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Article

Enhancing Sialidase Production from the Oerskovia paurometabola O129 Strain by the Optimization of Fermentation Parameters and the Addition of Stimulative Compounds

by
Yana Gocheva
*,
Ekaterina Krumova
,
Irina Lazarkevich
,
Rumyana Eneva
and
Stephan Engibarov
Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. Georgi Bonchev Str., Bl. 26, 1113 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2025, 5(2), 50; https://doi.org/10.3390/applmicrobiol5020050
Submission received: 13 May 2025 / Revised: 23 May 2025 / Accepted: 23 May 2025 / Published: 25 May 2025
(This article belongs to the Special Issue Current Trends in the Applications of Probiotics and Other Beneficial Microbes)
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Abstract

:
Sialidases are gradually entering various areas of human practice—in medicine and pharmacy, as antiviral, antitumor, diagnostic, and vaccine preparations; for the chemoenzymatic synthesis of regioselective sialoglycoconjugates; and for the structural analysis of sialoglycoproteins. Optimizing the synthesis conditions of these commercially important enzymes would be beneficial for enhancing their production and expanding potential applications. Since sialidase producers are often pathogenic microorganisms, the use of saprophytic bacteria could be an alternative to reduce the health risk when working with them. So far, the topic has not been widely discussed. By a single-factor optimization method, the most suitable fermentation conditions for achieving maximum sialidase production by the non-model strain Oerskovia paurometabola O129 were established. The dynamics of enzyme accumulation during the growth phases and the optimal physicochemical parameters for cultivation were determined (30 °C, pH 8.0, agitation at 200 rpm, for 28 h). The addition of various inducers and surfactants to improve enzyme yield was also investigated. The effect of surfactants on bacterial sialidase production was tested for the first time. Maximum enzyme production (98.3 U/mL), representing about a three-fold increase compared to non-optimized conditions, was obtained by culturing the strain under optimal conditions and by the synergistic action of glucomacropeptide and Tween 80. A new, simple, and cost-effective laboratory model for optimizing sialidase production by the saprophytic strain O. paurometabola O129 in submerged fermentation was proposed. Future work may involve scaling up the process and exploring genetic or metabolic enhancements for targeted biomedical and industrial applications.

1. Introduction

Sialidases (neuraminidases, exo-alpha sialidases, N-acylneuraminosylglycohydrolases, E.C. 3.2.1.18) are enzymes that cleave terminal sialic acids from a number of glycoproteins, glycolipids, oligosaccharides, mucins, etc. [1,2,3]. Sialic acids are amino sugars that participate in various intercellular and intermolecular interactions, and serve as receptors for a number of viruses, bacteria, lectins, hormones, and antibodies [1]. They are found in almost all tissues in the deuterostome lineage of animals and in certain viruses, bacteria, protozoa, and fungi [2,3,4]. So far, sialidase has been found in some animals, microorganisms, and viruses [3]. A large proportion of bacterial sialidase producers are pathogens, such as Clostridium perfringens, Streptococcus pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, Erysipelothrix rhusiopathiae, Corynebacterium diphtheriae, Pasteurella multocida, and Salmonella typhimurium [3,5,6,7,8]. Sialidases contribute to pathogenicity in a variety of ways, including degradation of the mucous layer, adhesion to host cells, loosening of intercellular tight junctions, disclosure of cryptoreceptors, biofilm formation, and evasion of host immune defenses [2,3,9,10,11]. Some commensals that meet their energy needs by breaking down mucin also have sialidase expression [3]. In contrast to pathogens, sialidase production has been reported less frequently in soil saprophytes, for example in representatives of the genera Streptomyces, Arthrobacter, Oerskovia, and Micromonospora [12,13,14,15,16].
It should be noted that bacterial sialidases are gradually entering various areas of human practice. Some of them are of interest in medicine and pharmacy, as they have potential or have already been approved as antiviral [17,18,19], antitumor [20,21,22], diagnostic [23,24,25], and vaccine preparations [26,27]. Bacterial sialidases are also used for the chemoenzymatic synthesis of a number of regioselective sialoglycoconjugates [28,29,30,31]; for the conversion of polysialogangliosides to monogangliosides [32,33,34]; and for the structural analysis of various sialoglycoproteins [35,36]. To our knowledge, studies addressing the issue of optimizing sialidase production in bacteria are few [6,37,38]. In this regard, optimizing the conditions for the synthesis of bacterial sialidases would lead to the production of a larger quantity of these commercially important enzymes with potential application in practice. It is also necessary to emphasize the use of saprophytic bacteria with high sialidase activity in order to increase safety and as a successful alternative to pathogenic producers.
Oerskovia, previously identified as Nocardia, are Gram-positive, rod-shaped bacteria, primary environmental inhabitants that can be found in soil, water, grass, decaying plant material, sewage, compost, feces of invertebrates, etc. [16,39]. Although rare, they can be associated with certain diseases in immunocompromised patients (peritonitis, bacteremia, eye and prosthetic joints infections, etc.) and some animals [39,40,41,42,43,44]. Reports regarding sialidase production in Oerskovia species are few [16,33,45,46].
Our previous studies have focused on the isolation, purification, and characterization of sialidase from the Oerskovia paurometabola O129 strain [16,46,47,48]. There, a sialidase production of 31 U/mL was obtained without any optimization [46]. In other laboratory experiments involving the optimization of conditions, enzyme productions of 42 U/mL and 47 U/mL were achieved in Arthrobacter nicotianae and V. cholerae non-O1/13, respectively [15,38]. In this context, the current study aims to systematically optimize the cultivation conditions (pH, temperature, aeration, addition of inducers and surfactants) for O. paurometabola O129 in order to maximize sialidase production. This bacterial species has not been studied in this respect. Moreover, to our knowledge, the use of surfactants to optimize the production of bacterial sialidase has not been performed before. We hypothesize that targeted optimization will significantly enhance enzyme yield, positioning O. paurometabola O129 as a viable, non-pathogenic candidate for industrial sialidase production.

2. Materials and Methods

2.1. Strain and Culture Conditions

In the present study, the Oerskovia paurometabola strain O129 (NBIMCC9093) was used. The strain was identified by 16S RNA gene sequencing and deposited in the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC), Sofia, Bulgaria [16]. Stock cultures were stored at −80 °C by adding 200 μL of 100% sterile glycerol to 800 μL of bacterial culture.
Meat-Peptone Broth (MPB) (HiMedia, Laboratories Pvt. Ltd., Mumbai, India) was used for bacterial growth. Erlenmeyer flasks of 100/20 mL sterile liquid medium (pH 7.0) were inoculated with 2% (v/v) bacterial culture and incubated at 30 °C (200 rpm agitation) for 24 h. All experiments were carried out with a fresh inoculum with optical density (OD) 0.180 at 600 nm. The OD of the samples was measured using a Spectrophotometer UV-VIS 75 (Laborbio, Sofia, Bulgaria).

2.2. Extracellular Sialidase Activity Assay

The sialidase activity was quantitatively assessed using the colorimetric thiobarbituric acid method described by Uchida et al. (1977) [49]. As a substrate for determining enzyme activity, a glucomacropeptide (GMP) isolated from milk whey, a waste product from the dairy industry, was used [50]. One unit of sialidase activity is defined as the amount that releases 1 μmol of sialic acid (Neu5Ac) for 1 min under standard conditions.
Samples of 2 mL were taken and their OD600 was measured. Then they were centrifuged at room temperature, 7000 rpm for 15 min, and the enzyme activity of the supernatant was determined.

2.3. Effect of Different Conditions on Cell Growth and Enzyme Production

Biomass and enzyme production were monitored under the following parameters: inoculum size; temperature, aeration, and pH of the medium; the presence of inducers and surfactants. A single-factor optimization method was chosen [51]. The following baseline values of the individual parameters were chosen for the control samples: 2% inoculum, cultivation at pH 7.0, 30 °C, and 200 rpm agitation on a shaker. The optimization experiments were carried out with variations in a given parameter and the remaining parameters unchanged.

2.3.1. Dynamics of Enzyme Accumulation During Growth Phases

The growth curve and enzyme production of O. paurometabola O129 were monitored in 50 mL culture medium for 72 h. Samples were taken at 4 h intervals during the exponential phase and at 8 h intervals after entering the stationary phase.

2.3.2. Effect of Inoculum Size

To assess whether the inoculum size has an impact on biomass and enzyme production of O. paurometabola O129, Erlenmeyer flasks of 100/20 mL MPB were inoculated with 0.5%, 1%, 2%, 3%, and 4% (v/v) fresh culture.

2.3.3. Effect of Physicochemical Parameters—Temperature, pH, and Aeration

Biomass and sialidase production of O. paurometabola O129 were evaluated at temperature 25 °C, 30 °C, and 35 °C.
To establish the optimal pH for sialidase production, the culture broth was adjusted to different initial pH values (5.0, 6.0, 7.0, 8.0, 9.0) using 1 N HCl or 1 M NaOH.
The influence of aeration on bacterial growth and sialidase production was studied by cultivation under microaerophilic conditions (achieved by adding sterile liquid paraffin after inoculation), static cultivation in a thermostat, and agitation at 100 rpm or 200 rpm on a wrist shaker.

2.3.4. Effect of Inducers and Surfactants

To study the effect of inducers on enzyme synthesis, glucomacropeptide (GMP) [50], horse serum (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) and casamino acids (Oxoid, Thermo Scientific™, Waltham, Massachusetts, US were used. Horse serum and casamino acids were added in concentrations of 0.12%, 0.25%, and 0.5% w/v to the culture medium. GMP was added only in a concentration of 0.12% w/v. Since its role as an inducer at a concentration of 0.5% has been demonstrated in a previous study [46], the aim was to determine the inducing effect at a lower concentration. The best inducer was tested by adding it at 16 h of growth in order to determine the effect of the addition of time.
Since sialidase is an extracellular enzyme, its production was also assessed in the presence of surfactants, which permeabilize the cell membrane. Tween 80 and Triton X-100 (Fluka Chemie GmbH, Buchs, Switzerland) were added separately in concentrations of 0.012%, 0.025%, and 0.05% v/v to the culture medium. Rhamnolipid-biosurfactant (RL) isolated from Pseudomonas sp. PS-17 [52] was added in concentrations of 0.001%, 0.0025%, and 0.005% w/v.

2.3.5. Effect of Combined Optimal Conditions

Once the most favorable values of multiple variables on sialidase production were established, a final experiment was conducted in which they were applied simultaneously. Specifically, the identified most effective inducer and surfactant, GMP 0.12% and Tween 80 0.025%, were applied simultaneously at fermentation parameters that demonstrated maximum individual contributions to sialidase activity: temperature 30 °C, pH 8, shaking speed 200 rpm, inoculum size 2%, and incubation time 28 h. The hypothesis was that the combination of favorable chemical additives under optimized physical conditions would lead to a cumulative or synergistic enhancement of enzyme production. Cultures without inducers or surfactants and those under non-optimized conditions were used as controls.

2.4. Statistical Analysis

The results were evaluated from at least three repeated experiments using three parallel runs, and reported values represent the mean. The error bars indicate the standard error (SE) of the mean triplicate experiments. The data were analyzed using one-way analysis of variants (ANOVA), followed by Tukey’s test.

3. Results

3.1. Dynamics of Enzyme Accumulation During Growth Phases

The obtained results clearly indicated that the highest sialidase activity (35.9 U/mL) of O. paurometabola O129 occurred at 28 h of cultivation, during the latter part of the logarithmic growth phase (Figure 1). It remains relatively constant until the end of the stationary phase, after which it declines in correlation with the reduction in biomass. Based on this observation, 2 mL samples were collected at 28 h of cultivation for all subsequent analyses.

3.2. Effect of Inoculum Size

It has been demonstrated that the inoculum size in the studied range (0.5–4%) does not have a significant impact on biomass accumulation, but it noticeably affects sialidase production (Figure 2a). There was no significant difference in enzyme activity when 0.5 to 2% inoculum is added (47.5–48.2 U/mL). Further increase in inoculum size led to the opposite effect and enzyme secretion diminished to 35.7 U/mL at 4% inoculum.

3.3. Effect of Physicochemical Parameters—Temperature, pH, and Aeration

3.3.1. Effect of Temperature

The dependence of the level of sialidase synthesis by O. paurometabola O129 on the cultivation temperature was observed (Figure 2b). The optimal temperature for sialidase production appeared to be 30 °C (42.8 U/mL). Although at 25 °C there is no significant difference compared to cell growth at 30 °C, enzyme production is much lower (32.1 U/mL). At 35 °C, bacterial growth was very weak, and there was a corresponding loss in enzyme production over 80% (5.7 U/mL) compared to that at the optimal temperature (30 °C).

3.3.2. Effect of pH

The influence of pH on the sialidase production of O. paurometabola O129 was studied in the range 5.0–9.0. At both pH 5.0 and 9.0, cell growth was weak, and, respectively, enzyme production was reduced. Although the strongest growth was observed in cultivation at pH 7.0, the highest sialidase secretion was recorded at pH 8.0 (59.6 U/mL) (Figure 2c).

3.3.3. Aeration

Aeration had a noticeable effect on both biomass accumulation and enzyme production (Figure 2d). Under microaerophilic conditions, there was very weak bacterial growth of O. paurometabola O129, and, respectively, weak sialidase activity (5.6 U/mL) was recorded. Although aeration apparently had a more pronounced beneficial effect on growth than on sialidase activity, increasing the shaking speed during cultivation resulted in greater enzyme production. The level of enzyme activity recorded at 200 rpm (50 U/mL) exceeded those at 100 rpm and under static conditions, which were almost identical.

3.4. Effect of Inducers and Surfactants

In order to increase the production of the enzyme, compounds stimulating sialidase biosynthesis were added. No effect of horse serum on bacterial growth was found, but a weak inducing effect on the sialidase production of O. paurometabola O129 was detected. However, increasing the concentration to 0.5% resulted in a 25% higher production (Figure 3a). Conversely, casamino acids and GMP stimulated both growth and enzyme production. For casamino acids, the effect was well expressed, with no difference in enzyme activity regardless of the concentrations tested (53 U/mL) (Figure 3b). GMP had the most pronounced stimulating effect. Even at a low concentration of 0.12%, bacterial growth was abundant, and an almost two-fold increase in enzyme activity was achieved (64.2 U/mL) (Figure 3c). Therefore, another experiment was conducted to determine the time when the inclusion of GMP into the medium would have an optimal effect on enzyme synthesis. It was added at the same concentration as mentioned above during the logarithmic phase (at the 16 h time point), when the initial production of the enzyme had already begun. The results showed that bacterial growth was affected in a similar way, but the yield increased significantly (85 U/mL) compared to that obtained when adding GMP at the very beginning of the experiment and was two-fold higher compared to the control.
Enzyme production was also tested in the presence of surfactants, which increase the permeability of the cell membrane. Tween 80 did not affect cell growth or enzyme production at a concentration of 0.012%. Although bacterial growth was only slightly attenuated at a higher concentration of 0.025%, enzyme production was enhanced (45.6 U/mL). However, increasing the concentration to 0.05% caused a suppression of the sialidase activity (27.3 U/mL), despite the lack of change in bacterial growth (Figure 4a).
Unlike Tween 80, the other non-ionic surfactant, Triton-X 100, exerted a clear inhibitory effect on both cell growth and sialidase activity. Meager bacterial growth was observed only at the lowest of the tested concentrations; however, some degree of preserved sialidase activity was detected (24.6 U/mL) (Figure 4b).
The enhanced sialidase activity of O. paurometabola O129 was noted in the presence of rhamnolipid, especially at concentrations of 0.0025% and 0.005% (45 U/mL). The biosurfactant did not appear to significantly affect bacterial growth (Figure 4c).

3.5. Effect of Combined Optimal Conditions for Cell Growth and Sialidase Production

Taking into account the results of all the experiments conducted, it was clarified that maximum sialidase production in O. paurometabola O129 was achieved when cultivated under the conditions summarized in Table 1.
Once the beneficial effect of GMP on sialidase production, as well as the effect of Tween 80 on the release of the enzyme into the medium, was established, the next step was to test the joint effect of the two compounds. It was found that an adequate operational strategy in order to obtain the maximum extracellular sialidase consisted of the simultaneous addition of GMP (0.12%) and Tween 80 (0.025%). Their combined effect was studied when they were added to the culture medium at the two time points—at the beginning of the experiment and at 16 h. In both cases, an enhancement in enzyme production was observed compared to the experiments when the compounds were applied separately. A remarkable increase in enzyme production was observed when both substances were added during the exponential phase (98.3 U/mL) (Figure 5).

4. Discussion

There is a continuous search for bacterial isolates capable of optimally producing enzymes for various industrial purposes. At the same time, approximately 99% of soil microorganisms still need to be studied in the laboratory and their biosynthetic potential clarified [53]. Some of the enzymes of microbial origin that have been most extensively studied and have the greatest commercial importance are proteases, amylases, and lipases [53,54,55]. Due to their properties and potential application in various fields, interest in sialidases is growing.
Apart from genetic predisposition, the efficiency of a microorganism as a producer of practically valuable metabolites depends largely on the physical and physicochemical factors of cultivation. By changing them, it is possible to stimulate and control production. Therefore, it is important to pay special attention to the composition of the medium and growth conditions for high yields and the cost-effective production of the desired metabolites [54]. Submerged fermentation (SmF) is considered the best method for cultivating bacteria because they require a high moisture content. It is often used when harvesting secondary metabolites such as enzymes that are secreted into the fermentation broth [56].
Each microbial strain requires specific conditions to achieve maximum enzyme production, including the composition of the nutrient broth, the physicochemical parameters for cultivation, and different additives such as inducers and surfactants [57]. A shorter period for maximal enzyme production is undoubtedly a positive aspect for economically viable production. In order to favor the enzyme secretion by O. paurometabola O129, the influence of some key variables such as culture conditions (i.e., temperature, pH, aeration) and the presence of inducers/surfactants) was assessed.

4.1. Dynamics of Enzyme Accumulation During Growth Phases

The maximum sialidase production in O. paurometabola O129 occurred during the second half of the logarithmic growth phase and remained at almost the same level throughout the stationary phase. This is consistent with findings in some streptococci and in Pseudomonas aeruginosa PAO1 of an increase during the logarithmic phase and a decline during the late stationary phase, which may be a consequence of protease secretion [58,59,60,61]. However, in other bacterial species, a peak in enzyme activity was observed during the middle or late stationary phase: Erysipelothrix rhusiopathiae [6], Pasteurella multocida [7], Vibrio cholerae non-O1/13 [37], Aeromonas sp. 40/02 [50]. A rather rare phenomenon is the maximum production of sialidase during the lag phase and a decrease in enzyme activity upon entering the exponential phase in some Arthrobacter strains [62].

4.2. Effect of Physicochemical Parameters—Temperature, pH, and Aeration

The available data in the literature indicate a wide range of optimal temperatures for sialidase production in different microorganisms. In the psychrophilic fungal strain Penicillium griseofulvum P29, enzyme activity was highest at 15 °C and significantly reduced at 30 °C [63]. In group A streptococci, an interesting temperature dependence of sialidase production was observed—it was highest during the logarithmic phase, but decreased as the cells entered the stationary phase during incubation at 37 °C, while remaining parallel to the growth curve at 22 °C [59]. A similar trend of maximum enzyme production at 37 °C and lower yields at 22 °C and 40 °C was reported in Clostridium tertium [64] and in Pseudomonas aeruginosa PAO1 [61]. Zhang et al. (2011) also reported the strongest cell growth and sialidase activity of a soil bacterium Oerskovia xanthineolytica YZ-2 at 37 °C and a significant decrease at 40 °C [33]. Compared to these data, the huge sialidase production in mesophilic O. paurometabola O129 is favored by a lower temperature (30 °C) and is strongly suppressed when it is raised to 35 °C.
Many studies have focused on the pH optimum of the enzyme, but rarely address the effect of pH on sialidase production. Such research evidenced the highest extracellular sialidase activity at pH 7.0 in Propionibacterium acnes [65] and at pH 8.0 in O. xanthineolytica YZ-2 [33]. In O. paurometabola O129, pH 8.0 was also found to be optimal. Moreover, the sialidase activity at pH 8.0 was maximal compared to all other experiments conducted under equal conditions, without adding an inducer to the culture medium. pH appears to be one of the most important and determining factors for enhancing sialidase production.
Aeration has been manifested to have a contradictory role in obtaining maximum sialidase production in particular producers. Agitation during incubation has been shown to contribute to significantly higher biomass than in static cultures, as well as a 10% increase in sialidase production in the fungal strains P. griseofulvum P29, P. fimorum III 7-1, and Aspergillus niger A 3-2 [63]. Shaker cultivation has been also required for A. nicotianae, C. diphteriae, and P. aeruginosa [5,15,61]. Our results confirmed this trend. Undoubtedly, Oerskovia paurometabola O129 prefers good aeration for higher enzyme activity. Conversely, Eneva et al. (2011) [37] ascertained that the secretion of extracellular Vibrio cholerae non-O1/13 sialidase is most intense at low oxygen concentrations, but only traces are found with cultivation at 100 rpm. They also speculated that the microaerophilic induction of sialidase in clinically relevant vibrios could be viewed as an adaptation to anaerobic conditions in the host gut and function as an instrument of pathogenicity, whereas in the environment, the enzyme has only a trophic function. Other examples of maximal enzyme synthesis under static conditions include Aeromonas sp. strain 40/02, Streptococcus oralis, and Capnocytophaga canimorsus [50,66,67].

4.3. Effect of Inducers and Surfactants

In the present work, several compounds have been added to submerged cultures of O. paurometabola O129 in order to evaluate their ability to stimulate sialidase secretion. Most bacterial sialidases are indicated as inducible enzymes [68,69,70]. Rarely, some bacteria synthesize them constitutively, such as certain streptococci [58] and non-pathogenic representatives of the intestinal microflora [68]. The stimulation of the transcription of certain genes, thereby increasing the production of their corresponding products, is assisted by inducers, which are most often substrates or products of the particular enzyme or their structural analogs. Furthermore, the induction of enzyme biosynthesis can be triggered by the presence of the susceptible chemical bond (in particular case, the α-glycosidic bond). A number of sialoglycoconjugates, aminosugars, free sialic acid, etc., are described as effective sialidase inducers [68]. Other compounds, e.g., transferrin, fetuin, casamino acids, glucomacropeptide, and various animal blood sera, also have an inductive effect [6,50,71]. Sialidase synthesis in O. paurometabola O129 was affected differently in the presence of the tested inducers. The induction of sialidase by the application of horse serum was noticeable, although not particularly strong. Improved growth in various media supplemented with horse serum has been reported for different strains of E. rhusiopathiae [6]. The stimulating effect of the other two inducers was more pronounced. The influence of casamino acids on O. paurometabola O129 was comparable to that observed in E. rhusiopathiae [72] and exceeds that of V. cholerae non-O1/13 [38] and Aeromonas sp. [50]. The most prominent stimulation appeared to be with GMP. This confirms the findings from the E. rhusiopathiae [72], Aeromonas sp. [50], and V. cholerae non-O1/13 [38] sialidases that GMP showed a stronger inducing effect than casamino acids. In order to determine the effect of the addition of time on enzyme production, it is common practice to add the main components of the culture broth, such as inducers or carbon sources, at different time points during the cultivation depending on the authors’ opinion [73]. Furthermore, GMP supplementation during the logarithmic phase at the 16 h time point resulted in an even more pronounced stimulation of sialidase production in O. paurometabola O129. A cumulative effect was observed in both cell growth and enzyme production in this case. This is probably due to the fact that in the mid-logarithmic phase, the bacterial culture already has a built-up biosynthetic potential and mobilized metabolic resources. The use of GMP as an inducer has a certain advantage, since it is obtained from a waste product of the dairy industry. Its production is not associated with high costs and at the same time contributes to reducing environmental pollution [38].
Many studies have reported that the addition of surfactants to the culture medium could improve the production of various extracellular enzymes: amylase, protease, xylanase, cellulase, lipase, phytase [73,74,75,76,77,78]. This phenomenon has been attributed to changes in the permeability of the cell membrane, thus facilitating the passage of secretory enzymes outside the bacterial cell [79]. The integrity and permeability of the outer membrane are important properties for controlling the release of enzymes from the periplasmic space into the culture medium, which can be influenced by operating conditions. Developing of new strategies to increase extracellular secretion are desirable to simplify the downstream process and reduce economic costs [73]. However, surfactants do not always increase enzyme production, and the effect appears to depend on both their type and the strain tested [79]. The sialidase production of O. paurometabola O129 was tested in the presence of two non-ionic surfactants and rhamnolipid-biosurfactant. To our knowledge, the influence of surfactants on the production of sialidase as an extracellular enzyme has not been studied so far. Tween 80 (0.025%) and rhamnolipid (0.0025%) both led to a 20% increase in enzyme activity compared to the control. Both surfactants have been found to stimulate cell growth in bacteria, actinomycetes, and fungi and to enhance the extracellular production of amylase, carboxymethylcellulose enzymes (CMCase), and xylanase; therefore, treatment with Tween 80 and rhamnolipid accelerates the composting process by the faster degradation of cellulose and hemicellulose [80,81]. Rhamnolipids-biosurfactants, produced by microorganisms, have several advantages compared to synthetic surfactants, e.g., biodegradability, high environmental compatibility, strong surface activity, and lower toxicity [82]. Regarding their influence on enzyme production, they can alter cell membrane functions (fluidity, permeability, etc.), which promotes the secretion and release of extracellular enzymes [82].
The role of Tween 80 and Triton X-100 in terms of the impact on enzyme production seems controversial. For example, Tween 80 was proven to have an inducing effect on lipase production of Aspergillus terreus, Candida cylindracea, and Serratia marcescens [73], while it is not beneficial to Yarrowia lipolytica [79]. Some studies reported an improved production of certain enzymes in the presence of Triton X-100 [83,84,85], while others found reduced activity [75]. Triton X-100 introduced at the same concentrations as Tween 80 caused a drastic decrease in the biomass concentration of O. paurometabola O129. Despite the obvious inhibitory effect on the bacterial growth of O. paurometabola O129, the observed, albeit weak, sialidase activity is likely a consequence of the released amounts of enzymes as a result of cell lysis. Due to the ability of surfactants to dissolve the lipids and proteins of the cell membrane, its disruption is possible, provoking partial cell lysis. Tween 80 apparently caused less extreme damage to the cells than Triton X-100, which favors lysis, triggering the release of both membrane and intracellular proteins [75].
Finally, it should be noted that among the microbial producers described in the literature, the ability to synthesize sialidase is unevenly distributed and strain-specific [63]. Moreover, the activity quantitatively varies in a wide range in different microbial producers: M. viridifaciens (1.7 U/mL) [13], Aeromonas sp. 40/02 (7.8 U/mL) [50], P. griseofulvum P29 (14.9 U/mL) [63], A. nicotianae (42 U/mL) [15], and V. cholerae non-O1/13 (47 U/mL) [38]. The highest sialidase activity obtained under optimized conditions in this study was 98.3 U/mL. This exceeds the reported values for lab-scale procedures, indicating the potential of O. paurometabola O129 to be a high-yield sialidase producer. The synergistic combination of GMP and Tween 80 offers a successful approach for optimizing the microbial fermentation process.
One of the main strengths of this study is the use of a non-pathogenic saprophytic bacterium, which provides a safer alternative to traditional sialidase-producing strains. The flask-level optimization is a routine laboratory procedure, but it represents an essential preliminary step to subsequent large-scale studies. Furthermore, the combination of cost-effective inducers (e.g., GMP, obtained from dairy by-products) with simple process modifications represents a scalable strategy for enzyme production. The study also provides a systematic and detailed evaluation of fermentation parameters, offering a solid basis for process optimization.
However, some limitations must be acknowledged. The study was conducted at a laboratory scale and pilot validation is needed to confirm reproducibility and economic feasibility. The molecular mechanisms underlying the regulation of sialidase synthesis—especially in response to GMP and surfactants—also remain unclear.

5. Conclusions

This study provides new information on the optimal conditions for the enhanced extracellular production of sialidase by a strain of the saprophytic species Oerskovia paurometabola. The systematic investigation of key parameters of the fermentation process and the addition of stimulative compounds resulted in elevated levels of enzyme synthesis. Supplementation with glucomacropeptide, especially during the exponential phase, significantly increased sialidase production. This demonstrated its role as an effective inducer and proved that the timing of the addition is essential as well. Surfactants like Tween 80 and rhamnolipid also improved enzyme secretion. A synergistic effect was observed when combining GMP and Tween 80, leading to maximum enzyme yield.
The results provide a basis for a scalable approach for optimizing sialidase production by submerged fermentation and highlight the biotechnological potential of non-pathogenic environmental actinobacteria. They can be considered as new sources of important enzymes, offering the benefit of low biohazard risk. The advantages of using native producers include the simplicity of the production system and the cost-effectiveness.
Future perspectives include the development of bioprocess scale-up strategies, such as bioreactor-based production and fed-batch or continuous fermentation techniques. Further studies may also explore the genetic or metabolic engineering of O. paurometabola to enhance enzyme yield and stability. The purified enzyme could also undergo comprehensive biochemical and structural characterization to evaluate its potential applications in areas such as diagnostics, therapeutics, and glycoengineering. Uncovering the regulatory mechanisms controlling enzyme biosynthesis at the transcriptional and translational levels is also of interest for future research.

Author Contributions

Conceptualization, Y.G. and S.E.; methodology, Y.G., S.E., and R.E.; formal analysis, Y.G., S.E., R.E., I.L., and E.K.; investigation, Y.G., S.E., R.E., I.L., and E.K.; data curation, Y.G. and I.L.; writing—original draft preparation, Y.G., S.E., R.E., and I.L.; writing—review and editing, Y.G., S.E., R.E., I.L., and E.K.; visualization, Y.G. and I.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applmicrobiol 05 00050 g001
Figure 1. Cell growth and extracellular sialidase production in O. paurometabola O129 (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
Figure 1. Cell growth and extracellular sialidase production in O. paurometabola O129 (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
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Figure 2. Effect of the following: (a) inoculum size; (b) temperature; (c) pH; and (d) aeration (MA—microaerophilic conditions) on the cell growth and sialidase production of O. paurometabola O129 (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
Figure 2. Effect of the following: (a) inoculum size; (b) temperature; (c) pH; and (d) aeration (MA—microaerophilic conditions) on the cell growth and sialidase production of O. paurometabola O129 (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
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Figure 3. Sialidase synthesis from O. paurometabola O129 in the presence of inducers: (a) horse serum; (b) casamino acids; and (c) glucomacropeptide (GMP) (“A” means the addition at 16 h) (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
Figure 3. Sialidase synthesis from O. paurometabola O129 in the presence of inducers: (a) horse serum; (b) casamino acids; and (c) glucomacropeptide (GMP) (“A” means the addition at 16 h) (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
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Figure 4. Sialidase synthesis from O. paurometabola O129 in the presence of surfactants: (a) Tween 80; (b) Triton X-100; and (c) rhamnolipid (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
Figure 4. Sialidase synthesis from O. paurometabola O129 in the presence of surfactants: (a) Tween 80; (b) Triton X-100; and (c) rhamnolipid (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
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Figure 5. Sialidase synthesis from O. paurometabola O129 at optimal fermentation conditions, under the combined action of GMP (0.12%) and Tween 80 (0.025%), added at 0 h and 16 h (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
Figure 5. Sialidase synthesis from O. paurometabola O129 at optimal fermentation conditions, under the combined action of GMP (0.12%) and Tween 80 (0.025%), added at 0 h and 16 h (data are expressed as mean ± standard error (SE); significant differences considered p < 0.05).
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Table 1. Optimal conditions for maximal sialidase synthesis by O. paurometabola O129 in submerged fermentation.
Table 1. Optimal conditions for maximal sialidase synthesis by O. paurometabola O129 in submerged fermentation.
ParametersValue
Incubation period (hour)28
Inoculum size (%)2
pH8
Temperature (°C)30
Time point of supplementation of additives (hour)16
Agitation (rpm)200
Tween (%)0.025
GMP (%)0.12
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Gocheva, Y.; Krumova, E.; Lazarkevich, I.; Eneva, R.; Engibarov, S. Enhancing Sialidase Production from the Oerskovia paurometabola O129 Strain by the Optimization of Fermentation Parameters and the Addition of Stimulative Compounds. Appl. Microbiol. 2025, 5, 50. https://doi.org/10.3390/applmicrobiol5020050

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Gocheva Y, Krumova E, Lazarkevich I, Eneva R, Engibarov S. Enhancing Sialidase Production from the Oerskovia paurometabola O129 Strain by the Optimization of Fermentation Parameters and the Addition of Stimulative Compounds. Applied Microbiology. 2025; 5(2):50. https://doi.org/10.3390/applmicrobiol5020050

Chicago/Turabian Style

Gocheva, Yana, Ekaterina Krumova, Irina Lazarkevich, Rumyana Eneva, and Stephan Engibarov. 2025. "Enhancing Sialidase Production from the Oerskovia paurometabola O129 Strain by the Optimization of Fermentation Parameters and the Addition of Stimulative Compounds" Applied Microbiology 5, no. 2: 50. https://doi.org/10.3390/applmicrobiol5020050

APA Style

Gocheva, Y., Krumova, E., Lazarkevich, I., Eneva, R., & Engibarov, S. (2025). Enhancing Sialidase Production from the Oerskovia paurometabola O129 Strain by the Optimization of Fermentation Parameters and the Addition of Stimulative Compounds. Applied Microbiology, 5(2), 50. https://doi.org/10.3390/applmicrobiol5020050

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