A marine red yeast with high yield of fucoidan-degrading enzyme and application thereof

Abstract

The application discloses a marine red yeast with high yield of fucan sulfate degrading enzyme and application thereof, and belongs to the technical field of microorganisms. The marine red yeast is used as a starting strain, a high-yield mutant C5 is obtained through UV-NTG one-time compound mutagenesis, and the C5 is preserved in the China General Microbiological Culture Collection Center on May 28, 2026, with a preservation number of CGMCC No. 38933. Meanwhile, a Glu-FUC co-metabolic induction fermentation system matched with the physiological characteristics of the strain is established, efficient and stable production of fucan sulfate degrading enzyme is realized, and the marine red yeast has a very good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a marine red yeast strain that produces a high yield of brown algae polysaccharide sulfate-degrading enzyme and its applications. Background Technology

[0002] Fucoidan sulfate (FUC) is a sulfated polysaccharide unique to the cell walls of brown algae. It has been proven to possess a variety of important biological activities, including anti-inflammatory, immunomodulatory, anticoagulant, antithrombotic, antiviral, and antitumor effects, and has broad application prospects in functional foods, biomedicine, and wound repair materials. However, natural FUC generally suffers from problems such as excessively large molecular weight (typically >400 kDa), strong structural heterogeneity, and large batch-to-batch variability, resulting in low bioavailability and difficulty in controlling product quality, which seriously restricts its industrial application.

[0003] Currently, the main methods for reducing the molecular weight of fucoidan sulfate (FUC) include chemical degradation, physical degradation, and enzymatic degradation. Among these, enzymatic degradation has become the most promising technological route due to its mild reaction conditions, high substrate specificity, and ability to retain active structural units such as sulfate groups to the greatest extent. Fucoidanase, as the core of enzymatic degradation, directly determines the preparation efficiency and product quality of low molecular weight FUCs due to its source, yield, and properties.

[0004] The existing strains producing alginate sulfate-degrading enzymes are extremely limited in origin, with over 90% concentrated in marine bacteria, such as *Pseudomonas*, *Sphingosomalmonella*, and *Microbacterium sinense*. For example, *Microbacterium sinense* isolated from the nearshore waters of Dalian by Guo Jingjing et al. Sinomicrobium sp. Fuc19, after optimization, produced an enzyme activity of 143.60 U / mL (Guo Jingjing, Liu Shimeng, Han Ziqi, et al. Fuc19, a bacterium that degrades alginate sulfate). Sinomicrobium sp .Optimization of enzyme production and chemical composition of degradation products of Fuc19[J]. Journal of Dalian Ocean University, 2022, 37(2): 285-294.); Sphingosine monocytogenes isolated by Kim et al. Sphingomonas paucimobilisPF-1, with an enzyme production level of approximately 120 U / mL in shake flasks (Kim WJ, Kim SM, LeeY H, et al. Isolation and Characterization of Marine Bacterial Strain Degrading Fucoidan from Korean Undaria pinnatifida Sporophylls[J]. Journal of Microbiology and Biotechnology, 2008, 18(4): 616-623.). In contrast, research on marine yeasts, as an important marine microbial resource, for the production of alginate sulfate-degrading enzymes is almost nonexistent. Existing research on the genus *Rhodotorula* (*Rhodotorula*) is limited. Rhodotorula Research on this topic has mainly focused on the production of carotenoids, oils, and extracellular polysaccharides. Only a few reports mention that some red yeast strains have glycosidase activity, but marine red yeast strains are rarely mentioned. Rhodotorula sphaerocarpa This is a publicly reported strain that produces alginate sulfate-degrading enzymes.

[0005] To improve enzyme production levels, current research primarily employs traditional mutagenesis methods such as ultraviolet (UV) and nitrosoguanidine (NTG) to modify wild-type strains. However, existing mutagenesis techniques generally suffer from narrow mutation spectra and low positive mutation rates, with enzyme activity increases typically below 70% after mutagenesis, failing to meet the enzyme yield requirements of industrial production. Furthermore, high-yielding mutant strains obtained through existing mutagenesis generally exhibit poor genetic stability. Most reported mutant strains show enzyme activity decline exceeding 30% after 3-5 generations of continuous passaging, with some strains even completely losing their enzyme-producing ability, making long-term stable large-scale production impossible.

[0006] Current research on the fermentation of alginate sulfate-degrading enzymes is mostly limited to single-factor optimization in shake flasks, lacking a systematic fermentation regulation system targeting the physiological characteristics of inducible enzymes. After transferring from shake flask culture to fermenter scale-up, enzyme activity typically decreases by 20%-50% due to mismatches in parameters such as dissolved oxygen transfer, substrate mixing, and induction timing. For example, some bacterial strains can achieve enzyme activity of 200 U / mL in shake flasks, but after fermenter scale-up, it can only maintain 100-150 U / mL, resulting in high industrial production costs. Furthermore, existing studies often use a single FUC as both carbon source and inducing substrate, which not only leads to slow cell growth and long fermentation cycles, but also significantly increases system viscosity due to high concentrations of polysaccharide substrates, further deteriorating mass transfer conditions and limiting further improvements in enzyme production levels.

[0007] The fucoidan sulfate-degrading enzymes produced by existing strains generally have low degradation efficiency for natural high molecular weight fucoidan (FUC), typically requiring 24-48 hours to reduce the substrate molecular weight to below 200 kDa. Furthermore, the degradation products exhibit a wide molecular weight distribution and a large dispersion coefficient, and some enzymes lose key active structural units such as sulfate groups during degradation, making it difficult to prepare low molecular weight FUC products with well-defined structures and stable activity.

[0008] In summary, existing technologies suffer from core defects such as a single source of enzyme-producing strains, low enzyme production levels, poor genetic stability, difficulty in fermentation scale-up, and low degradation efficiency, which severely restrict the industrialization process of the brown alginate sulfate enzymatic low molecular weight technology. Summary of the Invention

[0009] To address the technical problems of existing fucoidan sulfate-degrading enzyme-producing strains, such as single source, low enzyme production level, poor genetic stability, and difficulty in fermentation scale-up, the present invention aims to provide a marine red yeast strain that produces high levels of fucoidan sulfate-degrading enzyme and its applications. This invention is the first to utilize marine red yeast (… Rhodotorula sphaerocarpa Using the strain as the starting material, a high-yielding mutant strain C5 was obtained through a one-time UV-NTG combined mutagenesis selection. A Glu-FUC co-metabolism induced fermentation system matching the physiological characteristics of this strain was established, achieving efficient and stable production of alginate sulfate-degrading enzyme.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] In a first aspect, the present invention provides a marine red yeast strain ( Rhodotorula sphaerocarpa C5 was deposited on May 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38933.

[0012] Secondly, the present invention provides a method for producing alginate sulfate-degrading enzyme by fermentation using the above-mentioned marine red yeast C5, wherein the method is selected from either method one or method two: Method 1: Includes the following steps: Inoculate the marine red yeast C5 into a fermentation medium and culture it at 25~35℃ and 100~300 r / min for 24~48 h to isolate and obtain the alginate sulfate degrading enzyme; Method 2: Includes the following steps: Inoculate the marine red yeast C5 into a seed culture medium and culture it at 25-35℃ and 100-300 r / min for 12-36 h. Inoculate the obtained seed liquid into the fermentation culture medium at an inoculation rate of 5-15% and culture it at 25-35℃, pH 6.0-7.0, 100-300 r / min, and an aeration rate of 1-5 vvm for 10-14 h. Add 10% of the fermentation broth volume of FUC at a concentration of 15-25 g / L as an inducing substrate and continue fermentation for 12-48 h to isolate and obtain the alginate sulfate degrading enzyme.

[0013] Based on the above technical solution, the fermentation medium described in Method 1 further comprises the following components: peptone 0.5~0.9 g / L, FUC 1.0~3.0 g / L, NaCl 15.0~25.0 g / L, CaCl2 0.05~0.15 g / L, MgSO4 0.1~0.3 g / L, Na2HPO4 0.5~0.7 g / L, FeSO4 0.01~0.03 g / L, and initial pH 6.0~7.0.

[0014] Based on the above technical solution, the cultivation conditions described in Method 1 are as follows: fermentation at 30℃ and 150 r / min for 36-42 h.

[0015] Based on the above technical solution, the seed culture medium described in Method 2 further comprises the following components: FUC 1.0~3.0 g / L, NaCl 15.0~25.0 g / L, CaCl2 0.05~0.15 g / L, MgSO4 0.1~0.3 g / L, Na2HPO4 0.1~0.3 g / L, and FeSO4 0.01~0.03 g / L.

[0016] Based on the above technical solution, the fermentation medium described in Method 2 further comprises the following components: peptone 0.5~0.9 g / L, glucose 5.0~15.0 g / L, NaCl 15.0~25.0 g / L, CaCl2 0.05~0.15 g / L, MgSO4 0.1~0.3 g / L, Na2HPO4 0.5~0.7 g / L, FeSO4 0.01~0.03 g / L, 0.05~0.15% (v / v) organosilicon defoamer, and initial pH 6.0~7.0.

[0017] Based on the above technical solutions, the specific separation process described in Method 1 and Method 2 is as follows: Centrifuge the fermentation broth, take the supernatant, add 1 to 3 times the volume of pre-cooled acetone and mix well, let stand at 1 to 5°C for 0.5 to 10 h, centrifuge again, discard the supernatant, and reconstitute the obtained precipitate with buffer solution.

[0018] Based on the above technical solution, the centrifugation conditions are further as follows: 1~5℃, 8000~15000 r / min for 5~60 min; the buffer solution includes 0.02 mol / L Tris-HCl buffer.

[0019] Thirdly, the present invention provides the application of the alginate sulfate-degrading enzyme prepared by the above method in the enzymatic hydrolysis of natural alginate sulfate to prepare low molecular weight alginate sulfate.

[0020] Based on the above technical solution, further, the number average molecular weight of the low molecular weight alginate sulfate is not higher than 200 kDa.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to develop a new resource for enzyme production from marine red yeast, filling a gap in the industry. It is the first to demonstrate that marine red yeast can efficiently secrete extracellular alginate sulfate-degrading enzymes, expanding the diversity of microbial sources for this type of enzyme, breaking the technical limitations of long-term reliance on marine bacteria, and opening up a new direction for the application of marine yeast in the field of seaweed polysaccharide biotransformation.

[0022] 2. Enzyme production levels were significantly improved, reaching a leading level among yeast-derived enzymes of the same type. The original strain C0 had an enzyme activity of 151.28 U / mL in shake flasks. After UV-NTG combined mutagenesis, the mutant strain C5 had an enzyme activity of 273.59 U / mL in shake flasks, an increase of 80.85%. After optimization of shake flask fermentation conditions, the enzyme activity was further increased to 343.53 U / mL. After scaling up in a 10 L fermenter, the enzyme activity reached 570.03 U / mL, which is the highest level of yeast-derived alginate sulfate-degrading enzymes reported to date, meeting the basic requirements for enzyme activity in industrial production.

[0023] 3. Excellent genetic stability, solving the industry pain point of easy degradation of mutant strains. After five consecutive passages, the enzyme activity of mutant strain C5 remained in the range of 245.38-273.59 U / mL, with a maximum degradation rate of only 10.3%, which is far lower than the degradation level of more than 30% of existing technologies. At the same time, there was no significant decrease in cell biomass, which can achieve stable fermentation in multiple batches, providing a reliable strain guarantee for industrial-scale production.

[0024] 4. The fermentation scale-up adaptability is good, significantly reducing production costs. The established Glu-FUC co-metabolism induction system solves the contradiction between cell growth and enzyme production. After scale-up in the fermenter, enzyme activity not only did not decrease, but actually increased by 65.9% compared with the shake flask optimization level. The fermentation cycle is shortened from 48-72 h in the existing technology to 36 h, and the production efficiency is increased by 33%-50%. Glucose is used as the carbon source for growth, replacing part of the expensive alginate sulfate substrate, reducing raw material costs by about 40%.

[0025] 5. High degradation efficiency and complete preservation of product active structure: The crude enzyme solution produced by mutant strain C5, with an enzyme addition of 10,000 U / g substrate, can degrade natural alginate sulfate with a number average molecular weight of 458.56 kDa to 181.54 kDa in just 12 hours, improving the degradation efficiency by 50%-75%; The product has a concentrated molecular weight distribution and a dispersion coefficient of 42.65, which is beneficial for the standardized preparation of subsequent low molecular weight products. Attached Figure Description

[0026] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0027] Figure 1 The colony morphology (A) and cell morphology (B) of the original marine red yeast strain C0.

[0028] Figure 2 This is the ITS phylogenetic tree of the original marine red yeast strain C0.

[0029] Figure 3 The image shows the comparison results of the crude enzyme activity of candidate mutant strains C1-C30 after shake-flask re-screening.

[0030] Figure 4 The figures show HPLC chromatograms of the degradation products of natural high molecular weight alginate sulfate in four experimental groups, where A is the enzyme-inactivating group, B is the blank group, C is the original group, and D is the experimental group. Detailed Implementation

[0031] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0032] All reagents used in all examples were commercially available analytical grade, and the original marine red yeast strain C0 was provided by the National Research Center for Seaweed Processing Technology (Dalian).

[0033] FUC seed culture medium (g / L): FUC 2.0 g / L, NaCl 20.0 g / L, CaCl2 0.1 g / L, MgSO4 0.2 g / L, Na2HPO4 0.2 g / L, FeSO4 0.02 g / L.

[0034] Shake flask fermentation medium (g / L): FUC 2.0 g / L, peptone 3.0 g / L, NaCl 20.0 g / L, CaCl2 (anhydrous) 0.1 g / L, MgSO4 0.2 g / L, Na2HPO4 0.2 g / L, FeSO4 0.02 g / L.

[0035] All culture media were autoclaved at 115 °C for 30 min and then cooled for later use.

[0036] Enzyme activity assay: Take 10 mL of fermentation broth and centrifuge at 4℃, 10000 r / min for 10 min, and collect the supernatant; add 2 volumes of pre-cooled acetone and mix well, let stand at 4℃ for 1 h, and then centrifuge again (4℃, 10000 r / min, 10 min), and discard the supernatant; reconstitute the precipitate with 0.02 mol / L Tris-HCl buffer (pH 8.0) and keep it at 4℃ for later use.

[0037] Prepare a 0.2% (w / v) fucose-FUC substrate solution using 0.02 mol / L Tris-HCl (pH 8.0). Mix 1.0 mL of crude enzyme solution with 1.0 mL of substrate solution and react at 30 °C for 5 min. Terminate the reaction by incubating in a water bath at 80 °C for 15 min and then cooling in an ice bath. Add 0.25 mL of potassium ferricyanide colorimetric reagent (50 mmol / L K3[Fe(CN)6] + 0.25 mol / L Na2CO3) to 0.25 mL of the reaction solution and develop the color at 80 °C for 15 min. After cooling to room temperature, dilute with 1.0 mL of pre-cooled ultrapure water, centrifuge (4 °C, 10000 r / min, 10 min), and measure the absorbance of the supernatant at 420 nm. Replace the crude enzyme solution with an equal volume of buffer solution for the blank, and follow the same steps. Perform triple replicates for each sample. Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of reducing sugar (in fucose equivalent) per minute under the above conditions, and the result is expressed in U / mL.

[0038] Fucose standard curve: Using 200 μg / mL fucose as a sample, a gradient concentration was established, and the absorbance was measured according to the potassium ferricyanide method for determining reducing sugars described above.

[0039]

[0040] y: (U / mL): Enzyme activity of the sample to be tested; xn (μmol / mL): Fucose content in blank (n=0) and sample (n=1); A n Absorbance at 420 nm for blank (n=0) and sample (n=1).

[0041] Example 1: (a) Molecular identification of the starting strain The starting strain used in this embodiment is the original marine red yeast strain C0, which was isolated from the surface of brown algae in the nearshore waters of Dalian by the National Research Center for Seaweed Processing Technology (Dalian). The original shake-flask fermentation enzyme activity was 151.28 U / mL.

[0042] Colony morphology: When cultured on FUC solid medium at 30℃ for 72 h, the colonies are orange-red, smooth, moist, plump, with neat edges, and are round or nearly round, with a diameter of 2-3 mm, without wrinkles or diffusion.

[0043] Cell morphology: Under an optical microscope, the cells are round or oval, measuring (3-5) μm × (4-6) μm, and are uniform in size. They reproduce by budding and do not form pseudohyphae.

[0044] Genomic DNA was extracted from the original C0 strain of *Rhodotorula marinum* using a DNA extraction kit (Servicebio, G3633). PCR was performed using ITS1 / ITS4 primers, and the amplified products were sequenced after 2% agarose gel electrophoresis (sequence amplification and sequencing were performed by Wuhan Saiwei Biotechnology Co., Ltd.). The sequenced sequences were aligned to GenBank (NCBI BLAST) and a phylogenetic tree was constructed using MEGA 11.0 software. The results are shown below. Figure 2 As shown, the comparison results indicate that strain C0 is similar to... Rhodotorula sphaerocarpa The reference sequence showed high homology, with a sequence similarity of 99.49%, and was also found to be similar to the reference sequence in the phylogenetic tree. R. sphaerocarpa They clustered into the same branch. Based on sequence alignment and phylogenetic analysis, this strain was identified as *Rhodotorula marinum*. Rhodotorula sphaerocarpa ).

[0045] (II) Methods for selecting and breeding strains This invention employs a single UV-NTG combined mutagenesis strategy, which differs from the single mutagenesis (UV only or NTG only) or stepwise mutagenesis (UV first, then NTG, with interval culture) commonly used in existing technologies. This single combined treatment broadens the mutation spectrum and increases the positive mutation rate. The specific steps are as follows: (1) Seed culture preparation: The C0 strain stored at -80℃ was inoculated into FUC seed culture medium and cultured at 30℃ and 150 r / min for 24 h until the logarithmic growth phase (OD200) was reached.600 =0.6-0.8).

[0046] (2) Pretreatment of bacterial suspension: Take 5 mL of logarithmic growth phase bacterial suspension, centrifuge at 4℃ and 6000 r / min for 10 min, discard the supernatant, wash the bacterial cells 3 times with sterile physiological saline, and finally resuspend in 0.05 mol / L Tris-maleic acid buffer (pH 9.0) and adjust OD. 600 Dilute to 0.7, then dilute with sterile saline to 10. -4 CFU / mL available for use.

[0047] (3) NTG chemical mutagenesis: Take 1 mL of diluted bacterial suspension, add NTG solution with a final concentration of 600 μg / mL, shake at 30℃ and 150 r / min for 30 min; immediately add an equal volume of 25% (w / v) sodium thiosulfate solution to terminate the reaction, centrifuge at 4℃ and 6000 r / min for 10 min, wash the bacterial cells 3 times with PBS buffer to completely remove residual mutagen.

[0048] (4) UV physical mutagenesis: Take 200 μL of the NTG-treated bacterial suspension and spread it evenly on FUC solid culture medium plates (add 20 g / L of agar to the FUC seed culture medium), place it under a 30 W UV lamp at 45 cm for 50 s, and immediately wrap the plate with double-layer black newspaper to prevent photoreactivation.

[0049] (5) Initial screening and secondary screening: After mutagenesis, the plates were cultured at 30℃ in the dark for 54 h. Single colonies with normal morphology, moderate size and uniform color were selected and inoculated into shake flask fermentation medium. The culture was carried out at 30℃ and 150 r / min for 36 h. The enzyme activity of the crude enzyme solution was determined by potassium ferricyanide method, and high-yield mutant strains were obtained by screening.

[0050] Candidate mutant strains C1-C30 were obtained under this screening pressure condition. Crude enzyme solution was prepared from the 36-hour fermentation broth, and enzyme activity was measured. Results are shown below. Figure 3Compared with the original strain C0, the enzyme activities of the candidate mutant strains showed significant differences, indicating that the combined mutagenesis could cause mutations on a large scale, resulting in significant differences in enzyme activity: the enzyme activity of the control strain C0 was (151.28±9.53) U / mL, while the enzyme activity of the candidate strains ranged from 42.53 to 273.59 U / mL. Among them, 15 mutant strains had enzyme activities higher than the control, accounting for 50% of the total number of candidate strains, and 10 mutant strains had an increase of 20% or more. This indicates that the lethal pressure can effectively enrich positive mutants while ensuring the number of survivors. Among the candidate strains, C5 showed the highest enzyme activity at (273.59±14.83 U) / mL, an increase of 80.85% compared to the control, thus being identified as the dominant mutant. Meanwhile, C14, C27, and C24 also exhibited high enzyme activities, at (231.14±17.88), (229.77±4.46), and (221.54±9.23) U / mL, respectively, representing increases of approximately 46%–53% compared to the control. This further demonstrates that combined strong pressure mutagenesis can achieve a stable increase in enzyme production in a certain proportion of mutant strains. On the other hand, some mutant strains showed a significant decrease in enzyme activity, such as C30 at (42.53±12.80) U / mL and C19 at (68.64±17.32) U / mL. Furthermore, some mutants, such as C3 and C10, showed significant fluctuations, reflecting that the mutagenesis process simultaneously introduced a negative mutation effect, causing damage to the strain that it could not repair itself. Overall, the population exhibited random mutagenesis characteristics, with most mutant strains closely resembling the control, a few showing significant increases and others showing marked decreases. This indicates that the screening process can effectively obtain strains with superior enzyme activity. Considering the magnitude of enzyme activity enhancement and the needs of subsequent research, mutant strain C5 was ultimately selected for genetic stability evaluation and fermentation optimization studies.

[0051] (6) Genetic stability verification: The high-yield strains obtained by screening were passaged 5 times. The enzyme activity and biomass of the fermentation broth were measured in each generation to evaluate the stability of the high-yield mutant strains.

[0052] The results are shown in Table 1. The mutant strain C5 maintained significantly higher enzyme activity levels than the original strain in each generation after mutagenesis. The enzyme activity of C5 in generations 0–5 ranged from 245.75 to 273.59 U / mL, which was 1.62–1.81 times that of the original strain. The average enzyme activity across generations was 255.89 U / mL, with minimal fluctuation (CV≈3.80%). The lowest generation still maintained 89.82% of the enzyme activity of generation 0, indicating that this high enzyme activity phenotype did not progressively decline during continuous passage and possessed good genetic stability. Meanwhile, the biomass of C5 in each generation ranged from 0.811 to 0.914, generally close to that of the original strain. The fact that enzyme activity remained at a high level for a long period without a significant increase in biomass suggests that the advantage of C5 mainly stemmed from improved enzyme production efficiency / secretion capacity per unit biomass rather than simple proliferative advantage. Based on the above results, it can be concluded that the key mutation of mutant strain C5 has strong genetic fixity, which meets the basic requirements for the stability of the working strain in subsequent fermentation optimization and scale-up studies.

[0053] Table 1. Genetic stability results of mutant strain C5

[0054] The applicant will use marine red yeast ( Rhodotorula sphaerocarpa C5 was deposited on May 28, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38933.

[0055] (III) Fermentation enzyme production process of the strain This invention establishes a glucose-fucoidan sulfate (Glu-FUC) co-metabolism induced fermentation system, which differs from the existing technology that generally uses a single FUC as both a carbon source and inducing substrate. By adopting a "grow bacteria first, then induce" strategy, it resolves the contradiction between cell growth and enzyme production, and achieves highly efficient enzyme production at the fermenter level.

[0056] 1. Optimized shake-flask fermentation process (1) Culture medium composition (g / L): peptone 0.7 g / L, FUC 2.0 g / L, NaCl 20.0 g / L, CaCl2 0.1 g / L, MgSO4 0.2 g / L, Na2HPO4 0.6 g / L, FeSO4 0.02 g / L, initial pH 6.6, C / N ratio 3:1.

[0057] (2) Culture conditions: 20 mL / 50 mL conical flask, 5% inoculum, 30℃ culture temperature, 150 r / min shaking speed, 36-42 h fermentation cycle.

[0058] (3) Enzyme production effect: Under these conditions, the enzyme activity of shake-flask fermentation reached 343.53 U / mL, which was 25.6% higher than before optimization.

[0059] 2. Scale-up process for a 10 L fermenter (1) Culture medium: The same shake flask optimized culture medium (FUC is not added to the culture medium initially, but 10 g / L of glucose is added as an energy carbon source), and 0.1% (v / v) organosilicon defoamer is added. (2) Seed culture: Marine red yeast C5 strain was inoculated into seed culture medium and cultured at 30℃ and 150 r / min for 24h to obtain the seed culture for the fermenter; (3) Fermentation control parameters: liquid volume 5L, inoculum 10%, culture temperature 30℃, pH automatically controlled at 6.6; (4) Induction strategy: After culturing for 12 h, 10% of the fermentation broth volume of FUC at a concentration of 20 g / L was added once as an induction substrate. (5) Oxygen supply parameters: stirring speed 150 r / min, air flow rate 6 L / min; (6) Fermentation cycle: 36 h; (7) Enzyme production effect: Under these conditions, the enzyme activity in the 10 L fermenter reached 570.03 U / mL, which is 65.9% higher than that in the shake flask, showing a significant scale-up effect.

[0060] (iv) Degradation of brown alginic acid sulfate by crude enzyme solution of marine red yeast C5 and determination of molecular weight The experimental group was divided into four groups: the blank group (without the addition of alginate sulfate-degrading enzyme), the enzyme-inactivated group (with the addition of alginate sulfate-degrading enzyme but after enzyme inactivation treatment), the original group (with the addition of alginate sulfate-degrading enzyme produced by the original strain C0), and the experimental group (with the addition of alginate sulfate-degrading enzyme produced by marine red yeast C5).

[0061] 1. Experimental Materials Crude enzyme solution: Take 10 L of marine red yeast C5 fermentation broth cultured in a fermenter for 36 h, centrifuge at 10000 r / min for 10 min at 4℃, and collect the supernatant; add 2 volumes of acetone pre-cooled to -20℃, let stand at 4℃ for 1 h, centrifuge at 10000 r / min for 10 min at 4℃, collect the precipitate, and reconstitute with 0.02 mol / L Tris-HCl buffer (pH 8.0) to obtain crude enzyme solution with an enzyme activity of 570 U / mL (using the alginate sulfate degrading enzyme produced by the original strain C0 as a control).

[0062] Substrate: Dalian kelp brown algae polysaccharide sulfate (FUC), all provided by the National Research Center for Seaweed Processing Technology (Dalian).

[0063] Reagents: 0.02 mol / L Tris-HCl buffer (pH 8.0), anhydrous ethanol, sodium chloride (chromatographic grade), dextran standards (6, 10, 40, 70, 100, 200, 500 kDa).

[0064] Instruments: LC-20A high performance liquid chromatograph (Shimadzu, Japan), TSK-gel G5000 PWXL gel chromatography column (7.8 mm × 300 mm, 5 μm), RID-10A differential refractive index detector, freeze dryer.

[0065] 2. Experimental Procedure (1) Enzymatic hydrolysis reaction: Prepare 0.2% (w / v) Dalian kelp FUC and add the above crude enzyme solution at an enzyme addition amount of 10000 U / g substrate. Shake at 30℃ and 150 r / min for 12 h. After the reaction is completed, immediately place it in a boiling water bath and heat for 15 min to terminate the enzymatic reaction.

[0066] (2) Preparation of enzymatic hydrolysis product: The enzymatic hydrolysate after the reaction was terminated was centrifuged at 4℃ and 10000 r / min for 10 min, the precipitate was discarded, and the supernatant was collected; 3 times the volume of anhydrous ethanol was added to the supernatant, and the mixture was allowed to stand at 4℃ overnight for alcohol precipitation; the next day, the mixture was centrifuged at 4℃ and 10000 r / min for 10 min, the precipitate was collected, and the mixture was freeze-dried to obtain the enzymatic hydrolysis product.

[0067] (3) Molecular weight determination: The molecular weight of the product was determined by high performance gel permeation chromatography (HPGPC). 10 mg of lyophilized sample was accurately weighed, 1 mL of 0.2 mol / L sodium chloride solution was added, and the sample was dissolved by ultrasonication. The solution was then filtered through a 0.45 μm microporous membrane, and the filtrate was tested by the instrument.

[0068] Chromatographic conditions: mobile phase was 0.2 mol / L sodium chloride solution, flow rate was 0.5 mL / min, column temperature was 30℃, injection volume was 20 μL, and detection was performed using a differential refractive index detector.

[0069] Standard curve plotting: Using 6, 10, 40, 70, 100, 200, and 500 kDa dextran standards as references, and under the same chromatographic conditions, a standard curve was plotted with the retention time of the standards on the x-axis and the logarithm of the molecular weight (log Mw) on the y-axis. The regression equation was y = -0.4164x + 12.426, R0. 2 =0.9637.

[0070] 3. Experimental Results HPLC chromatograms of the degradation products of natural high molecular weight fucoidan sulfate from the four experimental groups are shown below.Figure 4 As shown, the main peaks of the four sample groups were all distributed within the range of 12 to 15 minutes, but there were certain differences in peak shape and peak area, as well as peak elution time. Calculations showed that the number-average molecular weights of the blank group and the enzyme-inactivated group were 458 kDa and 459 kDa, respectively, which were quite close, indicating that the substrate molecular weight change was small under conditions without effective enzymatic reaction. In contrast, the molecular weights of the original group and the experimental group decreased to 241 kDa and 182 kDa, respectively, indicating that the enzyme action significantly degraded alginate sulfate, with the experimental group showing better degradation. Meanwhile, the dispersion coefficients of the original group and the experimental group were 32.996 and 42.648, respectively, higher than those of the blank group and the enzyme-inactivated group, indicating that different molecular weight fragments were generated in the samples after enzymatic hydrolysis. Enzymatic hydrolysis had a more significant effect on the cleavage of the large molecular chains of the substrate, which was conducive to the formation of low molecular weight enzymatic hydrolysates.

[0071] Table 2. Degradation effect of natural high molecular weight alginate sulfate in each experimental group

[0072] The above experimental results show that the crude enzyme solution produced by marine red yeast C5 of the present invention has a good degradation effect on alginate sulfate. It can reduce the number average molecular weight of natural high molecular weight alginate sulfate to below 200kDa in just 12 hours, and retain the bioactive structure of alginate sulfate to the greatest extent. It is suitable for industrial preparation of low molecular weight alginate sulfate.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of marine red yeast ( Rhodotorula sphaerocarpa C5, characterized in that, It was deposited on May 28, 2026 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38933.

2. A method for producing alginate sulfate-degrading enzyme by fermentation using the marine red yeast C5 according to claim 1, characterized in that, The method is selected from either Method 1 or Method 2 as follows: Method 1: Includes the following steps: Inoculate the marine red yeast C5 into a fermentation medium and culture it at 25~35℃ and 100~300r / min for 24~48 h to isolate and obtain the alginate sulfate degrading enzyme; Method 2: Includes the following steps: Inoculate the marine red yeast C5 into a seed culture medium and culture it at 25-35℃ and 100-300 r / min for 12-36 h. Inoculate the obtained seed liquid into the fermentation culture medium at an inoculation rate of 5-15% and culture it at 25-35℃, pH 6.0-7.0, 100-300 r / min, and an aeration rate of 1-5 vvm for 10-14 h. Add 10% of the fermentation broth volume of FUC at a concentration of 15-25 g / L as an inducing substrate and continue fermentation for 12-48 h to isolate and obtain the fucoidan sulfate degrading enzyme.

3. The method according to claim 2, characterized in that, The fermentation medium described in Method 1 has the following composition: peptone 0.5~0.9 g / L, FUC 1.0~3.0 g / L, NaCl 15.0~25.0 g / L, CaCl2 0.05~0.15 g / L, MgSO4 0.1~0.3 g / L, Na2HPO4 0.5~0.7 g / L, FeSO4 0.01~0.03 g / L, and initial pH 6.0~7.

0.

4. The method according to claim 2, characterized in that, The cultivation conditions described in Method 1 are: fermentation at 30℃ and 150r / min for 36-42h.

5. The method according to claim 2, characterized in that, The seed culture medium described in Method 2 has the following composition: FUC 1.0~3.0 g / L, NaCl 15.0~25.0 g / L, CaCl2 0.05~0.15 g / L, MgSO4 0.1~0.3 g / L, Na2HPO4 0.1~0.3 g / L, FeSO4 0.01~0.03 g / L.

6. The method according to claim 2, characterized in that, The fermentation medium described in Method 2 has the following composition: peptone 0.5~0.9 g / L, glucose 5.0~15.0 g / L, NaCl 15.0~25.0 g / L, CaCl2 0.05~0.15 g / L, MgSO4 0.1~0.3 g / L, Na2HPO4 0.5~0.7 g / L, FeSO4 0.01~0.03 g / L, 0.05~0.15% (v / v) organosilicon defoamer, and initial pH 6.0~7.

0.

7. The method according to claim 2, characterized in that, The specific separation process described in Method 1 and Method 2 is as follows: Centrifuge the fermentation broth, take the supernatant, add 1 to 3 times the volume of pre-cooled acetone and mix well, let stand at 1 to 5°C for 0.5 to 10 h, centrifuge again, discard the supernatant, and reconstitute the obtained precipitate with buffer solution.

8. The method according to claim 7, characterized in that, The centrifugation conditions are as follows: 1~5℃, 8000~15000r / min for 5~60 min; the buffer solution includes 0.02 mol / L Tris-HCl buffer.

9. The application of the alginate sulfate-degrading enzyme prepared by the method according to any one of claims 2-8 in the enzymatic hydrolysis of natural alginate sulfate to prepare low molecular weight alginate sulfate.

10. The application according to claim 9, characterized in that, The number average molecular weight of the low molecular weight alginate sulfate is not higher than 200 kDa.

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