Enzymatic degradation shows significant advantages in biological environments. It can achieve rapid decomposition of pollutants under mild conditions, significantly reducing energy consumption and treatment costs (Han et al., 2021; Payanthoth et al., 2024). Combined with microbial fermentation, it provides an efficient, precise, environmentally friendly and sustainable pollution control and material recycling solution (Xia et al., 2017). Numerous studies have investigated hyaluronidase, and in this study, we analyzed evolutionarily validated hyaluronidases through phylogenetic clustering (Figure 1) and selected four functionally characterized candidates for heterologous expression: HHya1 (EC 3.2.1.36) from H. nipponia (Jin et al., 2014), LHya2 from L. intermedia (Ferrer et al., 2013), SHya3 from S. horrida (Ng et al., 2005), and EHya4 from Enterobacterales. HHya1, identified as hyaluronate-3-glycanohydrolases (EC 3.2.1.36), acts as an endo-β-D-glucuronidase primarily degrading HA into tetrasaccharides (Jin et al., 2014; Wenshuang Wang and Li, 2017). The HHya1 can express in P. pastoris. The LHya2 is present in spider venom and has been shown to degrade hyaluronic acid to produce a small molecule of 29–45 kDa (Ferrer et al., 2013). SHya3, discovered in the venom gland of stonefish Synanceja horrida, marks the first hyaluronidase from an aquatic source, capable of breaking down HA less than 20 kDa into smaller oligosaccharides (Ng et al., 2005). HHya1, LHya2 and SHya3 are derived from eukaryotes. While HHya1 has shown good activity when expressed using Pichia pastoris (Kang et al., 2016). LHya2 and SHya3 have been successfully expressed in Escherichia coli, yielding inclusion bodies that are subsequently denatured, refolded in vitro, and transformed into active hyaluronidase, although with modest enzyme activity (da Silveira et al., 2007; Ng et al., 2005). To address this, we engineered LHya2 and SHya3 with sequence truncations and SUMO fusion tags to enhance solubility in P. pastoris, though activity improvements remained limited. In contrast, EHya4—a prokaryotic enzyme—achieved robust expression in E. coli without optimization. To determine the expression level of the recombinant proteins, the crude lysates supernatant activity of the four recombinant proteins was determined. Comparative analysis revealed superior expression levels and activities for HHya1 (138,467 U/mL) and EHya4 (135,733 U/mL) versus LHya2 (51,982 U/mL)/SHya3 (42,935 U/mL) (Figure 2; Supplementary Figure S1). Given industrial cost considerations, HHya1 and EHya4 were selected for subsequent S. zooepidemicus fermentation trials, balancing enzymatic efficiency with production feasibility for HA yield optimization.

Hyaluronic acid fermentation optimization requires precise control of enzyme additives to maintain system stability. Therefore, it is necessary to obtain highly active enzymes with the minimum addition amount. In this study, the fermentation process was optimized by adding purified enzymes with the same enzyme activity. In this study, evolutionary analysis of hyaluronidases (Figure 1) identified four candidates—HHya1 (H. nipponia), LHya2 (L. intermedia), SHya3 (S. horrida), and EHya4 (Enterobacterales)—for heterologous expression with N-terminal His-tags to facilitate purification. Ni-NTA affinity chromatography significantly enhanced enzyme purity (Figures 3a,b), boosting specific activity by 54-fold for HHya1 (138,467 U/mL) and 77-fold for EHya4 (135,733 U/mL) compared to crude extracts (Figure 3c). Functional characterization revealed distinct degradation profiles: HHya1 generated saturated HA oligosaccharides (Figure 3d), while EHya4 predominantly produced unsaturated disaccharides (ΔHA2) within 8 h, with minor tetrasaccharide (ΔHA4), hexasaccharide (ΔHA6), and octasaccharide (ΔHA8) byproducts (Figure 3e). Despite lower activities of LHya2 (51,982 U/mL) and SHya3 (42,935 U/mL), their inclusion highlighted mechanistic diversity in HA depolymerization. In this study, the kinetic parameters of four different hyaluronidases were determined (Table 1).

In Streptococcus zooepidemicus fermentations, purified HHya1 and EHya4 reduced broth viscosity by 53%, improved mass transfer efficiency, and achieved 10.3 g/L HA titers with a narrow molecular weight distribution (1 × 10⁴–2 × 10⁵ Da). While this study focused on yield optimization, the enzymatic specificity of HHya1 (saturated products) and EHya4 (unsaturated ΔHA2) positions them as versatile tools for tailored HA production in biomedical and cosmetic applications. Ongoing process refinements aim to standardize molecular weight control, underscoring the potential of enzymatic engineering to harmonize industrial scalability with product customization in HA biosynthesis.

The optimization of fermentation conditions, as the core bridge connecting laboratory scale and industrial large-scale production, determines the economic feasibility of the fermentation process, the stability of product quality and the key links of the entire biomanufacturing process (Kwaw et al., 2017; Wang et al., 2023). To assess the role of hyaluronidases in enhancing HA production during S. zooepidemicus fermentation, shake flask experiments were conducted with varying concentrations of HHya1 and EHya4. The different catalytic efficiency represented by the differences in kinetic parameters of different enzymes leads to differences in viscosity and DO levels during fermentation, which leads to differences in HA titer. It was found that the maximum HA production was categorized as 4.03, 4.27, 4.39, and 4.32 g/L when 500, 1,000, 1,500, and 2,500 U/L of HHya1 were added, respectively (Figures 4a–d). Among them, the highest yield of HA was obtained when 1,500 U/L was added, which increased the yield by 8% compared to the fermentation process without HA (Figure 5). On the other hand, the maximum yield of HA was categorized as 4.14, 4.33, 4.57, and 4.45 g/L when 500, 1,000, 1,500, and 2,500 U/L of EHya4 were added, respectively (Figures 4e–h). As with HHya1, the highest yield of HA was achieved when EHya4 was added at 1,500 U, with a 12% increase in yield compared to the fermentation process without HA (Figure 5). It was found that the highest hyaluronan production was achieved when the addition of both hyaluronans was controlled at 1,500 U/L, so hyaluronidase needs to be added moderately (Figure 4). Therefore, EHya4 can be used as the optimal enzyme to enhance the HA production capacity of S. zooepidemicus by adding hyaluronidase. The enzyme was used as the main ingredient to optimize the HA production process. Notably, hyaluronidase supplementation correlated with elevated biomass (OD600), suggesting enzymatic HA degradation alleviates broth viscosity, enhances nutrient diffusion, or releases growth-promoting oligosaccharides, thereby partially addressing the chronic challenge of low cell density in S. zooepidemicus fermentations. These findings reveal the dual role of hyaluronidases in boosting HA yield and microbial vitality, though the pathogenicity and metabolic constraints of the strain necessitate further exploration of novel enzymes or engineered systems. Future studies should prioritize hyaluronidases with enhanced thermostability or synergistic activity to amplify fermentation density and HA output, ultimately advancing scalable, cost-effective bioproduction of tailored HA for biomedical and cosmetic applications.

Building on shake flask experiments demonstrating the benefits of moderate hyaluronidase supplementation for hyaluronic acid (HA) production in S. zooepidemicus, this study systematically evaluated enzymatic strategies in a 15 L fermenter. Two fermentation strategies, timed addition (fixed intervals) and dissolved oxygen (DO)-responsive addition, were compared to optimize HA yield. The timed addition of 1,500 U/L hyaluronidase at predetermined intervals achieved a HA titer of 10.3 g/L, representing a 14.4% increase over non-supplemented fermentations. In contrast, DO-responsive addition, where enzyme dosing was triggered by dissolved oxygen thresholds, yielded 9.9 g/L HA, a 10% improvement (Figure 6). The superior performance of timed supplementation likely stems from sustained viscosity reduction and consistent nutrient availability, whereas DO-based delays in enzyme administration may allow transient HA accumulation to impede mass transfer. Notably, these results align with shake flask trends, confirming hyaluronidase’s role in mitigating viscosity-related metabolic constraints. However, this study focused solely on enzymatic supplementation, leaving other parameters (pH, feeding strategies, aeration) unoptimized. Further research integrating hyaluronidase dosing with multivariate process engineering could unlock additional yield gains. These findings underscore the industrial viability of timed enzymatic supplementation for HA bioproduction, while highlighting the need for holistic fermentation optimization to fully harness hyaluronidase’s potential in scalable systems.

The strains used in this study are P. pastoris GS115 (purchased from Bioon), S. zooepidemicus, and Enterobacteriaceae (both preserved in our laboratory). The genes were synthesized by Genscript Biotech Corporation (Supplementary Table S1). The amino acid sequences of each part are shown in Supplementary Table S2. The medium used in this study was: Yeast Extract Peptone Dextrose (YPD) medium: yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L. Yeast Nitrogen Base (YNB) medium 13.4 g/L, biotin 4 × 10⁻⁴ g/L, and agar 20 g/L. Buffered Glycerol-complex (BMGY) medium: yeast extract 10 g/L, peptone 20 g/L, K₂HPO₄ 3 g/L, KH₂PO₄ 11.8 g/L, amino acid-free yeast nitrogen source YNB 3.4 g/L, ammonium sulfate 10 g/L, biotin 4 × 10⁻⁴ g/L, glycerol 10 g/L. Buffered Methanol-complex (BMMY) medium: yeast extract 10 g/L, peptone 20 g/L, K₂HPO₄ 3 g/L, KH₂PO₄ 11.8 g/L, YNB 3.4 g/L, ammonium sulfate 10 g/L, biotin 4 × 10⁻⁴ g/L, methanol 10 mL/L. BSM medium: glycerol 40 g/L, K₂SO₄ 18 g/L, KOH 4.13 g/L, 85%H₃PO₄ 26.7 mL/L, CaSO₄·2H₂O 0.93 g/L, MgSO₄·7H₂O 14.9 g/L, 4.4 mL/L PTM1 for filtration and sterilization. PTM1:CuSO₄·5H₂O 6 g/L,KI 0.09 g/L,MnSO4·H₂O 3 g/L,H₃BO₃ 0.02 g/L,MoNa₂O₄·2H₂O 0.2 g/L, CoCl₂·6H₂O 0.92 g/L, ZnCl₂ 20 g/L, FeSO₄·7H₂O 65 g/L,biotin 0.2 g/L,H₂SO₄ 5.0 mL/L. Fermentation medium: peptone 16 g/L, yeast dip 2 g/L, glucose 10 g/L, monosodium glutamate 2 g/L, K₂HPO₄ 2 g/L, MgSO₄·7H₂O 0.7 g/L. Solid medium A: peptone 15 g/L, yeast maceration powder 5 g/L, glucose 5 g/L. K₂HPO₄ 2 g/L, MgSO₄·7H₂O 0.5 g/L, 15 g/L.

Cultivate P. pastoris GS115 on YPD agar plates at 30°C for 2 days, then pick a single colony and culture it in YPD medium at 30°C with shaking overnight. Inoculate the seed culture into YPD medium at a 1% inoculation ratio and culture with shaking until the OD₆₀₀ reaches between 0.5 and 0.8. Pre-cool on ice and then centrifuge at 3,000 g to collect the yeast cells. Wash the yeast cells three times with pre-cooled 1 M sorbitol solution. Resuspend the cells with an appropriate amount of pre-cooled 1 M sorbitol solution, divide into portions, and add 1–2 μg of linearized plasmid that has been digested with SalI enzyme and purified. Transform the plasmid into competent cells using electroporation (2,700 V, 200 Ω). After electroporation, spread on histidine-deficient MD plates for initial screening. Transfer the single colonies from the initial screening to YPD plates containing G418 (4 mg/mL) for secondary screening to select for P. pastoris/pPIC9K strains with high copy recombinant hyaluronidase genes.

Pick the successfully constructed strain and inoculate it into 50 mL of YPD medium, cultivate at 30°C and 200 rpm for 24 h to prepare the seed culture. Transfer the seed culture to 50 mL of the initial expression medium BMGY at a volume ratio of 10%, to enrich the biomass, and cultivate at 30°C and 200 rpm for 24 h. Collect the biomass by centrifugation, wash with sterile water, and then transfer to 40 mL of the induction expression medium BMMY, cultivate at 30°C and 200 rpm, and add methanol (containing 1.2% (v/v) PTM1) at a volume ratio of 1% to the fermentation flask every 24 h until the induction expression reaches 96 h. Collect the supernatant, use membrane packaging for concentration, and then carry out enzyme activity assays. Escherichia coli is used for protein expression with LB medium, cultivated at 37°C and 220 rpm for 8 h, and then the biomass is collected using a centrifuge; resuspend the collected biomass with 5 times the volume of PB buffer and disrupt it using a homogenizer to release the intracellular enzymes, then collect the supernatant by centrifugation to remove cell debris. Slowly add 5% ammonium sulfate to the fermentation broth, mix the solution with an overhead stirrer at 300 rpm, let it stand for 30 min, and then centrifuge with a vertical centrifuge (5,000 rpm, 20 min) to remove the precipitate and retain the supernatant; then use 30% ammonium sulfate precipitation, retain the precipitate in the same manner, and redissolve with PB buffer at a 1:1 ratio to obtain the protein solution. Use a 300 K hollow fiber to clarify the fermentation broth containing hyaluronidase, and select different columns for purification based on the type of hyaluronidase. Verify the purified hyaluronidase using polyacrylamide gel electrophoresis.

The unit of activity for hyaluronidase (U) is defined as the amount of enzyme required to release 1 μg of reducing sugar equivalent from the sugar chain of HA per hour under conditions of pH 5.5°C and 38°C. The reducing sugar is determined using the dinitrosalicylic acid (DNS) method, with the specific procedure as follows: Mix 0.8 mL (2 mg/mL) of HA (HA, 120 kDa) solution with 0.1 mL of supernatant and 0.1 mL of citrate buffer (pH = 5.5), react in a 38°C water bath for 15 min, then immediately boil for 5 min to stop the reaction, cool down, and after treatment, add 1 mL of the reaction mixture to 2 mL of DNS solution, mix well, and then boil in water for 10 min; after cooling in an ice water bath to room temperature, add 7 mL of deionized water, mix well, use analytical grade glucose for a standard curve, and measure the absorbance of the solution in the colorimetric tube at 540 nm with a UV-Vis spectrophotometer, and express the enzyme activity (U/mL) in terms of the amount of glucose.

The fermentation broth was treated with hyaluronidase and then tested for hyaluronic acid. For the detection of HA content, high-performance liquid chromatography (HPLC) is used. Hyaluronic acidase can act on the β-1,4-glycosidic bond of sodium hyaluronate, causing hydrolysis to produce N-acetylglucosamine-glucuronic acid disaccharides, and the content of N-acetylglucosamine-glucuronic acid disaccharide products is determined using HPLC. Detection is performed using an Agilent 1260 HPLC system (UV detector) with an MCI GEL CKO8EH column (8 mm × 300 mm, 5 μm); the mobile phase is 1% phosphoric acid; flow rate: 0.6 mL/min; injection volume: 20 μL; column temperature: 40°C; detection wavelength: 232 nm, with a peak time of 9.806 min. For the detection of HA content, high-performance liquid chromatography (HPLC) is used. Hyaluronic acidase can act on the β-1,4-glycosidic bond of sodium hyaluronate, causing hydrolysis to produce N-acetylglucosamine-glucuronic acid disaccharides, and the content of N-acetylglucosamine-glucuronic acid disaccharide products is determined using HPLC. Detection is performed using an Agilent 1260 HPLC system (UV detector) with an MCI GEL CKO8EH column (8 mm × 300 mm, 5 μm); the mobile phase is 1% phosphoric acid; flow rate: 0.6 mL/min; injection volume: 20 μL; column temperature: 40°C; detection wavelength: 232 nm, time: 9.806 min.

The enzymatic catalysis of hyaluronidase was assayed using sodium hyaluronate. 500 U of hyaluronidase was added to 0.1 g/mL of sodium hyaluronate solution and the reaction was carried out at 37°C. Samples were taken at 0, 1, 3, 4.5, and 8 h, and the reaction was allowed to stand at 100°C for 5 min to end the reaction, and then assayed using HPLC.

Glycerol bacteria were taken and stored at −80°C and streaked, single colonies were picked and streaked in solid medium A. After overnight incubation at 37°C, they were inoculated into shake flasks containing 50 mL of fermentation medium and incubated at 37°C and 160 rpm for 14–16 h. From the 8 h of incubation, appropriate amounts of hyaluronidase were added. Samples were taken every 4 h for the determination of OD₆₀₀ and HA content, OD₆₀₀ was detected using a spectrophotometer, and hyaluronic acid was detected using liquid chromatography. Each experiment was repeated three times.

The addition of hyaluronidase during the fermentation process will reduce the viscosity of the fermentation broth, thus affecting the growth state of the bacteria. Therefore, the fermentation conditions need to be optimized, including stirring speed, aeration, time and concentration of sugar supplementation. Fermentation conditions were coupled with hyaluronidase addition to determine two enzyme addition schemes: the first was dissolved oxygen associated stirring, enzyme addition started when dissolved oxygen decreased to 10%, and the dissolved oxygen level was controlled by adding different hyaluronidases; the second was hyaluronidase addition according to fermentation viscosity (time dependent), hyaluronidase was added from the 8 h, and added every hour, for a total of 5 times. Samples were taken at 4 h intervals and then the hyaluronic acid content was measured using HPLC.