Acremonium nanhaiense CGMCC 42655 and application thereof

Abstract

The application discloses a deep-sea fungus Acremonium nanhaiense CGMCC 42655 and application thereof. The application isolates a deep-sea fungus of the genus Acremonium from a deep-sea sediment sample, and names the deep-sea fungus Acremonium nanhaiense CGMCC 42655. The deep-sea fungus is preserved in the China General Microbiological Culture Collection Center on March 23, 2026, has a preservation number of CGMCC 42655, and the address of the preservation unit is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Xibaheyanli, Chaoyang District, Beijing.

Description

Technical Field

[0001] This invention relates to the field of microbial strains, and in particular to a deep-sea fungus, Acremonium nanhaiense CGMCC 42655, and its applications. Background Technology

[0002] Acremonium is a genus of filamentous fungi belonging to the family Hypocreaceae in the phylum Ascomycota. The name derives from the Greek word akremōn (twig), referring to the morphology of its conidiophores. This genus was long classified under the asexual form of "Cephalosporium," but modern molecular systematics has revised this classification, removing many species: for example, *A. recifei* was moved to *Xenoacremonium*, *A. kiliense* / *A. strictum* to *Sarocladium*, and *A. falciforme* was merged into *Neocosmospora*. Currently, about one hundred species remain in *Acremonium sensustricto*, most of which are saprophytic or weakly parasitic.

[0003] Ecologically, *Acremonium* is widely distributed, mainly isolated from soil, decaying plant debris, and indoor building materials, with most species being weak saprophytes. Medically, it has a "dual identity": on the one hand, *Acremonium chrysogenum* (formerly *Cephalosporium acremonium*) is the source of the discovery of cephalosporin C, initiating the entire family of cephalosporin antibiotics; on the other hand, many species are opportunistic pathogens, often causing local infections in immunocompetent hosts via wound inoculation, such as eumycetoma, fungal keratitis, and onychomycosis. In immunosuppressed individuals (neutropenia, organ transplantation, dialysis), it can progress to vitreous inflammation, osteomyelitis, peritonitis, endocarditis, and even disseminated bloodstream infections, with high mortality rates. Treatment-wise, this genus is inherently resistant to fluconazole and 5-fluorocytosine, often requiring voriconazole or amphotericin B selected based on drug sensitivity testing.

[0004] In terms of industrial and agronomic value, besides its main role as a cephalosporin C, another notable aspect of Acremonium is its endophytic relationship with grasses. Originally part of the Neotyphodium genus (now partially merged into the Epichloë complex, closely related to Acremonium), it acts as an endophyte in tall fescue and ryegrass, producing ergot alkaloids and sphagnum mossin. While these substances offer insect and stress resistance, they can also cause poisoning in livestock, making them an unavoidable issue in the forage and livestock industry. Furthermore, a few species produce collagenase and cellulase, finding sporadic applications in biotransformation. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a deep-sea fungus, Acremonium nanhaiense CGMCC 42655.

[0006] Another object of the present invention is to provide the application of the above-mentioned deep-sea fungus Acremonium nanhaiense CGMCC42655.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A deep-sea fungus, Acremonium nanhaiense, was isolated from deep-sea sediments and named Acremonium nanhaiense CGMCC 42655.

[0009] The deep-sea fungus Acremonium nanhaiense, with accession number CGMCC 42655, was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] The deep-sea fungus Acremonium nanhaiense has white to grayish-white colonies on PDA medium, with a denser, fluffy or powdery center and thinner, more regular edges; the reverse side is light cream to pale yellowish-brown, with more obvious coloring in the center and lighter coloring at the edges; the hyphae are slender, clearly septate, and grow in a branching manner.

[0011] The nucleotide sequence of the ITS of the deep-sea fungus Acremonium nanhaiense is shown in SEQ ID NO.4.

[0012] The nucleotide sequence of LSU of the deep-sea fungus Acremonium nanhaiense is shown in SEQ ID NO.8.

[0013] The nucleotide sequence of RPB2 of the deep-sea fungus Acremonium nanhaiense is shown in SEQ ID NO.12.

[0014] The deep-sea fungus Acremonium nanhaiense has the ability to degrade cellulose.

[0015] The deep-sea fungus Acremonium nanhaiense has the ability to degrade pectin.

[0016] The application of the deep-sea fungus Acremonium nanhaiense in the preparation of cellulase and pectinase.

[0017] The present invention has the following advantages and effects compared with the prior art:

[0018] This invention isolates a deep-sea fungus of the genus Acremonium from deep-sea sediment samples, named Acremonium nanhaiense CGMCC 42655, which was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC 42655). The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0019] Figure 1 This is the growth of the deep-sea fungus LX2313 colony on the culture medium in Example 2; where a to d are the growth of the strain on PDA, MEA, CMA and OA media, respectively; e is the flask peduncle and the conidial mass at its tip; f is the mycelium; g is a single conidium;

[0020] Figure 2 This is the growth of the deep-sea fungus LX2322 colony on the culture medium in Example 2; where a-d are the growth of the strain on PDA, MEA, CMA and OA media respectively; e-f are the flask peduncle and the conidial mass at its tip; g-i are mycelium; j is a single conidium;

[0021] Figure 3 This is the growth of the deep-sea fungus LX2319 colony on the culture medium in Example 2; where a-d represent the growth of the strain on PDA, MEA, CMA, and OA media, respectively; e-f represent the flask peduncle and the conidial mass at its tip; g-i represent mycelium; and j represents a single conidium.

[0022] Figure 4 This is the growth of the deep-sea fungus LX2317 colony on the culture medium in Example 2; where a to d are the growth of the strain on PDA, MEA, CMA and OA media, respectively; e is the flask peduncle and the conidial mass at its tip; f is the mycelium; g is a single conidium;

[0023] Figure 5 This is the multigene phylogenetic tree of the deep-sea fungus ITS+LSU+RPB2 in Example 2;

[0024] Figure 6These are the results of the enzyme production performance experiment of deep-sea fungi in liquid fermentation in Example 3; where A is the growth of the strain on the Congo red stained cellulose screening plate; B is the result of cellulase activity determination in 3.1; and C is the result of pectinase activity determination in 3.2. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0026] Example 1: Strain Isolation

[0027] The specific steps for screening bacterial strains from deep-sea sediment samples collected from the South China Sea are as follows:

[0028] Under aseptic conditions, 2 g of sediment sample was inoculated into freshly prepared 3% sea salt PDB medium (200 g peeled fresh potato, 20 g anhydrous glucose, 30 g crude sea salt, 1000 mL distilled water). The medium was incubated statically at room temperature in the dark for 2 days, then shaken at 180 rpm for 2 hours on a shaking plate. 100 µL of the culture solution was then evenly spread onto a 90 mm diameter plate of freshly prepared 3% sea salt PDB medium (200 g peeled fresh potato, 20 g anhydrous glucose, 30 g crude sea salt, 16 g agar powder, 1000 mL distilled water). The culture was prepared at 28°C for 7 days, with daily observation. After the fungal colony was formed, the hyphae from the edge of the fresh fungal colony were picked and inoculated into freshly prepared PDA medium. The above operation was repeated until a single colony was observed. Pure cultures were obtained, from which 4 strains of Acremonium-like deep-sea fungi were isolated and numbered LX2313, LX2317, LX2319 and LX2322, respectively. They were cryopreserved in glycerol PDA medium at ultra-low temperature.

[0029] Example 2 Identification of the strain

[0030] 2.1 Morphological characteristics of the strain

[0031] Preliminary identification of the strain based on its morphological characteristics was performed using the "Handbook of Fungal Identification".

[0032] The four deep-sea fungal strains isolated above were inoculated into PDA and MEA agar, respectively, using a center-point inoculation method, and cultured at 28°C in the dark for 14 days. Colony characteristics such as diameter, surface and reverse color, colony texture, and edge morphology were recorded, and colony photographs were taken for archiving. Fresh hyphae were prepared using the slide method and cultured at 28°C for 7 days to obtain coverslips. The microstructure of the strains was observed using an optical microscope, and photomicrographs were taken.

[0033] Table 1. Morphological comparison of different strains

[0034] strains LX2313 LX2319 / LX2322 Acremonium brachypenium Colony growth After culturing at 28°C for 2 weeks on PDA medium, the colony diameter can reach 55-58 mm. After culturing at 28°C for 2 weeks on PDA medium, the colony diameter can reach 25-29 mm. After culturing at 28°C for 10 weeks on PDA medium, the colony diameter can reach 16 mm. Colony morphology The center is denser, with a velvety or powdery texture, while the edges are thinner and more regular. Compact, with a fine, velvety texture and dense radial folds. From fluff to smooth hyphae The hyphae are slender, clearly septate, and branch, with a width of 1.2-2.4 μm. Transparent, septate, with smooth walls, 1.1-2.4 micrometers wide. Cartilaginous hyphae, 2.5-3.0 micrometers wide, tough Morphology of the sporophyte Slender lanceolate, straight or curved, 16.7–33.2 micrometers in length. Slender, with a length of 21.2-40.9 micrometers. Spike-like, 10-30 micrometers long Conidial morphology Most are elliptical or short cylindrical, relatively uniform in size, with clear edges, and a size of 2.6-6.8 × 1.2-2.8 μm. Elliptical to obovate, sometimes slightly fusiform, 2.5–4.3 × 1.4–2.4 micrometers Short spindle-shaped, 3.0-4.5 × 1.2-1.6 micrometers Habitat Deep-sea sediments in the South China Sea Deep-sea sediments in the South China Sea Dead coconut tree trunk

[0035] Colony photos and morphological identification results as follows Figures 1-4 As shown in Table 1, Acremonium brachypenium is a strain of Acremonium preserved in our laboratory. The identification results show that LX2319 and LX2322 are morphologically similar and should be closely related strains. They are quite different from other strains, and LX2313 and LX2319 / LX2322 have significant differences in appearance from existing Acremonium fungi.

[0036] 2.2 Molecular biological identification of the strain

[0037] 2.2.1 DNA Extraction

[0038] The pure culture strains were inoculated into the center of PDA medium and cultured at 28 °C for 7 days. Fungal hyphae were scraped using a 1000 µL pipette tip, placed in a mortar, and ground under liquid nitrogen. Fungal genomic DNA was extracted according to the instructions of the UNlQ-10 column-based fungal genomic DNA extraction kit (Shanghai Sangon Biotech, Shanghai, China) and then used for PCR amplification.

[0039] 2.2.2 PCR amplification and sequencing

[0040] Using genomic DNA as a template, ribosomal genes and protein-coding gene fragments commonly used for fungal identification, including ITS, LSU, and RPB2, were amplified. Each site was amplified using corresponding primers. Primer information is as follows:

[0041] Amplification of the internal transcribed spacer (ITS) region of fungi was performed using primers ITS1 / ITS4:

[0042] ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'

[0043] ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

[0044] Amplification of the large subunit rRNA (LSU) region of fungi was performed using primers LR0R / LR7:

[0045] LR0R: 5'-GTACCCGCTGAACTTAAGC-3'

[0046] LR7: 5'-TACTACCACCAAGATCT-3'

[0047] Amplification of the second large subunit (RPB2) region of fungal RNA polymerase II using primers fRPB2-5F / fRPB2-7cR:

[0048] fRPB2-5F: 5'-GAYGAYMGWGATCAYTTYGG-3'

[0049] fRPB2-7cR: 5'-CCCATRGCTTGYTTRCCCAT-3'

[0050] PCR reaction system (50μL): 2μL DNA template, 25μL PCR mix (Shanghai Sangon Biotech), 2μL each of forward and reverse primers, and 17μL dd water.

[0051] PCR reaction conditions:

[0052] Pre-denaturation: 94 °C for 10 min;

[0053] Denaturation at 94 °C for 2 min;

[0054] Annealing at 54 ℃ for 1 min;

[0055] Extended at 72 ℃ for 2 minutes;

[0056] The above three steps involve a total of 35 cycles;

[0057] Final extension at 72 °C for 10 min.

[0058] PCR amplification products were stored at -20 °C, and amplification quality was detected by agarose gel electrophoresis.

[0059] After the PCR products were verified and purified by electrophoresis, they were sent to Shanghai Sangon Biotech for bidirectional sequencing to obtain the sequences at each site. The ITS sequences of LX2313, LX2317, LX2322 and LX2319 are shown in SEQ ID NO.1 to 4, the LSU sequence is shown in SEQ ID NO.5 to 8, and the RPB2 sequence is shown in SEQ ID NO.9 to 12.

[0060] 2.2.3 Sequence alignment and phylogenetic analysis

[0061] The taxonomic identification of fungi differs from that of bacteria. While bacteria can be identified and classified relatively quickly using 16S rDNA, authoritative methods for fungal molecular taxonomy are still under investigation. A 2012 paper by Schoch et al. published in PNAS proposed… [1]The ITS regions (including ITS1, 5.8S, and ITS2) exhibited good distinguishing ability at the species level due to sufficient interspecies variation, while remaining relatively conserved within species, making them the internationally recognized standard for fungal barcoding at the time. However, with further research, recent studies have found that the accuracy of ITS is constrained by multiple factors. First, there is significant heterogeneity in the evolutionary rate of ITS across different fungal lineages. In some groups, such as some Basidiomycota or early clades, ITS may be too conserved to distinguish closely related species, or in other cases, its high variability may lead to excessive division. Second, ribosomal DNA exists in tandem repeats, and intragenomic variation may exist between different copies. This can interfere with PCR amplification and sequencing results, producing artificial products such as chimeric sequences, thus affecting classification. [2] A more serious problem is the misannotation in public databases. Studies have shown that in databases such as GenBank, up to 30% of ITS sequences may be incorrectly associated with the wrong taxonomic unit names. This directly leads to unreliable identification results based on rapid BLAST alignment using ITS. [3] .

[0062] To overcome the limitations of ITS as a single marker, multi-site phylogenetic analysis has become the best practice in fungal taxonomy. Within this framework, LSU (usually referring to the D1 / D2 hypervariable region of 28S rDNA) plays a crucial role. LSU sequences are more conserved than ITS, providing a more stable and reliable signal in reconstructing phylogenetic relationships at the genus, family, and even higher levels. It helps improve the sequence alignment quality of deep branches and provides more suitable evolutionary parameters for phylogenetic models, thus stabilizing the topology of the entire phylogenetic tree. Although LSU has limited resolution in distinguishing very closely related species, its combination with ITS can effectively compensate for the latter's shortcomings in higher-level classification. For example, in the study of the new species *Cerioporus yunnanensis*, researchers used a combination of ITS, LSU, and RPB2 data for phylogenetic analysis to ensure the accuracy of its taxonomic position. [4] Similarly, when describing new species in the families Fuscosporellaceae and Savoryellaceae, a five-gene matrix including LSU, SSU, ITS, RPB2, and TEF1α was also used. [5] Other studies have also examined the classification of fungi in the genus *Acremonium*. [6] The analysis was combined with ITS+LSU multi-gene phylogenetic analysis to identify the strains more accurately and systematically.

[0063] Therefore, this invention, referring to existing literature, employs the ITS+LSU multigene phylogenetic tree analysis method for the identification and classification of *Acremonium* strains. After sequencing the relevant sequences of the fungi to be tested, the ITS, LSU, and RPB2 sequences of fungal species in the *Acremonium* genus were downloaded from GenBank along with the sequenced fungal sequences. Phylosuit software was used to align, cut, and tandem these sequences before phylogenetic analysis. The phylogenetic analysis used maximum likelihood (ML) to construct a phylogenetic tree, and the partitioning substitution model was selected using ModelFinder based on the corrected Akaike information criterion (AICc) to screen for the optimal partitioning model. Subsequently, maximum likelihood analysis was performed in IQ-TREE v2.4.0, using 10,000 ultrafast bootstrap iterations to assess support. In the phylogenetic tree, a branch with an ML bootstrap value ≥75% was considered to have strong support.

[0064] The resulting evolutionary tree is as follows Figure 5As shown, the phylogenetic analysis included 111 taxa, including the four newly isolated strains obtained in this study. The assembled ITS-LSU sequence matrix contained 684 sites (including deletion / insertion sites), with ITS at 293 bp and LSU at 391 bp. For maximum likelihood (ML) analysis, the optimal alternative model was selected in ModelFinder based on the corrected Akaike Information Criterion (AICc), and the optimal model for both LSU and ITS was GTR+F+I+G4. For Bayesian inference (BI), the model was selected in ModelFinder based on the Bayesian Information Criterion (BIC), and the optimal model for both LSU and ITS was GTR+I+G4. The resulting phylogenetic tree showed that the species most closely related to Acremonium sp. LX2313 is Acremonium sclerotigenum. Combining the genetic distance and topological position of the two on the phylogenetic tree, and considering the morphological identification results, this fungus was identified as Acremonium sclerotigenum LX2313. The closest species to Acremonium sp. LX2317 is Acremonium brunneisporum. Based on their genetic distance and topological position on the phylogenetic tree, and combined with morphological identification results, this fungus was identified as Acremonium brunneisporum LX2317. Isolates LX2319 and LX2322 form a branch with high support (ML expansion value 97%, BI posterior probability = 0.99) and are closely related to Acremonium brachypenium. Significant differences exist in the gene molecules, with ITS similarity at 93.7%, far less than 97% (the threshold given by Schoch et al. in 2016), and LSU similarity at 98.7%, far less than 99.8% (the threshold given by Vu et al. in 2019). Furthermore, we found that *A. nanhaiense* sp. nov. LX2319 / LX2322 differs significantly from its close relative *A. brachypenium* based on colony morphology and microstructure (as shown in Table 1). Combining the phylogenetic tree and the microstructure of the fungus, *A. nanhaiense* sp. nov. X2319 / LX2322 is a newly discovered species, tentatively named *A. nanhaiense* sp. nov.

[0065] The strain Acremonium sclerotigenum LX2313 was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42651. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0066] The strain Acremonium brunneisporum LX2317 was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42654. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0067] The strain Acremonium nanhaiense LX2319 was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42655. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0068] The strain Acremonium nanhaiense LX2322 was deposited on March 23, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 42653. The address of the depository is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0069] Example 3: Verification of fungal enzyme production capacity by liquid fermentation

[0070] Initial screening of the strains using Congo red stained cellulose screening plates revealed that the deep-sea fungi screened in this invention all possessed a certain cellulose degradation ability. To further verify their effectiveness, liquid fermentation was subsequently carried out and their enzyme activity was measured.

[0071] 3.1 CMC-Na-induced liquid fermentation for cellulase production

[0072] 3.1.1 Induction medium formulation

[0073] CMC-Na 10 g, (NH4)2SO4 1.4 g, KH2PO4 2.0 g, MgSO4·7H2O 0.3 g, CaCl2·2H2O 0.3 g, trace element solution: 1 mL (FeSO4·7H2O 5 g / L, MnSO4·H2O 1.6 g / L, ZnSO4·7H2O 1.4 g / L, CoCl2·2H2O 2.0 g / L), pH 5.5, dissolved in water and brought to a final volume of 1000 mL.

[0074] 3.1.2 Fermentation and preparation of crude enzyme solution

[0075] Add 50 mL of induction medium to a 250 mL Erlenmeyer flask, inoculate with spore suspension at 1% (v / v), and culture at 28℃ and 180 rpm for 7 days.

[0076] After the culture was completed, the fermentation broth was centrifuged at 8000 rpm / min for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the crude fungal enzyme solution, which was then stored at 4℃.

[0077] 3.1.3 Establishment of glucose standard curve

[0078] Take glucose standard solution (1.0 mg / mL) and gradually dilute it to concentration gradients of 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 mg / mL. Take 0.5 mL of glucose solution from each tube, add 1.0 mL of DNS, boil in a water bath for 5 min, cool to room temperature, and then add sterile water to make up to 10 mL. Measure the A540 value using a UV-Vis spectrophotometer and plot the standard curve using Excel software.

[0079] 3.1.4 DNS method for determining CMC-Na enzyme activity

[0080] Prepare a 1% (w / v) CMC-Na substrate solution using 50 mM citrate-sodium citrate buffer (pH 4.8). Mix 0.5 mL of the substrate with 0.5 mL of the crude enzyme solution and react in a water bath at 50°C for 30 min. Immediately after the reaction is complete, add 1.0 mL of DNS reagent to terminate the reaction. After boiling in a water bath for 5 min, cool to a boil and then bring the volume to 10 mL with sterile water. Measure the absorbance (A540) using a UV spectrophotometer.

[0081] Blank control: First, mix 0.5 mL of crude enzyme solution with 1.5 mL of DNS reagent, then add 0.5 mL of substrate and 0.4 mL of buffer solution, and boil in a water bath for 5 minutes to subtract background absorbance. Meanwhile, use the fungus *Acremonium brunneisporum* CGMCC 42654, isolated from the same deep-sea sediment, as a control; the following experiments are the same.

[0082] Enzyme activity is defined as the amount of enzyme required to catalyze the release of 1 μmol of galacturonic acid from CMC-Na substrate per minute under the specified assay conditions. One unit of enzyme activity, U, is defined as the amount of enzyme required to catalyze the release of 1 μmol of galacturonic acid from CMC-Na substrate per minute.

[0083] Substitute the values ​​into the glucose standard curve to calculate the amount of reducing sugar produced, and then calculate the CMC-Na enzyme activity using the following formula.

[0084] CMCase (U / L) = Amount of reducing sugar (μmol / mL) / Reaction time (min) × 1000

[0085] 3.1.5 DNS method for determining filter paper enzyme activity (reducing sugar release)

[0086] Weigh 50 mg of filter paper as the model substrate, add 1 mL of citrate buffer (pH 4.8) and 0.5 mL of crude enzyme solution, incubate at 50 °C for 1 h, centrifuge and take the supernatant, add 1 mL of DNS, boil in a water bath for 5 min, cool to room temperature and add water to make up to 10 mL, measure and calculate the reducing sugar concentration using A540 to evaluate the hydrolysis effect; at the same time, set up a blank control to subtract the background of reducing sugar in crude enzyme solution.

[0087] 3.2 Liquid fermentation to induce pectinase production

[0088] 3.2.1 Induction medium formulation

[0089] 10g citrus pectin, 2g NaNO3, 1g K2HPO4, 0.5g MgSO4·7H2O, 0.5g KCl, 15g agar, diluted to 1000 mL with artificial seawater at natural pH.

[0090] 3.2.2 Extraction of crude enzyme solution

[0091] Prepare crude enzyme solution according to the method in 3.1.2.

[0092] 3.2.3 Plotting the Standard Curve

[0093] Accurately weigh D-galacturonic acid (D-Galacturonicacid) standard and prepare a series of standard solutions with concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg / mL). Take 1.0 mL of each standard solution and add 1.5 mL of DNS reagent. Heat in a boiling water bath at 100°C for 5 minutes. Quickly cool with ice water, add distilled water to a final volume of 10 mL, and measure the absorbance at 540 nm (A540) using a spectrophotometer. Plot a standard curve.

[0094] 3.2.4 Determination of enzyme activity

[0095] Take 0.5 mL of 1% pectin substrate solution and preheat it (50°C) for 5 minutes in a test tube. Add 0.1 mL of crude enzyme solution and 0.4 mL of citrate-sodium citrate buffer (pH 4.8), and mix quickly. React precisely for 30 minutes in a constant temperature water bath. Quickly add 1.5 mL of DNS reagent to terminate the enzyme reaction, and heat in a boiling water bath for 5 minutes. After cooling to room temperature in an ice bath, add distilled water to a final volume of 10 mL and measure the absorbance at 540 nm.

[0096] Blank control: First, mix 0.1 mL of crude enzyme solution with 1.5 mL of DNS reagent, then add 0.5 mL of substrate and 0.4 mL of buffer solution, and boil in water for 5 minutes to subtract background absorbance.

[0097] Enzyme activity is defined as the amount of enzyme required to catalyze the release of 1 μmol of galacturonic acid from a pectin substrate per minute under the specified assay conditions. One unit of enzyme activity, U, is defined as such.

[0098] The amount of reducing sugar released is calculated by subtracting the absorbance of the blank from the absorbance of the sample and substituting the result into the D-galacturonic acid standard curve.

[0099] Pectinase activity (U / L) = Amount of reducing sugar (μmol / mL) / Reaction time (min) × 1000

[0100] Table 2 Results of bioenzyme activity assay (U / L)

[0101] LX2313 141.93 ± 1.06 49.33 ± 1.68 881.65 ± 57.07 LX2317 99.36 ± 1.92 36.44 ± 1.06 398.66 ± 11.89 LX2322 97.41 ± 1.34 26.74 ± 1.08 287.89 ± 26.88 LX2319 87.12 ± 1.92 28.39 ± 1.48 420.70 ± 13.65

[0102] Experimental results are as follows Figure 6 As shown in Table 2, the experimental results show that the CMC-Na enzyme activity of Acremonium sclerotigenum LX2313 can reach 140.9 U / L, the filter paper cellulase activity can reach 50.6 U / L, and the pectinase activity can reach 881.65 U / L, indicating that LX2313 has excellent enzyme production capacity and has broad application prospects in papermaking and other fields.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0104] References:

[0105] [1]Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge,J. L., Levesque, C. A., Chen, W., Bolchacova, E., Voigt, K., Crous, P. W.,Miller, A. N., Wingfield, M. J., Aime, M. C., An, K.-D., Bai, F.-Y., Barreto,R. W., Begerow, D., … Schindel, D. (2012). Nuclear ribosomal internaltranscribed spacer (ITS) region as a universal DNA barcode marker for Fungi.Proceedings of the National Academy of Sciences, 109(16), 6241–6246. https: / / doi.org / 10.1073 / pnas.1117018109.

[0106] [2] Zhou, J.-L., Xu, J., Jiao, A.-G., Yang, L., Chen, J., Callac, P.,Liu, Y., & Wang, S.-X. (2019). Patterns of PCR Amplification Artifacts of theFungal Barcode Marker in a Hybrid Mushroom. Frontiers in Microbiology, 10.https: / / doi.org / 10.3389 / fmicb.2019.02686.

[0107] [3] Hofstetter, V., Buyck, B., Eyssartier, G., Schnee, S., & Gindro,K. (2019). The unbearable lightness of sequenced-based identification. FungalDiversity, 96(1), 243–284. https: / / doi.org / 10.1007 / s13225-019-00428-3.

[0108] [4] Yang, Y., Li, R., Jiang, Q., Zhou, H., Muhammad, A., Wang, H., &Zhao, C. (2024). Phylogenetic and Taxonomic Analyses Reveal Three New Wood-Inhabiting Fungi (Polyporales, Basidiomycota) in China. Journal of Fungi, 10(1), 55. https: / / doi.org / 10.3390 / jof10010055.

[0109] [5] Du, H.-Z., Yang, J., Liu, N.-G., Cheewangkoon, R., & Liu, J.-K.(2022). Morpho-Phylogenetic Evidence Reveals New Species of Fuscosporellaceaeand Savoryellaceae from Freshwater Habitats in Guizhou Province, China.Journal of Fungi, 8(11), 1138. https: / / doi.org / 10.3390 / jof8111138.

[0110] [6] Zhao L, Groenewald JZ, Hou LW, Starink-Willemse M, Beek B, Grum-Grzhimaylo OA, Summerbell RC, Crous PW. New insights into acremonium-likefungi in Hypocreales: A taxonomic and phylogenetic perspective. Stud Mycol.2026 Feb 6;113:1-71. doi: 10.3114 / sim.2026.113.01. PMID: 41993082; PMCID:PMC13081845.

Claims

1. A deep-sea fungus, *Acremonium nanhaiense*, characterized by: The specimen, named Acremonium nanhaiense CGMCC 42655, was derived from a deep-sea sediment sample. The deep-sea fungus Acremonium nanhaiense, with accession number CGMCC 42651, was deposited on March 23, 2026, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The deep-sea fungus *Acremonium nanhaiense* according to claim 1, characterized in that: The deep-sea fungus Acremonium nanhaiense has white to grayish-white colonies on PDA medium, with a denser, fluffy or powdery center and thinner, more regular edges; the reverse side is light cream to pale yellowish-brown, with more obvious coloring in the center and lighter coloring at the edges; the hyphae are slender, clearly septate, and grow in a branching manner.

3. The deep-sea fungus *Acremonium nanhaiense* according to claim 1, characterized in that: The deep-sea fungus Acremonium nanhaiense has the ability to degrade cellulose.

4. The deep-sea fungus *Acremonium nanhaiense* according to claim 1, characterized in that: The deep-sea fungus Acremonium nanhaiense has the ability to degrade pectin.

5. The use of the deep-sea fungus Acremonium nanhaiense according to any one of claims 1 to 4 in the preparation of cellulase and / or pectinase.

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