activity, peptone was the optimal nitrogen source. We then optimized the enzyme production using the peptone concentration, and the experimental results are shown below. Figure 17 As shown in (B), peptone concentration promoted bacterial growth and reproduction, indicating that using peptone as a nitrogen source was reasonable. The maximum enzyme activity was achieved at a peptone concentration of 2%. At a concentration of 0.5%, the relative enzyme activities of the three enzymes were 53.50%, 30.14%, and 56.33%, respectively. At concentrations below 1.5%, the relative enzyme activities increased continuously with increasing peptone concentration, and the relative enzyme activities of the three enzymes were not significantly different between concentrations of 1.5% and 2%. However, at concentrations above 2%, the relative enzyme activities of the three enzymes showed a decreasing trend with increasing peptone concentration. Therefore, a peptone concentration of 2% was most beneficial for enzyme production in the HF strain.

[0182] 2.2.9 Effect of DMF on enzyme production

[0183] DMF, as an excellent inducer for high cellulase production, primarily induces a significant increase in intracellular Ca2+ levels and triggers the Ca2+-CRZ1 signaling pathway to induce enzyme gene transcription, thereby promoting enzyme activity. By adding different concentrations of DMF to the enzyme-producing medium, enzyme yield was increased; the experimental results are as follows... Figure 18 As shown, DMF significantly promoted the growth of the HF strain, and the highest enzyme activity was achieved at a DMF concentration of 0.5%, with relative enzyme activities of CMC enzyme and xylanase reaching 123.42% and 110.72%, respectively. However, at a DMF concentration of 1.0%, the relative enzyme activity of FPA enzyme reached its maximum.

[0184] 115.06%; When the concentration is greater than 1.0%, the relative enzyme activity decreases continuously with increasing DMF concentration, and the relative enzyme activity is lower than that of the control group. This may be because excessively high DMF concentrations inhibit the enzyme production capacity of the bacteria. Therefore, a DMF concentration of 0.5% is most favorable for enzyme production in the HF strain.

[0185] 2.2.10 Effect of bovine serum albumin on enzyme production

[0186] Bovine serum albumin (BSA) is often used as an enzyme stabilizer. Its strong hydrophobicity allows it to form ordered molecular aggregates—micelles—in solution, accelerating the movement of substrates, enzymes, and products, promoting enzyme desorption, and enhancing the effective adsorption of substrates and enzymes, thereby significantly increasing enzyme activity. Results are as follows... Figure 19 As shown, the addition of bovine serum albumin (BSA) to the enzyme-producing medium had little effect on the growth of the HF strain. When the BSA concentration was less than 1%, the enzyme activities of the three enzymes gradually increased with increasing BSA concentration. At a concentration of 1%, the strain exhibited the highest enzyme activity, with relative activities of CMC enzyme, FPA enzyme, and xylanase of 215.57%, 206.97%, and 192.23%, respectively. Compared with the control group, the activities of the three enzymes increased approximately twofold. When the concentration was greater than 1%, the enzyme activities decreased with increasing BSA concentration, and the decreasing trend of FPA enzyme activity was slower than that of CMC enzyme and xylanase activities. Therefore, a BSA concentration of 1% was most favorable for enzyme production by the HF strain.

[0187] 2.2.11 Effect of Tween-80 on enzyme production

[0188] The hydrophilic end of Tween-80 can adsorb cellulase, increasing enzyme-substrate binding and accessibility, thus making the cellulase component system more stable. Results are as follows... Figure 20 As shown, the addition of Tween-80 to the enzyme-producing medium significantly affected the growth and reproduction of the HF strain. When the Tween-80 concentration was less than 0.5%, the yields of the three enzymes gradually increased with increasing concentration. At a concentration of 0.5%, the strain exhibited the highest enzyme activity, with relative enzyme activities of CMC enzyme, FPA enzyme, and xylan at 123.42%, 114.02%, and 110.70%, respectively. When the concentration was greater than 0.5%, the enzyme activity decreased with increasing Tween-80 concentration. Furthermore, the relative enzyme activity of xylanase was found to be higher than that of CMC enzyme and FPA enzyme, suggesting that Tween-80 is more beneficial for xylanase production by the HF strain. Therefore, a Tween concentration of 0.5% is most favorable for enzyme production by the HF strain.

[0189] 2.2.12 Effect of PEG6000 on enzyme production

[0190] PEG6000 can alter the structure of substrates, increasing the accessibility of cellulases, and also making enzymes more stable, preventing them from denaturing during production; for example... Figure 21As shown, PEG6000 had little effect on the growth of the HF strain. At concentrations between 0.5% and 1.5%, the relative enzyme activities of the three enzymes were similar, but all were higher than those of the control group. When the concentration exceeded 1.5%, the relative enzyme activities of all three enzymes showed a decreasing trend, with the FPA enzyme activity decreasing most significantly. When the PEG6000 concentration was 2%, the relative enzyme activities of the three enzymes were 89.42%, 61.59%, and 74.86%, respectively. Therefore, a PEG6000 concentration of 0.5% was most beneficial for enzyme production in the HF strain.

[0191] 2.2.13 Effects of different metal ions on enzyme production

[0192] Some metal ions can act as enzyme activators, promoting enzyme activity, while others can interact with the active groups of enzymes, thus inactivating them. For example... Figure 22 The figure shows the effect of a final metal ion concentration of 10 mM on the activities of three enzymes in the crude enzyme solution of HF strain. As can be seen from the figure, Zn... 2+ Mg 2+ Cu 2+ It promotes the CMC enzyme activity of HF strains, including Zn 2+ It significantly promoted the activity of all three enzymes, with relative enzyme activities of CMC, FPA, and xylanase reaching 118.65%, 117.49%, and 122.18%, respectively, while Al... 3+ Ca 2+ K + Fe 3+ Fe 2+ It inhibits enzyme activity but promotes bacterial growth, among which Hg... + Ag + It completely inhibited enzyme activity and bacterial growth. Mn 2+ The effect on enzyme activity was not significant. Finally, 10 mM zinc chloride was added to the enzyme-producing medium.

[0193] 2.2.14 Optimization of enzyme production conditions using response surface methodology

[0194] The Box-Behnken experimental group was created using Design Expert 10, and a total of 29 experiments were conducted. The results are shown in Table 5.

[0195] Table 5 Summary of Box-Behnken Test Results

[0196]

[0197]

[0198] By performing quadratic polynomial fitting on 29 sets of data from the Box-Behnken experiment results table, the quadratic polynomial regression equations relating the activity values ​​of the three enzymes to four factors were obtained as follows:

[0199] Y1=50.40+1.61X1+1.77X2-0.22X3+0.81X4+1.18X1X2+0.69X1X3-1.67X1X4+0.27X2X3+0.54X2

[0200] X4 + 0.12X3X4 - 4.17X 12 -4.88X 22 -1.42X 32 -2.55X 42 (1)

[0201] Y2=22.82+1.38X1+0.74X2+0.43X3-0.038X4-0.62X1X2-0.42X1X3-0.083X1X4+0.035X2X3+0.29

[0202] X2X4+0.30X3X4-1.49X 12 -0.62X 22 -1.38X 32 -170X 42 (2)

[0203] Y3=91.76+2.34X1+2.61X2+1.15X3-0.52X4+1.59X1X2-0.61X1X3-0.80X1X4+5.75×10 -4 X2X3-0.3

[0204] 8 x 2 x 4 + 1.36 x 3 x 4 - 5.10 x 12 -2.23X 22 -4.10X 32 -5.45X 42 (3)

[0205] In the formula: Y1 is CMC enzyme activity (U / mL), Y2 is FPA enzyme activity (U / mL), Y3 is xylanase activity (U / mL), and X1, X2, X3, and X4 are peptone concentration, bovine serum albumin concentration, initial pH of the culture medium, and culture temperature, respectively.

[0206] Table 6 of the CMC enzyme activity results table shows that the model P < 0.0001, indicating that the model selected in this experiment has extremely significant differences, suggesting that the data is reasonable; the F-value of the model is 43.74, and less than 1% of the probability cannot be explained by this model, indicating that the model is significant; the mean squared error of the model is 23.64, and the variance is 330.92, indicating that the model is significant; the lack of fit P = 0.1917 > 0.05, indicating that the difference in the lack of fit of the model is not significant, indicating that the regression model has a high degree of fit with the experimental data, which meets the requirements of response surface optimization; as shown in Table 7, the correlation coefficient R2 of the model is 0.9776, the Std. Dev is 0.74, and the coefficient of deviation is 1.63, indicating that the model fits the actual situation well; the corrected coefficient of determination RAdj = 0.9553 and Pred R-Squared = 0.8840, indicating that the quadratic equation has high reliability.

[0207] Table 6. Analysis of Variance of Box-Behnken Experiment: CMC Enzyme Activity Results

[0208]

[0209]

[0210] Analysis of variance (F-value) revealed that the order of influence of the four factors on the experiment was: X2 > X1 > X4 > X3, i.e., bovine serum albumin concentration > peptone concentration > culture temperature > initial pH of the culture medium. Among these, bovine serum albumin concentration and peptone concentration had the main effects, while culture temperature and initial pH of the culture medium had secondary effects. If quadratic and interaction terms are considered, the order of influence of each factor on the experiment becomes: X 22 >X 12 >X 42 >X 32 >X1X4>X1X2>X1X3>X2X4>X2X3>X3X4.

[0211] Table 7. Box-Behnken Experiment Variance Analysis: Other Parameters of CMC Enzyme Activity

[0212]

[0213] Next, the response surface methodology for CMC enzyme activity was analyzed, yielding contour lines and response surface plots of the corresponding interactions. The results are as follows: Figure 23 As shown.

[0214] Depend on Figure 23It can be seen that at pH 6.4 and culture temperature 37℃, the effects of peptone concentration and bovine serum albumin (BSA) concentration increase with increasing dosage. Within a certain range, CMC enzyme activity increases with increasing dosage. After peptone concentration exceeds 2% and BSA concentration exceeds 1%, CMC enzyme activity begins to decrease steadily. The effects of peptone concentration and BSA concentration are significant, and the response surface is relatively steep. The contour lines of the response surface show that the optimal conditions are within the range of peptone concentration 1.9-2.3% and BSA concentration 0.9-1.3%.

[0215] The quadratic polynomial regression equation was analyzed using the software SAS. First, the first-order partial derivative of the equation was calculated and required to be equal to 0. The optimal level values ​​of the four factors were obtained, namely X1 = 0.199207, X2 = 0.211417, X3 = -0.003275, and X4 = 0.115813. That is, the peptone concentration, bovine serum albumin concentration, initial pH of the culture medium, and culture temperature were 2.0996 (%), 1.1057 (%), 6.399, and 37.3474 (℃), respectively. Substituting the obtained data into equation (1), the predicted value of the maximum CMC enzyme activity Y1 = 50.7932 U / mL was obtained.

[0216] As shown in Tables 8 and 9 of the FPA enzyme activity results from the analysis of variance, the model P < 0.0001, indicating that the differences in the selected model are extremely significant; the F-value of the model is 32.00, and less than 1% of the probability cannot be explained by this model, which also indicates that the model is significant; the lack-of-fit term P = 0.7248 > 0.05, and the variance of the lack-of-fit term is 1.33, indicating that the difference in the lack-of-fit term is not significant, indicating that the regression model fits the experimental data relatively well; the root mean square error of the model is 0.39, the mean response is 20.67, and R² = 0.9697, indicating that the model fits the actual situation well and the data accuracy is high; the coefficient of deviation is 1.88 and the correction determination coefficient RAdj = 0.9394, indicating that the equation has high reliability.

[0217] Table 8. Analysis of Variance for Box-Behnken Experiment: FPA Enzyme Activity Results

[0218]

[0219] Table 9. Box-Behnken Experiment Variance Analysis: Other Parameters of FPA Enzyme Activity

[0220]

[0221] Analysis of variance (ANOVA) comparing F-values ​​revealed that the order of influence of the four factors on enzyme activity during fermentation in the enzyme-producing medium was: X1 > X2 > X3 > X4, i.e., peptone concentration > bovine serum albumin (BSA) concentration > initial pH of the medium > culture temperature. Among these, peptone concentration and BSA concentration had the primary effects, while initial pH and culture temperature had secondary effects. Furthermore, considering the results of the quadratic and interaction terms, the sum of squares (Type I) of the quadratic term (33.14) was significantly greater than the sum of squares (Type I) of the interaction term (2.93). Therefore, the order of influence of the four factors on the experiment is: X... 42 >X 12 >X 32 >X 22 The response surface methodology for FPA enzyme activity was analyzed, yielding contour lines and response surface plots of the corresponding interactions. The results are shown below. Figure 24 As shown.

[0222] observe Figure 24 It was found that when the bovine serum albumin concentration was 1% and the culture temperature was 37℃, the CMC enzyme activity increased with increasing peptone concentration within the range of 1.5-2.1%. The effects of peptone concentration and culture medium pH were significant, resulting in a steep response surface. The contour lines of the response surface indicate that the optimal conditions were between a peptone concentration of 2.0-2.4% and a culture medium pH of 6.3-6.5%. By using the software SAS to perform typical analysis on the response surface of the regression equation based on the code, the derivative of equation (2) is obtained and the result is set to 0. The optimal critical values ​​of the four factors are obtained, namely X1 = 0.3599, X2 = 0.431425, X3 = 0.110514, and X4 = 0.026381, which are 2.1780 (%), 1.2157 (%), 6.444, and 37.0791 (℃) of peptone concentration, bovine serum albumin concentration, initial pH of culture medium, and culture temperature, respectively. Substituting the obtained data into equation (2), the predicted value of the maximum FPA enzyme activity Y2 = 23.2528 U / mL can be obtained. From Tables 10 and 11 of the ANOVA results for xylanase activity, we can see that the model P < 0.0001, indicating that the differences in the selected model are extremely significant; the model F value is 26.76, and less than 1% of the probability cannot be explained by this model, which also indicates that the model is significant; the lack-of-fit term P = 0.2025 > 0.05, and the variance is 18.43, indicating that the difference in the lack-of-fit term is not significant, indicating that the regression model has a relatively high fit with the experimental data; the correlation coefficient R² = 0.9640, the mean response is 83.87, and the root mean square error is 1.24, indicating that the model fits the reality well; the corrected coefficient of determination RAdj = 0.9280 and the predicted coefficient of determination are 0.8138, indicating that the equation has high reliability.

[0223] Table 10: Results of Xylanase Activity from Box-Behnken Experiment (Analysis of Variance)

[0224]

[0225] Table 11 Other parameters of xylanase activity in the Box-Behnken experiment variance analysis.

[0226]

[0227] The F-values ​​from the analysis of variance show that the order of influence of the four factors on the experiment is: X2 > X1 > X3 > X4, i.e., bovine serum albumin concentration > peptone concentration > initial pH of the culture medium > culture temperature. Among these, bovine serum albumin concentration and peptone concentration have the main effects, while initial pH of the culture medium and culture temperature have secondary effects. If we consider the sum of squares of the quadratic and interaction terms, as well as the F-values, the order of influence of the four factors on enzyme production is: X 42 >X 12 >X 22 >X 32 The xylanase activity response surface methodology was analyzed by plotting and analyzing the results, yielding corresponding interaction contour lines and response surface plots. The results are as follows: Figure 25 As shown. By Figure 25 It can be seen that at pH 6.4 and culture temperature 37℃, the effects of peptone concentration and bovine serum albumin (BSA) concentration increase the CMC enzyme activity in a gradient manner within a certain range with increasing addition amounts; when the peptone concentration exceeds 2.4% and the BSA concentration exceeds 1.3%, the CMC enzyme activity begins to decrease steadily. The effects of peptone concentration and BSA concentration are significant, and the response surface is relatively steep. The contour lines of the response surface show that the optimal conditions are within the range of peptone concentration 2.0-2.3% and BSA concentration 1.0-1.4%.

[0228] Finally, based on the integrated analysis and differentiation of the encoded data using SAS software, four optimal levels affecting xylanase activity can be obtained, namely X1 = 0.283208, X2 = 0.348730, X3 = 0.107619, and X4 = -0.066976, which are 2.1416 (%), 1.1744 (%), 6.4430, and 36.7991 (℃) of peptone concentration, bovine serum albumin concentration, initial pH of culture medium, and culture temperature, respectively. Substituting the obtained data into equation (3), the maximum predicted value of xylanase activity Y3 = 92.63 U / mL can be obtained. In summary, under the optimal conditions combining the three enzymes, the actual fermentation process using a peptone concentration of 2.14%, a bovine serum albumin concentration of 1.17%, an initial culture medium pH of 6.44, and a culture temperature of 37℃ yielded average CMC, FPA, and xylanase activities of 49.8977 U / mL, 22.9328 U / mL, and 90.7770 U / mL, respectively. The relative errors between these activities and the predicted values ​​were 1.76%, 1.38%, and 2.00%, respectively. Furthermore, compared to the enzyme activities before optimization, the activities of the three enzymes were approximately 9.712 times, 6.602 times, and 9.634 times higher, respectively.

[0229] (1) The optimal external conditions for enzyme production by the HF strain were determined: rotation speed 200 r / min, culture medium volume 200 mL / 500 mL, initial pH of the culture medium 6.4, inoculum size 15%, fermentation time 3 days, and culture temperature 37℃. (2) The enzyme production culture medium conditions for the HF strain were optimized. The optimal carbon source was CMC-Na, with an optimal CMC-Na concentration of 2%. The optimal nitrogen source was peptone, with an optimal peptone concentration of 2%. The optimal DMF concentration was 0.5%, the optimal bovine serum albumin concentration was 1%, the optimal Tween-80 concentration was 0.5%, and the optimal PEG6000 concentration was 0.5%. Zn 2+ Mg 2+ Cu 2+ It promotes the activity of CMC enzymes, among which Zn 2+ The promoting effect on the activity of the three enzymes was most obvious, while Al 3+ Ca 2+ K + Fe 3+ Fe 2+ It has an inhibitory effect on enzyme activity, among which Hg + Ag + The enzyme activity was completely inhibited. Mn 2+(3) Under single-factor conditions, a response surface methodology with four factors and three levels was used to optimize the enzyme activity. The results showed that the optimal conditions were: peptone concentration of 2.14%, bovine serum albumin concentration of 1.17%, initial pH of the culture medium of 6.44, and culture temperature of 37℃. Under these conditions, the CMC, FPA, and xylanase activities were measured to be 49.8977 U / mL, 22.9328 U / mL, and 90.7770 U / mL, respectively. The measured values ​​were not significantly different from the predicted values. Compared with the enzyme activities before optimization, the activities of the three enzymes were approximately 9.712 times, 6.602 times, and 9.634 times higher, respectively.

[0230] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Cellulomonas aegyptiacus ( Cellulomonas iranensis Application of strain LG2020 in the preparation of cellulase and / or lignin-degrading enzyme; enzyme-producing culture medium conditions for preparing cellulase and / or lignin-degrading enzyme: optimal CMC-Na concentration 2%, peptone concentration 2%, DMF concentration 0.5%, bovine serum albumin concentration 1%, Tween-80 concentration 0.5%, PEG6000 concentration 0.5%, 10 mM zinc chloride; the *Cellulostomia iridis* strain (… Cellulomonas iranensis The strain LG2020, with accession number GDMCC No. 62533, accession date: June 13, 2022, accession address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, China, accession institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC).

2. The application as described in claim 1, characterized in that: The Iranian cellulomonas ( Cellulomonas iranensis The strain LG2020 grew well on LB liquid medium; the colonies were golden yellow, about 1 mm in diameter, round, convex, with neat edges, opaque, smooth, glossy, and moist.

3. The application as described in claim 1, characterized in that: The enzyme production conditions for preparing cellulase and / or lignin-degrading enzyme are: rotation speed of 200 r / min, culture medium volume of 200 mL / 500 mL, initial pH of culture medium of 6.4, inoculum size of 15%, fermentation time of 3 days, and culture temperature of 37℃.

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