Partitioning purification, biochemical characterization, and milk coagulation efficiency of protease from a newly Streptomyces sp. isolate

Abstract

An actinobacteria strain was isolated from an olive waste mill and tested for protease production on skimmed milk media. The strain identification was achieved through both 16 S rDNA sequencing and phenotypic characterization. The enzyme was purified using the ammonium sulfate/t-butanol three-phase partitioning (TPP) method, followed by characterization to investigate the effect of pH, temperature, and various chemical agents. Subsequently, the enzyme was assessed for its milk coagulation activity. The strain belonging to the Streptomyces genera, exhibits significant phylogenetic and phenotypic differences from the aligned species, suggesting its novelty as a new strain. The enzyme was best separated in the TPP aqueous phase with a 5.35 fold and 56.25% yield. Optimal activity was observed at pH 9.0 and 60 °C, with more than half of the activity retained within the pH range of 7–10 over one hour. The protease demonstrated complete stability between 30 and 60 °C. While metallic ions enhanced enzyme activity, EDTA acted as an inhibitor. The enzyme displayed resistance to H₂O_2, SDS, Tween 80, and Triton X-100. Notably, it was activated in organic solvents (ethyl acetate, petroleum ether, and xylene), maintaining > 75% of its original activity in butanol, ethanol, and methanol. Additionally, the enzyme yielded high milk coagulant activity of 11,478 SU/mL. The new Streptomyces sp. protease revealed high activity and stability under a wide range of biochemical conditions. Its use in the dairy industry appears particularly promising. Further industrial process investigations will be valuable in determining potential uses for this enzyme.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-024-01386-y.

Introduction

Proteases, also known as proteinases, are enzymes responsible for protein hydrolysis into smaller polypeptides and amino acids [1]. They are produced by all living organisms, including animals, plants, and microbes, where they serve critical roles in various life cycle processes. However, owing to their diverse catalytic mechanisms, high specificity, stability, and ease of handling and production, microbial proteases are the preferred hydrolytic enzymes in industry, commanding a two-thirds share of the worldwide market for commercial proteases [1, 2]. These enzymes find extensive application in mediating various industrial transformations, playing pivotal roles across multiple sectors, including food, detergents, pharmaceutical, leather, paper, and chemical industries [3, 4]. Given that each catalytic process necessitates specific physicochemical conditions, proteases must exhibit suitable biocatalytic characteristics, especially activity spectra across different pH ranges and temperatures, stability, chemical resistance, and substrate specificity [3, 5–7]. Consequently, proteases with exceptional stability in diverse environments have become the focus of substantial investigations, aiming to meet the evolving demands of various industrial applications [8]. Presently, industrial demand for these enzymes continues to surge, constituting 40% of total enzyme sales [3, 8]. Therefore, research of novel proteases capable of adaptation to different industrial processes is imperative to address this substantial market demand. One of the most effective strategies is screening for novel microorganisms producing proteases with desirable characteristics.

Actinobacteria are gram-positive bacteria with a high G + C%. It is recognized as a reservoir for a large number of chemically different metabolites [9, 10]. It contributes significantly to the decomposition of organic polymers in various natural habitats through its enzymatic activity; therefore, bioprospecting and characterization of new strains are frequently requested for the discovery of new natural products, especially industrially important biocatalysts such as hydrolytic enzymes (amylases, cellulose, lipases, proteases, etc.) [10, 11]. The isolation and screening of actinobacteria from agro-industrial waste could open up new opportunities for the discovery of interesting hydrolytic extracellular enzymes.

Among these bacteria, the Streptomyces genus requires special attention as it is the most prevalent genus of actinobacteria. This predominance is attributed to its unique metabolism, which makes it a prime candidate for exploration as a valuable biosource of extracellular enzymes of industrial interest [12–15]. Studies carried out on proteases from Streptomyces strains have demonstrated high yields and interesting, applicable characteristics. Extracellular thermostable alkaline proteases with the ability to hydrolyze many substrates (casein, gelatin, albumin, keratin, hemoglobin, and fibrin) were produced from Streptomyces sp. and were highly promising for several applications, such as the detergent, leather industry, waste recycling, pharmaceuticals, biocontrol, etc. Due to these promising capacities, the screening of new Streptomyces strains is continually investigated; new proteases that possess height stability in various industrial processes are frequently described [14, 16–19].

This study aims to characterize a protease from a newly identified Streptomyces sp. and assess its efficiency and stability in various industrial processes. Herein, milk coagulation activity is showcased as an example of its potential application.

Materials and methods

Chemicals

All chemicals and solvents were of analytical quality; they were purchased from the following: EDTA, SDS, Triton x100, Tween 80, t-butanol, acetone, butanol, ethanol, ethyl acetate, methanol, petroleum ether, and xylene from ProLabo (Paris, France); CaCl₂, CoCl₂, CuCl₂, FeCl₂, NaCl, MgCl₂, MnCl₂, ZnCl₂, and ZnSO₄ from Biochem Chemopharma (Cosne sur Loire, France); citric acid, glycine, Na₂HPO₄, KH₂PO₄, NaOH, (NH₄)₂SO₄, casein, hemoglobin, gelatin, gluten, and ovalbumin from Sigma Chemical (St. Louis, USA).

Isolation and identification of the producing strain

The actinobacteria strain (coded PO3) was isolated from olive pomace using the decimal dilution technique and subsequently tested for protease production on a medium containing (%): skim milk 10, agar 15. Phylogenetic identification was established through 16 S rDNA sequencing. DNA extraction was performed using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies Sdn Bhd, Selangor, DE, Malaysia). PCR amplification was conducted using with primers 27 F: ‘5-AGA GTT TGA TCC TGG CTC AG-3’ and 1492R:‘5’-CCG TCA ATT CCT TTG AGT-3‘ [20]. Sequences were analyzed using a 3130 Genetic Analyzer Capillary Array (Applied Biosystems) and compared against NCBI Blat (http://blast.ncbi.nlm.nih.gov) for identification. The phylogenetic tree was constructed using Mega 11 (https://www.megasoftware.net/). The phenotypic characterization was performed by assessing cultural, morphological, and physiological characteristics on ISP₂ medium [21].

Protease production

A 24-hour pre-culture of the actinobacteria strain was prepared in ISP₁ broth [21], serving as the inoculums for a protease production medium optimized previously. This medium comprised (% w/v): wheat bran (25), NaCl (0.4), ZnSO₄.7H₂O (0.007), and CaCl₂ (0.015), adjusted to pH 9.0. The fermentation process was conducted under submerged conditions at 40 °C for 4 days. Following fermentation, the product was separated via centrifugation at 5000 x g and 4 °C for 30 min, yielding the crude enzyme extract.

Protease assay

Protease activity was determined according to Tsuchida et al. [22], with 1% casein as a substrate. The protease activity unit was defined as the amount of enzyme that released 1 mg of tyrosine per mL per minute under the assay condition.

Protease purification

The proteolytic enzyme was purified using the three-phase partitioning (TPP) method [23], by varying one parameter at a time: pH (ranging from 6 to 12), ammonium sulfate precipitation (from 20 to 80%), and t-butanol: crude extract ratio (from 0.25:1.0 to 1.0:2.0). The pH of the crude enzyme extract was adjusted to 9.0 using 0.5 M NaOH, followed by saturation at 25 °C with 60% (w/v) ammonium sulfate. Subsequently, t-butanol was added at a ratio of 1.5:1.0 (v/v), and the mixture was gently shaken every 10 min for one hour at 25 °C. Afterward, the mixture underwent centrifugation at 5000 x g for 10 min at 4 °C, resulting in the separation of three phases. The upper t-butanol phase was removed, and the aqueous and interfacial phases were carefully separated. The interfacial phase was dissolved in Glycine-NaOH buffer at pH 9.0. Protein content in the aqueous and interfacial phases was determined using the Lowry method [24]. Subsequently, purification parameters were calculated as follows:

Effects of pH and temperature

Protease activity was determined in various buffer solutions at different pH values: citric acid-Na₂HPO₄ (pH 4, 5, and 6), KH₂PO₄-Na₂HPO₄ (pH 7 and 8), Glycine-NaOH (pH 9 and 10), Na₂HPO₄-NaOH (pH 11 and 12). Additionally, enzymatic activity was examined over a wide range of temperatures (30, 40, 50, 60, 70, 80, 90, and 100 °C). Furthermore, protease stability was evaluated by incubating it for 1 h in different buffers and temperatures.

Inhibitory effect of chemical agents on enzyme activity

The enzyme was pre-incubated for 1 h with various chemical agents; 1 mM of metal ions (CaCl₂, CoCl₂, CuCl₂, FeCl₂, NaCl, MgCl₂, MnCl₂, and ZnCl₂); 10 mM of EDTA and SDS; 1% of Tween 80, Triton X-100, and H₂O₂; 50% of acetone, butanol, ethanol, ethyl acetate, methanol, petroleum ether, and xylene. Following incubation, the enzymatic reaction is considered complete. The negative control was prepared with an untreated enzyme.

Hydrolytic effect of protease on various substrates

The enzymatic reaction was carried out using various protein substrates at 1%: casein, gelatin, gluten, hemoglobin, and ovalbumin.

Milk-coagulation activity

The milk-coagulating activity was determined by the method of Arima et al. [25]. A 10% skimmed milk solution was prepared in sterile distilled water containing 0.01 M CaCl₂, pH adjusted at 6, and left overnight at 4 °C. The milk was placed in 5 mL tubes, pre-incubated for 10 min at 35 °C, and then 0.25 mL of the partially purified enzyme was added by twisting the tube. The coagulation time in seconds is noted as soon as the first milk clots appear. Milk coagulation activity (MCA) is defined in Soxhlet units (SU) and calculated by the equation:

T: coagulation time; D: enzyme dilution.

Statistical analysis

Data were expressed as means ± standard deviation (SD).A one-way analysis of variance (ANOVA) test was used to evaluate the effect of pH, temperature, chemical agents, and substrates on protease activity and stability. When the test was significant, a pairwise comparison was conducted using the Student-Newman-Keuls (SNK) post hoc test. The significant level was considered to be 0.05.

Results

Strain identification

A partial 16 S rDNA sequence of 1082 bp length was obtained from the isolate (Genbank accession number OQ726251); the top hit sequence similarity found by NCBI Blast was with Streptomyces griseorubens NBRC 12780T at 100%. The Neighbour-Joining phylogenetic tree constructed with the 16 S rDNA sequence of Streptomyces sp. PO3 and the type strains with more than 99% sequence similarity are shown in Fig. 1. The phenotypic characterization shows that the strain differs from its approach species by the following phenotypic characteristics: It has a well-developed substrate and aerial mycelium without fragmentation or sporangia appearance; aerial mycelium is white, then changes to pale gray during development, holding 25–30 rectiflexible spore chains; optimum growth occurs at pH 9.0 and 40 °C; and it is resistant to 11% NaCl.

Fig. 1.

Phylogenetic tree of Streptomyces sp. PO3 and type strains with more than 99% sequence similarity, constructed by the neighbor-joining method with 1000 bootstrap replication

Protease purification

The protease from Streptomyces sp. PO3 had the highest recovery in the aqueous phase TPP, with enhanced activity at 56.25% and 5.35 fold; there is also a reserved yield in the interfacial phase TPP (75%); however, specific activity is low; the purification profile is summarized in Table 1.

Table 1.

Purification parameters using the TPP method

	Total activity (U/mL)	Total protein (mg/mL)	Specific activity (U/mg)	Fold	Yield (%)
Crude extract	0.16	0.19	0.84	1	100
Interfacial phase TPP	0.12	0.16	0.75	0.89	75
Aqueous phase TPP	0.09	0.02	4.5	5.35	56.25

Optimum pH, temperature, and stability

The protease from Streptomyces sp. PO3 displayed > 77% activity at all pHs tested; the maximum is between pH 7.0 and 10. However, there is no statistically significant difference in the effect of the tested pH, according to one-way ANOVA analysis (Supplementary Table S1). The stability analysis demonstrates that the enzyme maintains > 50% of its activity for an hour within the range of pH 7 to 10. The ANOVA analysis revealed a significant effect (p < 0.001); and the SNK post hoc test showed that pH 9.0 providing the highest stability (Fig. 2a; Supplementary Table S2). The activity of the enzyme increases with temperature from 30 to 70 °C, then reduces gradually. Similarly, there were no significant differences between the tested temperatures. The enzyme remains completely operational for an hour in the range of 30–60 °C, and with a high level of activity (> 80%) until 100 °C (Fig. 2b). ANOVA analysis revealed a significant effect (p = 0.027) and the SNK post hoc test showed that the temperature of 60 °C provided the highest stability (Supplementary Tables S3 and S4).

Fig. 2.

Protease activity and stability at different pHs (a) and temperatures (b)

Effects of metal ions, surfactants, and organic solvents

For the tested chemical agents, the one-way ANOVA analysis revealed a highly significant effect (p < 0.001), and the SNK post hoc test showed that CuSO₄ provided the highest protease activity (Supplementary Tables S5 and S6). Among the metal ions tested, the majority (Ca²⁺, Cu²⁺, Fe²⁺, Hg⁺, Mg²⁺, Mn²⁺, and Na⁺) were found to increase protease activity (between 100 and 130%), while only Zn²⁺ inhibited the activity lightly (< 6%); furthermore, the metal chelator EDTA decreased activity by > 10%. After an hour of incubation, the enzyme demonstrated good resistance to 1% H₂O₂ and Triton X-100. Nevertheless, significant activity (> 80%) was maintained with Tween 80. In the presence of SDS, the activity decreased slightly (< 10%). Protease was more active (> 112%) in the presence of acetone, ethyl acetate, xylene, and petroleum ether and retained > 75% of its original activity with butanol, ethanol, and methanol (Table 2).

Table 2.

Effect of different chemical agents on protease activity

Effector	Concentration (mM)	Relative activity (%)*	Effector	Concentration (%)	Relative activity (%)*
CaCO3	1	121.13 ± 0.07	H2O2	1	109.88 ± 0.01
CuSO4	1	129.69 ± 0.09	Triton X-100	1	105.18 ± 11.53
FeCl2	1	108.01 ± 4.92	Tween 80	1	82.53 ± 4.76
HgSO4	1	102.91 ± 0.01	Acetone	50	119.87 ± 7.49
MgSO4	1	102.19 ± 6.74	Butanol	50	94.45 ± 5.36
MnSO4	1	106.47 ± 8.65	Ethanol	50	76.52 ± 0.02
NaCl	1	110.20 ± 0.01	Ethyl acetate	50	114.79 ± 6.28
ZnSO4	1	93.62 ± 5.46	Methanol	50	80.59 ± 0.01
EDTA	10	88.65 ± 1.12	Petroleum ether	50	109.61 ± 4.07
SDS	10	90.29 ± 5.35	Xylene	50	112.01 ± 0.01

* Relative activity was calculated with respect to the untreated enzyme

Substrate specificity

Various natural protein substrates were tested with the enzyme. The results showed a significant difference between the tested substrates (p = 0.028; Supplementary Table S7). The SNK post hoc test revealed that the enzyme had the most hydrolytic effect on casein; similar effects were obtained with hemoglobin, gelatin, and ovalbumin, while the least effect was on gluten (Fig. 3; Supplementary Table S8).

Fig. 3.

Effect of protease on different substrates

Milk coagulation activity

The coagulation time necessary for the appearance of the first milk clots is 23 s. The milk clotting activity of Streptomyces sp. PO₃ protease is 11,478 SU/mL.

Discussion

The constant demand for innovative industrial products drives the ongoing search for novel enzymes. Keeping this in mind, a new actinobacteria strain was isolated from agro-industrial waste and evaluated for its ability to produce proteolytic enzymes. Phylogenetic analysis revealed that the isolate PO3 formed a distinct lineage within the same clade as Streptomyces labedae NBRC 15864T, S. erythrogriseus NBRC 14601T, S. griseoincarnatus LMG 19316T, S. variabilis NBRC 12825T (42% bootstrap support), and S. griseorubens NBRC 12780T (55% bootstrap support). Phenotypic characterization over 21-day growth period on ISP₂ medium demonstrated that the strain displays typical Streptomyces traits but differs from its five closest phylogenetic relatives in certain phenotypic characteristics, when compared with the literature [9, 26]. This phylogenetic and phenotypic divergence leads us to classify the PO3 isolate as a novel strain within the genus Streptomyces.

The proteolytic Streptomyces PO3 enzyme was efficiently purified within a short timeframe using the TPP method in the aqueous phase. This method relies on the salting-out mechanism in the presence of an organic solvent. There are three main factors involved in the separation: pH, ammonium sulfate concentration, and t-butanol volume. Below the isoelectric pH of protein, the cationic charges form a bond with the sulfate ions, giving them an overall positive charge and enforcing the salting-out mechanism [27]. Sulfate ion binding reinforces enzyme conformation and, at higher salt levels, improves the hydrophobic interactions between proteins, leading them to precipitate [27, 28]. t-butanol is a ramified alcohol that does not bind to protein molecules with rearranged structures and does not cause protein denaturation [29]. It also allows the removal of organic solvent-soluble contaminants in the fractionation process [27]. Several microbial enzymes have previously been efficiently separated in the interfacial or aqueous phase by the TPP method, such as protease, cellulose, lipase, and laccase [27, 29–31]. Streptomyces proteases generally require several purification steps (ultrafiltration, ammonium sulfate precipitation, and chromatography), which are time-consuming and costly methods, as in the case of proteases from Streptomyces sp. M30, S. rutgersensis SCSIO 11,720, S. koyangensis TN650, and Streptomyces sp. 2M21; furthermore, the purification yields were lower than our study (15.5, 19, 35, and 36.1%, respectively) [13, 18, 32, 33].

pH and temperature are important parameters in regulating enzyme-driven reactions. The produced PO3 protease from, Streptomyces is alkaline, which is in line with the trend seen in the majority of Streptomyces proteases, known to lean towards neutrality or alkalineity with optimal pH resembling our observed value (pH 9.0). Similar optimal pH was reported with S. fungicidicus [16], Streptomyces sp. M30 [18], and Streptomyces sp. 2M21 [32]. The notable effectiveness of the PO3 isolate protease under alkaline conditions is an extremely important feature given the potential of future commercial applications, particularly in detergent formulations typically operating within pH ranges around 7–11 [3]. Furthermore, this protease demonstrates resilience across a wide pH range (4–12), underscoring its stability. This particularity renders it suitable for a multitude of industrial uses.

Hence, the protease under investigation showed excellent thermostability, another highly desirable feature in industrial applications. The enzyme’s ability to withstand thermal inactivation makes it particularly attractive for various industrial processes. Throughout the literature, thermotolerant proteases produced by several Streptomyces species have been effective in diverse applications such as detergent formulaions [16] and medical treatments [18]. Likewise, proteases from Streptomyces parvulus DPUA 1573, Streptomyces sp. CS684, and S. koyangensis TN650 have an optimum temperature of 40 ºC and exhibit thermostability in the temperature range of 30–60 °C [13–15].

The determination of enzyme structure often involves analyzing its response to metal ions and inhibitors. Enhanced activity in the presence of certain metal ions, along with inactivation by chelators such as EDTA, indicates the involvement of a metal cofactor at the enzyme’s active site [6], classifying it as a metalloproteases. The presence of metal ions can increase enzyme stability; for instance, Ca²⁺ and several divalent ions have been shown to increase the thermostability and preserve the three-dimensional active conformation of microbial proteases [3, 6, 18, 32–34]. However, the presence of Zn²⁺ at alkaline pH can lead to the formation of Zn (OH)₂, altering the medium’s structure and reducing enzyme activity [32]. Previous studies have reported the inhibitory effect of Zn²⁺ on microbial alkaline proteases [7, 34]. Proteases from Streptomyces parvulus DPUA 1573, S. koyangensis TN650, Streptomyces sp. 2M21, and Streptomyces sp. M30 were partially inhibited by Zn²⁺ [13, 15, 18, 32], as discovered in this work.

The investigation of enzyme activity in the presence of oxidants, ionic, and non-ionic surfactants aims to verify its stability during industrial processes. Non-anionic surfactants, by reducing the surface tension of the enzyme’s reaction medium, enhance its resistance to oxidation [27]. The reduction in enzyme activity observed with SDS indicates that the enzyme’s active conformation relies, in part, on non-covalent interactions. Microbial proteases are usually less stable in anionic surfactants [27, 34, 35]. The enhanced activity in the presence of surfactants suggested that the enzyme is adaptable for detergent applications [3, 7, 34]. The protease activity from Streptomyces sp. PO3 remains completely active by adding Triton X100 and H₂O₂, and is slightly inactivated by SDS (9.91%) and Tween 80 (19.47%). Such high resistance is also obtained with Streptomyces parvulus DPUA 1573 and Streptomyces sp. M30 [15, 18].

Organic solvent-stable enzymes are more suitable for a wide range of industrial applications, especially in non-aqueous media, where they favor biocatalysis by increasing the hydrophobic substrate’s solubility, mainly for the production of peptides and esters [3, 5]. Stability in the presence of organic solvents was observed with Streptomyces koyangensis TN650 and Streptomyces sp. 2M21 [13, 32]. In contrast, activity can be inhibited by organic solvents like Streptomyces sp. M30 protease [18].

The enzyme can be applied on different protein substrates; this diverse specificity proves its potential applications in various industries, especially detergents, medicine, and food. Previous research found similar Streptomyces protease effects; Streptomyces sp. 2M21, Streptomyces sp. M30, and S. rutgersensis SCSIO 11,720 showed the most notable activity on casein [18, 32, 33].

The ongoing demand for cheese production necessitates the continuous use of calf rennet for coagulation. However, economic and environmental constraints limit its long-term viability in satisfying commercial needs. As a potential alternative, microbial milk coagulation proteases are being investigated. While fungal acid proteases have been reported as effective enzymes for milk coagulation [17, 36, 37], recent investigation on bacterial proteases has demonstrated even better coagulant activity. This superiority is mainly due to their thermotolerance, wide pH range of activity, and stability, as in the case of Bacillus subtilis (natto) [38], Paenibacillus spp. BD3526 [39], B. subtilis SMDFS 2B [40], and B. subtilis MK775302 [41], of which MCA ranges between 600 and 6500 SU/mL. In the present study, Streptomyces sp. PO₃ protease have a high milk clotting activity compared with literature, which makes them suitable for cheese manufacturing.

Conclusion

The new Streptomyces sp. PO3 isolate is a bioresource for an important proteolytic enzyme. In effect, the biochemical characterization approved that the protease is operational and has shown high stability over 1 h over a wide range of pH (4–12) and temperature (30–100 °C), and is still active with oxidants, surfactants, and organic solvents. The enzyme can also catalyze the hydrolysis of various protein substrates. These properties are attractive and allow for broad application in a variety of industrial processes, including detergents, pharmaceuticals, and foods. Notably, the pronounced milk-clotting activity (11,478 SU/mL) of this enzyme merits further investigation in cheesemaking.

Electronic supplementary material

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Author contributions

Methodology and data analysis were performed by Abdelouahab Mebarki and Oussama Sinacer. The draft preparation was written by Faiza Boughachiche, Kounouz Rachedi, and Amel Ait Kaki. All authors read and approved the final manuscript.

Funding

The authors received no specific funding for this work.

Declarations

Ethical approval

Ethics is not necessary for this investigation.

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

Authors are responsible for correctness of the statements provided in the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.