An endo-beta-1,3-glucanase and uses thereof

Abstract

The present application provides a kind of endo beta-1, 3-glucanase and its application, the amino acid sequence of the endo beta-1, 3-glucanase is SEQ ID NO:1.The endo beta-1, 3-glucanase of the present application can efficiently hydrolyze laminarin and curdlan, its enzyme activity can still be kept above 80% in the range of 40-60 DEG C and pH 4.5-6.0, and it shows good stability under low temperature conditions, which is conducive to reduce the energy consumption in the process of practical application.At the same time, the enzyme has significant high salt tolerance, and has certain resistance to 5%-15% ethanol.The endo beta-1, 3-glucanase of the present application has obvious inhibitory effect on the formation of Candida albicans biofilm, and the laminarin oligosaccharide generated by hydrolyzing laminarin also shows good antioxidant capacity, indicating that the enzyme has broad application prospect and significant practical value in the fields of antibacterial, biological medicine and the like.

Description

Technical Field

[0001] This invention belongs to the field of functional gene screening application technology, specifically relating to an endogenous β-1,3-glucanase and its application. Background Technology

[0002] β-glucan is a high-molecular-weight polymer composed of D-glucose molecules linked by β-glycosidic bonds. β-1,3-glucan, with its β-1,3-glycosidic bonds as the main chain, possesses unique helical structures that exhibit immunomodulatory, antitumor, antioxidant, anti-infective, and metabolic regulatory functions, attracting widespread attention in the food and medical fields. β-1,3-glucan is mainly found in cereal crops, brown algae, and mushrooms, as well as in the cell walls of almost all fungi and some bacteria. Common polysaccharides containing β-1,3-glycosidic bonds include laminarin, gelatin, shiitake mushroom polysaccharide, and yeast glucan. Barley and oat β-glucan also contain some β-1,3-glycosidic bonds. However, the high molecular weight, poor water solubility, and low bioavailability of β-1,3-glucan limit its development value in practical applications.

[0003] β-1,3-glucan oligosaccharides are low-polymerization oligosaccharides generated from the degradation of β-1,3-glucan. They have better water solubility and more pronounced physiological activity, exhibiting effects such as cholesterol regulation, immune response activation, and anti-tumor activity. Currently, the degradation methods for β-1,3-glucan mainly include physical, chemical, and enzymatic methods. Physical degradation methods are energy-intensive and produce heterogeneous products. While chemical degradation methods are lower in cost, they easily generate byproducts and pollute the environment. Enzymatic methods, due to their mild reaction conditions, high specificity, and controllable products, have become a highly promising degradation method. β-1,3-glucanases are classified into endoglucanases and exoglucanases based on their hydrolysis mode. Endoglucans can randomly cleave β-1,3-glycosidic bonds from within the sugar chain to produce a mixture of oligosaccharides with different degrees of polymerization, while exoglucans cleave glucose or single oligosaccharides from the non-reducing ends of the sugar chain. Endoglucanase is a key enzyme in the preparation of β-1,3-glucan oligosaccharides. Its catalyzed reaction products have high purity and few side reactions, making it a core tool for achieving green manufacturing of β-1,3-glucan oligosaccharides.

[0004] Besides preparing β-1,3-glucan oligosaccharides, endoβ-1,3-glucanase has broad application prospects in other fields. In agricultural biological control, it can inhibit the growth and reproduction of pathogenic fungi by degrading glucan components in the cell walls, induce plant immune responses, and enhance crop disease resistance. Furthermore, in brewing or food fermentation, this enzyme can serve as a process modifier, effectively reducing the glucan content and viscosity in fermentation broth, generating oligosaccharides with prebiotic functions, and improving filtration efficiency. Endoβ-1,3-glucanase can also be used for yeast cell wall hydrolysis in yeast extract production and as a feed additive to eliminate the anti-nutritional component β-glucan in feed.

[0005] Most of the currently discovered endo-β-1,3-glucanases have an optimal temperature range of 30–60 °C, but they exhibit poor stability, making it difficult to maintain efficient and stable catalytic performance in complex reaction systems. Poor stability leads to decreased catalytic efficiency during continuous production, affecting product consistency and process stability, thus limiting their industrial application. Summary of the Invention

[0006] The purpose of this invention is to provide an endogenous β-1,3-glucanase and its applications, namely, an enzyme derived from the deep sea that exhibits better stability at medium and low temperatures (40-60℃) and high salt tolerance compared to other endogenous β-1,3-glucanases, and its applications in biotechnology fields such as oligosaccharide preparation, yeast cell wall hydrolysis, biocontrol (antifungal), and feed processing.

[0007] This invention first provides an endopeptidase β-1,3-glucanase Ls_LamA, wherein the endopeptidase β-1,3-glucanase comprises:

[0008] 1) Enzymes with the amino acid sequence SEQ ID NO:1:

[0009] (SEQ ID NO:1);

[0010] 2) An enzyme derived from 1) by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence of SEQ ID NO:1;

[0011] One specific nucleotide sequence of the gene encoding the aforementioned endonuclease β-1,3-glucanase Ls_LamA is SEQ ID NO: 2.

[0012]

[0013] In another aspect, the present invention provides a recombinant expression vector in which a nucleic acid fragment encoding the aforementioned endonuclease β-1,3-glucanase Ls_LamA is inserted;

[0014] The recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0015] Another aspect of the present invention provides a genetically engineered strain, wherein the genetically engineered strain is transformed with the above-mentioned recombinant expression vector;

[0016] The genetically engineered strain can be a eukaryotic engineered strain or a prokaryotic engineered strain; as a specific example, it is an Escherichia coli engineered strain.

[0017] The present invention also provides the application of the described endo-β-1,3-glucanase Ls_LamA in the degradation of polysaccharides to prepare oligosaccharides.

[0018] The present invention also provides a method for preparing oligosaccharides, which involves using the aforementioned endo-β-1,3-glucanase Ls_LamA to degrade polysaccharides.

[0019] The polysaccharides mentioned herein, as described in this specification, are kelp polysaccharides, gel polysaccharides, barley β-glucan, oat β-glucan, or lichen polysaccharides.

[0020] The present invention also provides a laminarin oligosaccharide, which is prepared by degrading laminarin polysaccharide using the above-mentioned endo-β-1,3-glucanase;

[0021] The present invention also provides one use of the kelp oligosaccharide described therein for the preparation of antioxidant products.

[0022] The present invention also provides another use of the described endo-β-1,3-glucanase in the preparation of articles for inhibiting pathogenic bacteria;

[0023] The pathogenic bacteria are fungi with β-1,3-glucan components, and as a specific example, Candida albicans is described.

[0024] The present invention also provides another use of the aforementioned endo-β-1,3-glucanase for hydrolyzing yeast cell walls to extract yeast contents;

[0025] The present invention also provides another use of the described endo-β-1,3-glucanase for removing the anti-nutritional component β-1,3-glucan from feed.

[0026] The endo-β-1,3-glucanase Ls_LamA of this invention exhibits excellent catalytic activity and environmental adaptability, enabling efficient hydrolysis of laminarin and gelatinose. Its enzyme activity remains above 80% within a temperature range of 40–60℃ and pH 4.5–6.0, and it demonstrates good stability under medium and low temperature conditions, which is beneficial for reducing energy consumption in practical applications. Simultaneously, this enzyme exhibits significant high-salt tolerance, with no significant decrease in activity under 0.5–5 M NaCl and 0.5–3 M KCl conditions, and it also shows some tolerance to 5%–15% ethanol, making it suitable for various complex environmental systems. Furthermore, the β-1,3-glucanase of this invention significantly inhibits the formation of Candida albicans biofilms, and the laminarin oligosaccharides generated from its hydrolysis of laminarin also exhibit good antioxidant capacity, indicating that this enzyme has broad application prospects and significant practical value in antibacterial, biomedical, and other fields. Attached Figure Description

[0027] Figure 1 This is an SDS-PAGE electrophoresis image of the purified enzyme protein.

[0028] Figure 2 Graphs showing the effects of enzymes on the hydrolysis of different substrates and the analysis of enzyme hydrolysis products;

[0029] Figure 3 The graph shows the effect of different temperatures on enzyme activity and enzyme stability.

[0030] Figure 4 The graph shows the effect of different pH values ​​on enzyme activity.

[0031] Figure 5 The graph shows the effect of different metal ions on enzyme activity.

[0032] Figure 6 The graph shows the effect of different concentrations of NaCl and KCl on enzyme activity.

[0033] Figure 7 The effect of different concentrations of ethanol on enzyme activity;

[0034] Figure 8 Figure showing the effect of enzymes on Candida albicans biofilm formation.

[0035] Figure 9 A graph showing the ability of the enzyme's hydrolysis products to scavenge ABTS free radicals. Detailed Implementation

[0036] This invention is derived from Levonorkhylon ( LeeuwenhoekiellaA novel endo-β-1,3-glucanase, Ls_LamA, was obtained from the genome sequencing data of sp. BCO5. Its amino acid sequence is SEQ ID NO:1, and the nucleotide sequence of the encoding gene is SEQ ID NO:2.

[0037] This invention also protects an endonuclease β-1,3-glucanase isolated from Levonorkhylonium, which has high homology with the enzyme having the amino acid sequence SEQ ID NO:1, comprising:

[0038] 1) An enzyme with the amino acid sequence SEQ ID NO:1;

[0039] 2) An enzyme derived from 1) by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence SEQ ID NO:1, which has similar function to the enzyme in 1);

[0040] A gene encoding the above-mentioned enzyme has a specific nucleotide sequence as SEQ ID NO:2, but it can also be other nucleic acid sequences that can encode enzymes having the above-mentioned amino acid sequence, optimized according to the host bacterium.

[0041] The endo-β-1,3-glucanase provided by this invention has the enzymatic digestion effect of conventional endo-β-1,3-glucanase, but this enzyme has excellent high salt tolerance. Its activity did not decrease significantly in 0.5-5M NaCl solution and 0.5-3M KCl solution, and it also has a certain tolerance to 5-15% ethanol.

[0042] The endo-β-1,3-glucanase screened in this invention has the effect of randomly cleaving β-1,3 glycosidic bonds within the sugar chain to produce oligosaccharides with different degrees of polymerization. It can be applied to the following aspects, but in addition to the specific ones listed below, it can also be used for other applications of its degradation of polysaccharides.

[0043] 1) Preparation of oligosaccharides: Oligosaccharides with functions such as anti-oxidation, moisturizing, prebiotic, and promotion of bifidobacteria growth are prepared by using laminarin, gel polysaccharide, barley β-glucan, oat β-glucan or lichen polysaccharide.

[0044] 2) Extracting yeast contents by hydrolyzing yeast cell walls: In the preparation process of yeast extract, it is used in combination with β-1,6-glucanase or chitinase to hydrolyze yeast cell walls;

[0045] 3) Biological control: This method achieves the effect of controlling fungi by hydrolyzing the cell walls of fungal pathogens;

[0046] 4) Application in feed processing: Removing anti-nutritional components such as β-glucan from feed to improve its nutritional value;

[0047] 5) Applications in other biological fields, such as hydrolyzing fungal cell walls to prepare protoplasts.

[0048] Based on the discovery of the endogenous β-1,3-glucanase of the present invention, a method for preparing oligosaccharides from polysaccharides was established. The polysaccharide can be any polysaccharide that can be enzymatically cleaved by the existing endogenous β-1,3-glucanase to prepare oligosaccharides. In the examples of the present invention, kelp polysaccharide, gel polysaccharide, barley β-glucan, oat β-glucan or lichen polysaccharide are specifically described.

[0049] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0050] Example 1: Preparation of endo-β-1,3-glucanase Ls_LamA

[0051] Escherichia coli ( Escherichia coli DH5α and BL21(DE3) strains (purchased from Shanghai Sangon Biotech); expression vector pET28a-SUMO (purchased from Novagen); FastPure Bacteria DNAIsolation Mini Kit (purchased from Nanjing Novizan Biotech); ApexHF HS DNA Polymerase FS Master Mix (purchased from AG Biotech); restriction endonucleases Bam HI and Xho I (purchased from TransGen); Ultra-rapid cloning kit (purchased from Nanjing Novizan); LB medium (containing 10g peptone, 5g yeast extract, and 10g NaCl per liter); Kanamycin (purchased from Shanghai Sangon Biotech); Binding buffer (1×PBS buffer: 10mM phosphate, pH 7.4); Wash buffer (500mM NaCl, 20mM / 50mM imidazole, 20mM phosphate, pH 7.4); Elution buffer (500mM NaCl, 500mM imidazole, 20mM phosphate, pH 7.4); Protein Iso ®Ni-NTA Resin (purchased from TransGen); 30kDa ultrafiltration tubes (purchased from Taijing Company); 3,5-dinitrosalicylic acid (DNS) (purchased from Shanghai Sangon Biotech); Glucose (G) (purchased from Shanghai Sangon Biotech); Laminaribiose (L2) and Laminarin (derived from brown algae) standards (purchased from Aladdin Company); Laminaritriose (L3), Laminaritetraose (L4), and Laminaripentaose (L5) standards (purchased from Megazyme Company); Barley β-glucan, Lichenan, and Sodium carboxymethylcellulose (CMC-Na) (purchased from Megazyme Company); Oat β-glucan (Oat β-glucan and curdlan (purchased from Pusitang Pharmaceuticals); Total antioxidant capacity assay kit (ABTS method) (purchased from Beyotime Pharmaceuticals). The LB medium containing kanamycin used in this invention had a kanamycin concentration of 50 μg / mL.

[0052] Lewinsky bacteria ( Leeuwenhoekiella sp. BCO5 was isolated by the inventors from deep-sea sediments in the western Pacific Ocean. The mature protein gene of the endo-β-1,3-glucanase was obtained by analyzing the genome sequencing data of *Lewinsky koraiensis* BCO5. Ls_LamA Its nucleotide sequence is SEQ ID NO:2, and the sequence of the translated protein is SEQ ID NO:1. Analysis using NCBI's online BlastP tool revealed that the mature protein sequence of the endo-β-1,3-glucanase Ls_LamA shares no more than 84% homology with its homologous protein (compared to proteins derived from...). Leeuwenhoekiella The amino acid sequence identity of the protein of the family 16 glycosylhydrolase sp. A2 was 83.46%, indicating that the enzyme screened in this invention is a novel endo-β-1,3-glucanase.

[0053] The specific process of recombinant expression of this enzyme is described below.

[0054] 1) Based on Ls_LamA Primers for amplifying the mature protein gene of endo-β-1,3-glucanase were designed and synthesized, and their sequences are as follows:

[0055] Upstream primer LamA-F: 5′-acagagaacagattggtggatccTGTCAGGATGATGATCAGACATTTGGAG-3′ (SEQ ID NO:3)

[0056] Downstream primer LamA-R: 5′-agtggtggtggtggtggtgctcgagCTATTGATAAACTCTTACATAATCAACTTCCATG-3′ (SEQ ID NO:4)

[0057] 2) PCR amplification and construction of recombinant plasmids

[0058] Genomic DNA was extracted from *Lewinsky Kocide* BCO5 using a bacterial genomic DNA extraction kit. The extracted BCO5 genomic DNA was used as a template for amplification using LamA-F and LamA-R primers. The amplification system (50 μL) was as follows: 1 μL LamA-F (10 µM), 1 μL LamA-R (10 µM), 1 μL genomic DNA, 25 μL 2×ApexHF HS DNA Polymerase FS MasterMix, and 22 μL ddH2O. The amplification conditions were as follows: 94℃ pre-denaturation for 30 s; 98℃ for 10 s, 55℃ for 5 s, and 72℃ for 20 s, for 30 cycles. The PCR products were recovered from the gel, and the pET28a-SUMO vector was desorbed using restriction endonucleases. Bam Linearization with HI was performed, and the PCR product and the enzyme-digested vector were ligated using an ultra-rapid cloning kit. The ligation product was then fused with *E. coli*. E. coli DH5α competent cells were mixed, placed on ice for 30 min, heat-shocked at 42℃ for 90 s, and then 150 µL of LB liquid medium was added. The mixture was then incubated at 37℃ and 100 rpm for 1 h. After centrifugation, the mixture was spread onto LB solid medium containing kanamycin and incubated overnight at 37℃. Recombinant plasmids were screened and then double-digested with enzymes. Bam HI and Xho I) and sequencing verification yielded the gene containing the β-1,3-glucanase endonuclease. Ls_LamA The recombinant plasmid.

[0059] (3) Induction of gene expression and preparation of crude enzyme solution

[0060] Transform the recombinant plasmid into E. coli A recombinant strain containing the β-1,3-glucanase gene was obtained from BL21(DE3). The recombinant strain was inoculated into 5 mL of LB broth containing 50 μg / mL kanamycin and cultured overnight. Then, it was inoculated at a 1% inoculation rate into 250 mL of LB broth containing kanamycin and cultured at 37°C and 180 rpm until OD500. 600The concentration was approximately 0.6. IPTG was added to achieve a final concentration of 0.5 mmol / L for induction. Expression was induced overnight at 18°C ​​and 180 rpm. The cells were collected by centrifugation at 8000 rpm for 20 min. The cells were then resuspended in 25 mL of binding buffer, and an appropriate amount of protease inhibitor was added. The cells were sonicated at 300 W with a work / interval time of 3 s / 5 s for 30 min. The cells were then centrifuged at 4°C and 4000 rpm for 10 min, and the supernatant was collected to obtain the crude enzyme solution.

[0061] (4) Enzyme purification

[0062] The prepared crude enzyme solution was added to a pre-equilibrated His purification column (Ni-NTA Resin) and mixed at 4°C for 2-3 hours, then the waste liquid was discarded. Next, the solution was washed twice with 10 ml of rinsing buffer containing 20 mM imidazole, then twice with 10 ml of rinsing buffer containing 50 mM imidazole. After rinsing, the solution was eluted four times with elution buffer containing 500 mM imidazole (4 mL each time). The eluent was collected in batches, and its purity was determined by SDS-PAGE electrophoresis. Ultrafiltration was performed using a 30 kDa ultrafiltration tube to remove imidazole and high concentrations of NaCl, yielding the purified enzyme solution. Figure 1 ).

[0063] Example 2: Enzymatic Properties Detection of Endo-β-1,3-glucanase Ls_LamA

[0064] 1) Method for determining the activity of endoβ-1,3-glucanase

[0065] The reducing sugar content was determined using the DNS method. 20 μL of appropriately diluted enzyme solution and 80 μL of 1% laminarin substrate were mixed and reacted at 55 °C for 10 min. After the reaction was complete, 200 μL of DNS reagent was added, and the mixture was boiled for 5 min. It was immediately cooled in ice water, briefly centrifuged, and the supernatant was collected for OD measurement. 540 The reducing sugar content and enzyme activity were calculated based on the standard curve (using inactivated enzyme solution as a control). Enzyme activity unit definition: Under the above test conditions, the amount of enzyme required to hydrolyze laminarin per minute to produce 1 µmol of reducing sugar is defined as one enzyme activity unit (U).

[0066] (2) Analysis of enzyme substrate specificity and hydrolysis products

[0067] Enzyme activity was determined at 55℃ and pH 5.5 using 1% barley β-glucan, oat β-glucan, lichen polysaccharide, laminarin, gel polysaccharide, and sodium carboxymethyl cellulose as substrates. The enzyme activity measured using laminarin as substrate was 100%. The relative activities of the endo-β-1,3-glucanase in hydrolyzing different substrates were calculated. The results showed that the enzyme effectively hydrolyzed gel polysaccharide and laminarin, and also had some hydrolytic activity against barley β-glucan, oat β-glucan, and lichen polysaccharide, but could not hydrolyze sodium carboxymethyl cellulose. This indicates that the enzyme is a β-1,3-glucanase. (See attached figures). Figure 2 A.

[0068] Thin-layer chromatography (TLC) was used to analyze the β-1,3-glucanase products. 15 μL of purified enzyme was added to 50 μL of 50 mM Na-acetate buffer (pH 5.5) containing 1% laminarin, and the mixture was incubated overnight at 30 °C and 55 °C, respectively. The reaction mixture was then heated at 100 °C for 10 min, followed by centrifugation at 5000 rpm for 2 min. The supernatant was collected for TLC analysis, with a sample loading volume of 2 μL. The developing solvent was ethyl acetate:acetic acid:water (2:2:1, v / v / v), and the colorimetric reagent was 10% sulfuric acid in ethanol. The mixture was heated to 130 °C for colorimetric detection. The results showed that the final product of the enzyme hydrolysis of laminarin was mainly a mixture of oligosaccharides of laminarin tetrasaccharides and higher, indicating that this enzyme is an endoglucanase with potential applications in the development and utilization of brown algae resources and the preparation of laminarin oligosaccharides. See below for details. Figure 2 B.

[0069] (3) Enzyme action temperature and stability

[0070] Enzyme activity was measured using 1% laminarin prepared with 50 mM Na-acetate buffer (pH 5.5) as substrate within the range of 5–70 °C. The maximum enzyme activity was taken as 100%, and the relative enzyme activity at different temperatures was calculated. The results showed that the optimal reaction temperature for this enzyme was 55 °C. The enzyme activity was high within the range of 40–60 °C, with its relative enzyme activity remaining above 80%. Within the range of 20–65 °C, its relative enzyme activity remained above 50%. Figure 3 A).

[0071] The enzyme solution was incubated at 10℃, 20℃, 30℃, and 40℃ for different times. Then, a 1% laminarin solution prepared with 50mM Na-acetate buffer (pH 5.5) was used as the substrate for the reaction at 55℃ and pH 5.5. The relative activities of the untreated enzyme were calculated as 100%. The results showed that the enzyme activity did not decrease after incubation at 10℃, 20℃, 30℃, and 40℃ for 168 h, indicating good stability of the enzyme under medium and low temperature conditions. (See attached figures). Figure 3 B.

[0072] (4) The role of enzymes pH

[0073] Enzyme activity was measured at 55℃ using 1% laminarin prepared with different buffers (50mM Na-acetate buffer, pH 4.0–6.0; 50mM Na-phosphate buffer, pH 6.0–8.0) as substrates. The relative activity of the enzyme at different pH values ​​was calculated with maximum enzyme activity as 100%. The results showed that the optimal pH for this enzyme was 5.5. Enzyme activity was high within the pH range of 4.5–6.0, maintaining above 80%; within the pH range of 4.5–7.5, enzyme activity remained above 50%; enzyme activity was low below pH 4.5 or above pH 7.5. Under the same pH conditions, Na-acetate buffer was superior to Na-phosphate buffer. (See attached results). Figure 4 .

[0074] (5) Effects of metal ions on enzyme activity

[0075] At 55℃, using 1% laminarin prepared with 50mM Na-acetate buffer (pH 5.5) as the substrate, different metal ions were added to the reaction system at final concentrations of 1mM and 5mM, respectively. Enzyme activity was then measured, and the relative enzyme activity under the influence of different metal ions was calculated with the relative enzyme activity under the condition of no added metal ions as 100%. The results showed that Mn... 2+ It promotes enzyme activity; low concentrations of Ca 2+ and Mg 2+ It also promotes enzyme activity, but high concentrations of Ca... 2+ and Mg 2+ Inhibits enzyme activity; Na + K + Fe 2+ Ba 2+ It has little effect on enzyme activity; Zn 2+ Partially inhibits enzyme activity, Cu 2+ The enzyme activity was significantly inhibited by more than 85%. Figure 5 ).

[0076] (6) Effects of NaCl and KCl on enzyme activity

[0077] Under conditions of 55℃ and pH 5.5, 1-5M NaCl or 0.5-3M KCl were added to the reaction system, with the relative enzyme activity under the condition of no additional salt ions as 100%. The relative enzyme activity under different salt ion concentrations was calculated. The results showed that different concentrations of NaCl or KCl had varying degrees of promoting effect on enzyme activity or had no effect. 1-4M NaCl could increase enzyme activity by more than 18%, and 2-3M KCl could increase enzyme activity by more than 13%. Figure 6 ).

[0078] (7) Effect of ethanol on enzyme activity

[0079] At 55℃ and pH 5.5, ethanol was added to the reaction system at final concentrations of 5%, 10%, 15%, 20%, and 25%, respectively. Enzyme activity was then measured, with the relative enzyme activity without ethanol added as 100%. The effect of different concentrations of ethanol on enzyme activity was calculated. The results showed that the enzyme exhibited some tolerance to 5–15% ethanol; 5% ethanol had little effect on enzyme activity, retaining over 70% of the activity; and even with the addition of 10% ethanol, the enzyme activity still retained over 50%. Figure 7 ).

[0080] Example 3: Application of endo-β-1,3-glucanase Ls_LamA

[0081] 1) Inhibits biofilm formation by Candida albicans

[0082] The effect of endoβ-1,3-glucanase on Candida albicans biofilm formation was investigated using a microplate method. Overnight cultures of Candida albicans were inoculated at a 1% inoculum into LB broth and transferred to sterile 96-well polystyrene microplates (200 µL / well). The plates were incubated statically at 37°C for 96 hours. During this period, enzyme solution at a final concentration of 10 mg / mL was added to cultures at the early biofilm formation stage (6 h) and the mature biofilm formation stage (48 h). Cultures without enzyme solution served as positive controls, and culture media without enzyme solution served as negative controls. After incubation, the absorbance of the culture medium was measured at 600 nm. Floating bacterial culture was then removed, and the biofilm was washed three times with ultrapure water and stained with 200 μL of 0.1% crystal violet solution for 20 min. After staining, the staining agent was removed, and the cells were washed three times with ultrapure water. The biofilm was then dissolved in 200 μL of 95% ethanol, and biofilm formation of *Candida albicans* was detected at a wavelength of 590 nm. The results showed that the enzyme significantly inhibited biofilm formation of *Candida albicans*, especially during the early biofilm formation stage, where the enzyme inhibited biofilm formation by over 90%. Adding the enzyme during the mature biofilm formation stage also reduced the biofilm formation rate of *Candida albicans* by over 50%. Figure 8 ).

[0083] 2) Detection of antioxidant capacity of enzyme hydrolysis products

[0084] The total antioxidant capacity of the enzyme hydrolysates was determined using an Absorbable Antioxidant Synthesis (ABTS) assay kit. 100 µL of pure enzyme solution was added to 2 mL of a 20% laminarin solution prepared with 1×PBS buffer. After mixing, the mixture was reacted at 40 °C. Samples were taken at 12 h, 24 h, and 36 h, with a sample volume of 500 µL. The samples were ultrafiltered using a 10 kDa ultrafiltration tube, then boiled and centrifuged to remove enzyme protein, obtaining a high-concentration hydrolysate solution. An inactivated enzyme solution of the same concentration was added to the laminarin solution and the above treatment was repeated; the resulting sample served as a control. The results showed that the enzyme hydrolysates exhibited significant antioxidant function. Compared with the control group, the scavenging efficiency of the hydrolysates against ABTS free radicals was significantly improved at different reaction times. The scavenging efficiency of the hydrolysates against ABTS free radicals at 12 h, 24 h, and 36 h of reaction was increased by 10.2%, 19.8%, and 22.4%, respectively. Figure 9 ).

[0085] In summary, the endo-β-1,3-glucanase Ls_LamA of this invention can effectively hydrolyze laminarin and gelatinose, with an optimal temperature of 55°C and an optimal pH of 5.5. The enzyme activity remains above 80% within the temperature range of 40–60°C and pH range of 4.5–6.0, and exhibits better stability under medium and low temperature conditions. Furthermore, this enzyme demonstrates excellent high-salt tolerance, showing no significant decrease in activity in 0.5–5M NaCl and 0.5–3M KCl solutions, and also exhibits some tolerance to 5–15% ethanol. Additionally, this enzyme significantly inhibits biofilm formation in Candida albicans, and the laminarin oligosaccharides generated from the hydrolysis of laminarin by this enzyme possess significant antioxidant capabilities.

Claims

1. An endoglucanase, characterized in that, The amino acid sequence of the endo-β-1,3-glucanase is SEQ ID NO:

1.

2. A gene characterized in that, The nucleotide sequence of this gene is SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the gene sequence as described in claim 2.

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

5. A genetically engineered strain, characterized in that, The genetically engineered strain is transformed with the recombinant expression vector of claim 3.

6. The application of the endo-β-1,3-glucanase of claim 1 in the degradation of polysaccharides to prepare oligosaccharides.

7. A method for preparing an oligosaccharide, characterized in that, The method described in claim 1 involves using the endo-β-1,3-glucanase to degrade polysaccharides to prepare oligosaccharides.

8. The method as described in claim 7, characterized in that, The polysaccharides mentioned are kelp polysaccharide, gel polysaccharide, barley β-glucan, and oat β-glucan.

9. The use of the endo-β-1,3-glucanase of claim 1 in the preparation of articles for inhibiting fungi.

10. An antibacterial product, characterized in that, The product contains the endo-β-1,3-glucanase as described in claim 1.

11. The use of the endo-β-1,3-glucanase according to claim 1 in the extraction of yeast contents by hydrolyzing yeast cell walls.

12. The use of the endo-β-1,3-glucanase according to claim 1 in removing the anti-nutritional component β-1,3-glucan from feed.

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