Dual Esterase and Protease Activities of Bacillus subtilis TIM27 Subtilisin E and Biosurfactant for Advanced Laundry Detergents

Abstract

This study investigated the purification and characterization of an extracellular protease (ProT27) and a lipopeptide biosurfactant (BiosT27) from Bacillus subtilis TIM27 for their potential use as laundry detergent additives. The crude ProT27 exhibited both protease and esterase activities, primarily due to the presence of a 39.4 kDa alkaline subtilisin E identified by mass spectrometry. ProT27 demonstrated its highest proteolytic activity at pH 8 and 50 °C, conditions similar to typical laundry processes, and showed stability in the presence of the commercial detergent, underscoring its potential as a laundry additive. BiosT27 was produced at 673 mg L^-1, with a 66.6% emulsion index and the ability to reduce water surface tension to 25.3 mN m^-1, maintaining its performance at pH 11 and 2 M sodium chloride (NaCl). When used as laundry additives, ProT27 and BiosT27 showed cleaning performance comparable to commercial detergent in removing egg yolk stains from cotton fabric. The addition of crude ProT27 to heated commercial detergent enhanced stain removal, outperforming the endogenous enzymes of the detergent, highlighting the significant potential of these biomolecules in detergent formulations.

Keywords:
subtilisin; lipopeptide; surfactin; lipolytic; tributyrin

Introduction

The increasing demand of modern society for biofriendly cleaning products has motivated the search for natural additives that are biodegradable and environmentally safe.¹ One example is the use of enzymes, which are used in several industrial sectors in the replacement of hydrolysis and oxidation reactions, which are mainly accomplished using unspecific chemicals and produce vast volumes of waste. In fact, microbial hydrolases are already found in many detergents in the supermarket shelves, facilitating the removal of tissue and surface stains.

Hydrolases, which include proteases and esterases, are enzymes demanded not only by the detergent industry, but also by the food, biodiesel, cosmetics and beverages, among others.² Proteases account for the largest share of sales of industrial enzymes in the world (60% of the global market). Compared to proteases from animals and plants, microbial proteases have a greater commercial interest due to their great production capacity, stability, and catalytic activity.³ Esterases are enzymes that target ester bonds in water-soluble substances. They exhibit high specificity, typically do not require cofactors, and are stable in organic solvents.⁴ Inclusion of proteases and esterases in detergent formulations improves their efficiency, reduces energy consumption and chemical usage, minimizes toxic by-products, and decreases environmental pollution.⁵

Building on the idea of producing a new generation of eco-friendly cleaning products, replacement of synthetic surfactants with biosurfactants is proposed alongside the use of microbial enzymes. Most commercial detergents today still rely on non-biodegradable synthetic surfactants derived from petroleum. In contrast, microbial surfactants offer not only excellent surface activity but also key advantages such as low toxicity, high biodegradability, and biocompatibility.⁶

In addition to being excellent producers of biosurfactants,⁷ Bacillus spp. have the largest share of industrial enzyme production on the current market.⁸ Subtilisins (EC 3.4.21.62) are extracellular proteases produced by various microorganisms and belong to the second largest family of serine proteases. Its name derives from Bacillus subtilis, from which the enzyme was isolated for the first time.⁹ Commercially, alkaline proteases, including subtilisin, with optimal activity at alkaline pH values (8-10), are ideal for applications as additives in commercial detergents due to their stability in this pH value range and in the presence of surfactants and oxidizing agents present in these products.¹⁰

In this context, this study aimed to evaluate the production and characterization of an enzyme (named ProT27), obtained from the ammonium sulfate enriched fraction (F40-70%) and lipopeptide biosurfactants (BiosT27) produced by a new strain of Bacillus subtilis TIM27, isolated from mangrove sediments. Given the stability under extreme washing conditions, including highly alkaline pH and elevated temperatures, ProT27 and BiosT27 demonstrate strong potential as additives in laundry detergent formulations, enhancing their cleaning efficiency and performance.

Experimental

Source of the strain Bacillus subtilis TIM27 and screening for proteases production

The strain Bacillus subtilis TIM27 was previously isolated by Farias et al.¹¹ from mangrove sediments from Timonha in Ceará state, Brazil, according to the relevant environmental agency (A982E7D-SisGen). TIM27 belongs to the collection of bacteria from the Laboratory of Microbial Ecology and Biotechnology (Lembiotech) of the Federal University of Ceará (UFC), Brazil.

The culture stored at −80 °C was reactivated in nutrient broth (composition per liter: 5 g of peptone; 5 g of sodium chloride; 1.5 g of meat extract and 1.5 g of yeast extract) and incubated (Tecnal^® incubator, model TE-420) at 30 °C, 150 rpm for 16 h. Then, it was inoculated on nutrient agar containing 0.1% tributyrin or 1% skim milk to detect esterase and protease activity, respectively. The plates were incubated at 30 °C for 24 h and the formation of hydrolytic zones around the colonies was monitored.

Enzyme production and purification

The enzyme production was carried out by inoculating a 24-h TIM27 culture (5% v/v), with cell density adjusted to 1 at 600 nm (Thermo Fischer Scientific, model Genesys™ 6, Madison, WI, United States of America) in 2 L flasks with 400 mL of nutrient broth supplemented with 0.2% glycerol. The flasks were incubated at 150 rpm for 48 h at 30 °C. Subsequently, the biomass was removed by centrifugation at 8,000 g, 4 °C for 10 min and the supernatant was subjected to precipitation of ammonium sulfate in three fractions (F0-40%; F40-70% and F70-90%) at 10 °C. The resulting precipitate in each fraction was dissolved and dialyzed against distilled water. Enzymatic activities were evaluated on agar plates as previously reported and protein content was determined according to the Bradford method,¹² using bovine serum albumin (BSA) as standard.

Production and purification of biosurfactants (BiosT27)

The production of biosurfactants was carried out by inoculating TIM27 in production medium described by Morán et al.¹³ with modifications, containing the following composition per liter: glucose, 10 g; yeast extract, 5 g; NaCl, 27 g; (NH₄)₂SO₄, 1 g; Na₂HPO₄, 6 g; KH₂PO₄, 3 g; and MgSO₄·7H₂O, 0.6 g. The medium was adjusted to pH 7 and autoclaved at 110 °C for 15 min. After that, it was supplemented with 0.1% (v/v) of a micronutrients solution containing 10.95 g of ZnSO₄·7H₂O; 5 g of FeSO₄·7H₂O; 1.54 g of MnSO₄·H₂O; 0.39 g of CuSO₄·5H₂O; 0.25 g of Co(NO₃)₂·6H₂O; and 0.17 g of Na₂B₄O₇·10H₂O per liter of water, and sterilized by 0.22 µm membrane filtration (Millipore, United States of America). The production medium was inoculated with 10% of a 24-h TIM27 culture with cell density adjusted to 0.1 at 600 nm and incubated with continuous shaking at 150 rpm, at 30 °C for 48 h. Subsequently, the culture was centrifuged at 7,000 g for 15 min, 4 °C. The cell-free supernatant was subjected to acid precipitation according to the methodology proposed by Pereira et al.¹⁴ For that, the supernatant was adjusted to pH 2 with HCl 12 M and incubated for 24 h at 4 °C. The precipitate was collected by centrifugation at 15,000 g, 4 °C for 30 min and washed twice with acidified distilled water (pH 2), under centrifugation at the same conditions. The precipitate was dissolved in sufficient demineralized water and the pH was adjusted to 7 with 1 M NaOH. This concentrated solution of biosurfactants was then lyophilized (Analytical, Christ alpha 1-2 LD plus). The composition of biosurfactants was analyzed by Farias et al.¹¹ using a high-performance liquid chromatography (HPLC) system (Agilent Technologies 1290 series Infinity System LC, Santa Clara, United States of America) coupled to a quadrupole time-of-flight (Q-ToF) iFunnel (Agilent Technologies 6550) mass spectrometry fitted with an electrospray ionization (ESI) source for liquid chromatography-mass spectrometry (LC-MS) analysis.

Emulsification index (E24) and surface tension assessment

Emulsification activity was determined according to Iqbal et al.¹⁵ by adding 2 mL of kerosene to the same volume of biosurfactant solutions at different concentrations (50, 100, 200, 300, 400, and 500 ppm) in glass test tubes in triplicate. The tubes were mixed using a vortex at high speed for 2 min and the mixture was left to settle for 24 h. After that, the emulsification index (E24) was calculated by dividing the height of the emulsified layer by the total height of the liquid column and then multiplying by 100 to express it as a percentage. The aspects of the emulsions produced were evaluated under an optical microscope with 10× magnification.

The surface tension was measured using Krüss K6 Tensiometer (Krüss GmbH, Hamburg, Germany) equipped with a 1.9-cm Du Noüy platinum ring. Measurements were performed at 25 °C using a biosurfactant solution at 50 ppm. Ultrapure water was used to calibrate the tensiometer. The experiments were carried out in triplicate and were repeated three times.

Effects of pH and NaCl on biosurfactant activities

To evaluate the effect of different pH levels on surface tension, 300-ppm solutions of biosurfactants were adjusted to pH 7, 8, 9, 10, and 11 and tested. The pH adjustments were made using 50 mM sodium phosphate buffer (pH 7), 50 mM Tris-HCl (pH 8), 50 mM sodium borate (pH 9), and 50 mM glycine-NaOH for pH 10 and 11.

To assess the impact of NaCl on the emulsification activity, 300-ppm solutions of the biosurfactant were prepared with varying NaCl concentrations (0.5, 1.0, 2.0, 3.0, and 4.0 M) and tested as previously described.

Purification of proteases the fraction F40-70%

The purification of proteases was performed with the help of the AKTA protein purification system (GE Healthcare, United Kingdom). First, 7 mg of the fraction 40-70% (F40-70%) was loaded onto the 1 mL HiTrap Q HP anion exchange column (GE Healthcare, United Kingdom) previously equilibrated with 50 mM glycine-NaOH pH 9.6. The bounded proteins were then eluted with a stepwise gradient of NaCl (200, 400, 600, 800, 1,000 mM) at a flow rate of 1 mL per min with a column volume of 7 times for each concentration. Tubes containing fractions exhibiting protease and lipase activities were loaded into a HiTrap Phenyl HP 5 mL column (GE Healthcare, United Kingdom) previously equilibrated with 50 mM sodium phosphate buffer, pH 7, supplemented with 1 M ammonium sulfate. Bonded proteins were eluted with a linear and decreasing gradient of 1-0 M ammonium sulfate at a flow rate of 5 mL per min. After that, the volume of each peak was dialyzed against ultrapure water and the enzymatic activities were verified. The samples were also analyzed by Tricine-SDS-PAGE¹⁶ stained with silver nitrate¹⁷ and Coomassie Blue G-250.¹⁸ The bands of interest were excised from the gel, digested with trypsin (15 µg mL ^-1 prepared in 25 nM NH₄CO₃ buffer, pH 8), and analyzed by mass spectrometry.¹⁹

Protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Tryptic peptides were separated in a reverse phase BEH300 C18 column (100 μm × 100 mm) using the nanoAcquity™ (Waters) system and eluted (600 mL min^-1) with an acetonitrile gradient (5-85%) supplemented with 0.1% formic acid. The eluates were analyzed on a hybrid mass spectrometer (LC-MS/MS) (Synapt HDMS, Waters Corp. MA, United States of America). The mass spectrometer was operated in positive mode using a source temperature of 90 °C and capillary voltage of 3.5 kV. The instrument was calibrated with phosphoric acid clusters, and the Lock mass used during the acquisition was the m/z 686.8461 ion. The LC-MS/MS procedure was performed according to the data-dependent acquisition (DDA) method, selecting MS/MS doubly to triply charged precursor ions. Ions were fragmented by collision-induced dissociation using argon as the collision gas and ramp collision energy that varied according to the charge state of the selected precursor ion. Data acquisition was performed at an m/z range of 300-2100 for the MS survey (1 scan per s) and at an m/z range of 50-2500 for MS/MS.

The data were then collected with MassLynx 4.1²⁰ software and processed using Protein Lynx GlobalServer 2.4²¹ and were converted to peak list text files and submitted to a database search using Mascot MS/MS ion search tool²² against the NCBI non-redundant database using a significance threshold of p < 0.05. The searches parameters were: allowed of one missed trypsin cleavage, experimental masses of peptides was monoisotopic, carbamidomethylation of cysteine was included as a fixed modification, whereas oxidation of methionine was included as possible variable modifications.²³

Sequence alignment and homology modeling

The peptide sequence VAVIDSGIDSSHPDLNVR, identified through mass spectrometry, was subjected to a Basic Local Alignment Search Tool (BLAST) search against the complete genome of Bacillus subtilis TIM27 (unpublished data). First, the open read frames (ORFs) were searched in the genome using the ORF finder and protein prediction was performed using BLASTP. This search enabled alignment of the peptide with the genomic data, resulting in the identification of the complete subtilisin sequence, designated ProT27, as shown in the Supplementary Information (SI) section ( Figure S2). The ProT27 peptide sequence was submitted for alignment of multiple sequences using the program Clustal W,²⁴,²⁵ neighbor joining²⁶ method with bootstrap based on 1000 replications. Furthermore, the sequence was also submitted to the SWISS-MODEL server (automated comparative protein modeling sever)²⁷ for comparative structural modeling.²⁸ The 3D structure of ProT27 was generated using the crystal structure of a mutant of subtilisin E from Bacillus subtilis 168 (PDB: 3whi.1)²⁹ as a modeling template. Further manipulations, structural alignments, and 3D figures were performed with PyMOL.³⁰

Protease activity assay

The protease activity was determined by incubating 250 µL of azocasein 1% (m/v) with 50 µL of crude ProT27 for 30 min at 37 °C as described by Sarath et al.³¹ The reaction was terminated by adding 300 µL of 12% (v/v) trichloroacetic acid (TCA) and centrifuged for 5 min at 12,000 g. Subsequently, 250 µL of the obtained supernatant was added to 750 µL of 2 M NaOH, and the absorbance in 420 nm (A_420nm) was measured against a reference tube, prepared separately for each sample by adding trichloroacetic acid stop solution immediately after mixing the enzyme solution with the substrate. The experiments were carried out in triplicate. A unit of proteolytic activity was defined as the amount of enzyme that produced an increase in A_420nm of 0.01 in 30 min at 37 °C.

Effect of pH, temperature and NaCl concentration on ProT27 activities

The effects of pH values on crude ProT27 were determined by incubating the reaction mixture in a wide pH value range of 6 to 11 at 37 °C for 30 min. The buffers used were sodium acetate (pH 6), sodium phosphate buffer (pH 7), Tris-HCl buffer (pH 8 and pH 9), sodium borate buffer (pH 10) and glycine-NaOH buffer (pH 11). The optimal temperature was determined in the range of 20-80 °C in 50 mM Tris-HCl buffer pH 8 with a pre-incubation step for 20 min at each temperature. The influence of salt on crude protease was evaluated at wide concentrations of NaCl (0.5, 1.0, 1.5, 2.0 and 3.0 M). The protease activity was evaluated as previously described, with the activity of the reference preparation set as 100%.

Effect of metal ions and inhibitors on ProT27

To evaluate the effect of metal ions, the crude ProT27 was incubated with K⁺, Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ at a concentration of 5 mM at 37 °C for 30 min. The effects of enzyme inhibitors such as phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (β-mer), were evaluated by incubating the crude protease with 1 or 5 mM of each inhibitor for 30 min at 30 °C. After that, protease activity was evaluated under standard assay conditions. Enzymatic activity without the addition of metal ions or enzyme inhibitors was considered 100%.

Effect of detergents and oxidizing agents on ProT27 activities

The stability of protease activity was assessed in the presence of the commercial solid detergent (7 mg mL^-1), 1% surfactants (Triton X-100, sodium dodecyl sulfate (SDS), and Tween 80), 1% oxidizing agent (H₂O₂) or 300 ppm of biosurfactant (the optimal emulsification concentration). For this, 50 µL of crude protease (105 U mL^-1) was incubated for 30 min at 37 °C. After incubation, protease activity was measured under standard conditions. Enzymatic activity without the addition of tested agents was considered as 100% relative activity.

Washing performance assay

The effectiveness of crude ProT27 as a detergent additive, along with its combined detergency activity with the biosurfactant BiosT27, was assessed using white cotton fabric pieces (3 × 3 cm) stained with 50 µL of egg yolk emulsion. The stained fabrics were left to dry at room temperature for 24 h before being subjected to different washing treatments: (i) distilled water only, (ii) commercial detergent (7 mg mL^-1), (iii) heated commercial detergent at 70 °C (7 mg mL^-1), (iv) BiosT27 (300 ppm), (v) BiosT27 (300 ppm) + crude ProT27 (1000 U), and (vi) heated commercial detergent at 70 °C (7 mg mL^-1) + crude ProT27 (1000 U). To inactivate the enzymes present in the commercial detergent, it was heat-treated at 70 °C for 1 h, following the method described by Rekik et al.³² and Banik et al.³³

After washing, the fabric pieces were rinsed three times with distilled water and dried at room temperature for 24 h. All experiments were conducted in duplicate, and the results were documented with photographs.

Results and Discussion

Isolation and characterization of proteases from a new strain of Bacillus subtilis TIM27

The production of extracellular enzymes by TIM27 was confirmed by the formation of hydrolytic zones around colonies grown on a medium containing tributyrin and skimmed milk as substrates (Figure 1a). The purification process using ammonium sulfate led to the enrichment of esterase and protease activities in the F40-70% fraction (Figure 1b).

Figure 1
Detection of esterase and protease in the cell-free supernatant of Bacillus subtilis TIM27 (a) and in ammonium sulfate fractions (from left to right: F0-40%, F40-70%, and F70-90%), assessed through agar plate assays using tributyrin and skimmed milk as substrates (b).

Protein quantification indicated a yield of 15 mg of total proteins in the F40-70% fraction per 400 mL of culture (Table 1).

Table 1
Concentration of proteins present in the TIM27 culture supernatant and in the fractions obtained by ammonium sulfate precipitation

	Volume / mL	Protein concentration / (mg mL-1)	Total protein / mg
Crude extract	400	0.12	48.0
F0-40%	7.0	0.7	4.9
F40-70%	7.8	1.92	15.0
F70-90%	7.5	1.2	9.0

In ion exchange chromatography, both activities (esterase and protease) were verified in the elution fraction of 200 mM NaCl (data not shown). This fraction was then applied to the hydrophobic column and elution was carried out using a linear gradient of 1-0 M ammonium sulfate, obtaining both activities in tubes corresponding to an elution volume of 110 to 120 mL (Figure 2a). The electrophoresis gel of the tube corresponding to 110 mL showed the main presence of only two bands, called band A (BA) and band B (BB) (Figure 2b).

Figure 2
Hydrophobic chromatography and SDS-PAGE analysis of the purification steps of ProT27. The main peak from the HiTrap Q HP ion exchange column was loaded in a HiTrap Phenyl HP hydrophobic column and the obtained chromatogram (solid line) is shown. The inset shows in (a) an SDS-PAGE (run under reducing condition) of the major peak in the chromatogram (lane 1: molecular marker, lane 2: major peak from HiTrap Q HP and lane 3: main peak from the HiTrap Phenyl HP and in (b) the proteolytic and lipolytic activities detected by the formation of hydrolysis halo in the skimmed milk and tributyrin plate assays of the eluted pick shown in lane 3.

One of the most fascinating aspects of subtilisin, a serine protease, is its promiscuous activity.⁹ Herein, the subtilisin of Bacillus subtilis TIM27 exhibited protease and esterase activities. This dual functionality allows subtilisin to catalyze a wide range of reactions, enhancing its utility in biocatalysis and its application in the detergent industry.³⁴

Production and partial purification of lipopeptides biosurfactants (BiosT27)

To further improve enzymatic activity, we produced and purified the TIM27 biosurfactant (BiosT27) and evaluated its emulsification capacity and performance as a detergent additive. The acid precipitation of BiosT27 biosurfactant resulted in a yield of 673 mg L^-1 of biosurfactant. At 50 ppm, it was able to reduce the surface tension of the water from 71.2 ± 0.03 to 25.3 ± 0.05 mN m^-1. This preparation was also able to promote the emulsification of a water-kerosene mixture, with an emulsification index of 67% at a concentration of 50 ppm, although, with the aid of an optical microscope, smaller and more stable droplets were observed at 300 ppm (Figure 3). The droplet size in emulsions significantly influences their rheological properties and is essential for various applications.³⁵ Notably, 300 ppm of BiosT27 is considerably lower than the critical micelle concentration (CMC) of synthetic surfactants like SDS, which ranges between 2000 and 2500 ppm (mg L^-1).³⁶

Figure 3
Emulsification activity and aspects of emulsions produced in a water-kerosene mixture (10× magnification) by different concentrations of BiosT27.

According to Willumsen and Karlson³⁷ and Mulligan,³⁵ a value of the emulsification index above 50% and a reduction in the surface tension (ST) of water from 72 to 35 mN m^-1 correspond to a great emulsifying/surfactant agent. Therefore, TIM27 emerges as a promising strain for the exploration of its exceptional surfactant properties.

The Bacillus subtilis TIM27 was isolated from mangrove sediments in the estuary of Timonha, in the state of Ceará, northeastern Brazil. Mangroves are ecosystems located at the interface between the continent and the ocean, influenced by tidal regimes. They are characterized by muddy, anoxic, and saline sediments, where species adapted to these inhospitable conditions-unfavorable for most living organisms-are able to survive. Due to their strategic location, mangroves are highly sensitive to pollution from human activities, such as oil spills.³⁸ The unique characteristics of mangroves make them a hotspot for the discovery of extremophilic microorganisms that produce molecules of biotechnological interest, such as biosurfactants. Species of the Bacillus genus are known for simultaneously producing various types of biosurfactant under different environmental conditions, such as temperature, pH, aeration, carbon/nitrogen sources, and the presence of micronutrients.³ The main compounds in BioT27 were previously identified as lipopeptides by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) in studies conducted by our group. Their details are demonstrated in Table 2 and the MS spectra and peaks assignment are presented in the SI section ( Figure S1 and Table S1).¹¹

Table 2
Major peaks of biosurfactant (BiosT27) produced by Bacillus subtilis TIM27 detected by ESI-Q-TOF MS

Ion m/z	Lipopeptide
1044.66	C14 surfactin A [M + Na]+ / Leu7
	C15 surfactin B [M + Na]+ / Val7
1058.68	C15 surfactin A [M + Na]+ / Leu7
	C16 surfactin B [M + Na]+ / Val7
1072.69	C16 surfactin A [M + Na]+ / Leu7
	C17 surfactin B [M + Na]+ / Val7
1066.64	C14 iturin A [M + Na]+ / Asn1
	C13 iturin [M + K]+ / Asn1
1097.57	C16 bacillomycin D [M + K]+
1477.83	C17 fengycin A [M + H] / Ala6
	C15 fengycin B[M + H]+ / Val

Among the families of biosurfactants commonly produced by species of Bacillus are surfactin, iturin, and fengycin.³⁹,⁴⁰ Through ESI-Q-TOF MS analyses, a predominant production of surfactins was observed, specifically peaks related to the surfactin A and B isomers [M + Na]⁺ with more than 14 carbons in the fatty acid chain, identified by the ions of m/z 1044.66, 1058.68, 1072.6920 (Table 2). Surfactin is known for its exceptional surface activity, reducing the surface tension of water at 20 °C from 72 to 27 mN m^-1 at concentrations below 20 µM,⁴¹ in addition to reducing the interfacial tension of the water/hexadecane system from 43 mN m^-1 to values below 1 mN m^-1. Furthermore, surfactin has low toxicity, low skin irritation, high performance in a temperature range, and high biodegradability,⁴²,⁴³ making it a suitable additive for detergents.

Effects of pH value and NaCl on BiosT27 surface-activities

The tested pH values (7-11) did not affect emulsification activity (Figure 4a); however, an increase in surface tension was observed with rising pH values (Figure 4b). The maximum reduction in surface tension was 25.3 ± 0.03 mN m^-1, when BiosT27 was adjusted to pH 7. Regarding the effect of NaCl on emulsifying activity, a slight decrease in E24 was observed at 0.5 and 1 M concentrations. At 2 M, there was a 25% reduction in E24, and a complete loss of activity occurred at 3 M (Figure 4c). These results confirm the stability of BiosT24 at a much higher salt concentration than that typically used in detergents, which ranges from 2-3% by weight.

Figure 4
Effect of pH on emulsification activity (a) and surface tension reduction (b), and effect of NaCl concentration on emulsification activity (c) of BiosT27.

Fei et al.⁴⁴ studied the application of a purified surfactin from Bacillus subtilis HSO21 in detergent formulations. The results demonstrated low toxicity (median lethal dose (LD₅₀) > 5000 mg kg⁻¹, median lethal concentration (LC₅₀) > 1000 mg kg⁻¹) and a low rate of skin irritation, as indicated by the primary irritation index (PII = 0). Furthermore, surfactin exhibited excellent surface and interfacial properties for emulsification and wettability, high compatibility and stability over a wide range of temperatures, pH value, and hard water conditions, and acceptable biodegradability and foam-forming capacity. The use of biosurfactants in the detergent industry requires that the surfactant can maintain its surface activity under a variety of conditions such as extremely alkaline pH values and high salt concentrations.⁴⁵ BiosT27 was stable in all pH values analyzed and in the presence of up to 1 M NaCl, with an emulsion index greater than 50%.

Protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The BA and BB bands, corresponding to protease and esterase, respectively, obtained during the purification of the F40-70% fraction (Figure 2b), were excised from the gel and subjected to trypsin digestion. The resulting tryptic peptides were analyzed by LC-MS/MS, leading to the identification of two distinct proteins. The first, BA, was identified as subtilisin E (designated ProT27) from Bacillus subtilis, with 4% coverage and a score of 87. The second, BB, was identified as a β-glucanase, with 15% coverage and a score of 251 (Table 3).

Table 3
Identification of proteins BA and BB, isolated from the extracellular proteases of Bacillus subtilis TIM27, using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Band	Protein	Molecular weight / kDa	pI	NCBI accession number	Score	Identified peptide sequences
BA	subtilisin E (ProT27)	39.4	9.04	P35835	87	VAVIDSGIDSSHPDLNVR
BB	β1-3,1;-4;-glucanase	26.08	5.61	CAA81093.1	93	ADGYSDGNMFNCTWR
					80	LALTSPSYNK
					78	ANNVSMTSLGEMR

• pI: isoeletric point.

Regarding extracellular enzyme production, the TIM27 strain exhibited both protease and esterase activities. In fact, species of the Bacillus genus are widely known for producing a diverse range of enzymes and demonstrating a high capacity for secreting them into the extracellular medium.⁴⁶ Subtilisins are extracellular serine endopeptidases that are considered the most important additive in modern detergents. They help in the removal of protein-based stains from fabric, such as eggs, milk, or blood.³⁴ In this study, we isolated a subtilisin E, named ProT27, which exhibits both protease and esterase activities. Certainly, some subtilisins have already been reported to possess esterase and amidase activities. This happens because subtilisins can catalyze peptide bond formation starting from an ester substrate by first forming an acyl enzyme intermediate, which then reacts with a primary amine to form the peptide product. Thus, this application requires high esterase activity to promote the formation of acyl enzymes.⁴⁷ Furthermore, this characteristic allows hydrolyzing of hydrophobic substrates such as elastin and collagen,³⁴ expanding the potential of ProT27.

Sequence alignment and homology modeling

The multi-alignment analysis of the Prot27 amino acid sequences together with homologous sequences is shown in Figure 5. This analysis reveals that subtilisins from Bacillus halotolerans (WP_105955331), Bacillus subtilis (WP_168780778), and Bacillus intestinalis (WP_061188300) share identities of 95.53, 99.21, and 97.90% with the target protein Prot27, respectively. The results showed that ProT27 contains a classical catalytic triad of proteases (Asp¹³⁸, His¹⁷⁰, and Ser³²⁷).²⁹

Figure 5
Amino acid sequence-based alignment of ProT27 with other subtilisin E (WP105955331); (WP168780778), (WP061188300). Detail of the classical catalytic triad of proteases (Asp¹³⁸, His¹⁷⁰, and Ser³²⁷) is shown in highlight residues.

The predicted ProT27 3D structure was constructed by homology modeling using Swiss-Model with the crystal structure of an subtilisin E mutant from Bacillus subtilis 168 as the template (PDB code 3whi.1.A 97,73% identity). Manipulation and visualization of the 3D structure in Pymol allowed confirmation of the catalytic triad (Figure 6).

Figure 6
Model structure of ProT27 based on mutant of subtilisin E from Bacillus subtillis 168 (PDB code 3whi.1.A 97.73% identity). (a) Cartoon diagram of ProT27 enzyme model pre-pared by using PyMOL graphics program and (b) predicted protease catalytic triad.

The α/β-hydrolase structural superfamily, as defined by Ollis et al.⁴⁸ includes a diverse range of enzymes whose activities primarily rely on a catalytic triad, typically composed of Asp, His, and Ser residues. Through multiple alignment analysis and the predicted tertiary structure of ProT27, we clearly identified the presence of this classical catalytic triad of proteases (Asp¹³⁸, His¹⁷⁰, and Ser³²⁷).²⁹ The Ser³²⁷ residue likely functions as a catalytic nucleophile, and together with Asp¹³⁸ and His¹⁷⁰, forms a typical catalytic triad also found in certain lipolytic enzymes, potentially granting ProT27 broad substrate specificity.⁴⁹

Subtilisin is a serine endopeptidase and the type example of the peptidase family S8. It lacks cysteine residues, although these are present in homologous enzymes. Variants include subtilisin BPN’ (also known as subtilisin B, subtilopeptidase B, subtilopeptidase C, nagarse, nagarse proteinase, subtilisin novo, bacterial proteinase novo) and subtilisin carlsberg (subtilisin A, subtilopeptidase A, alcalase Novo). Similar enzymes are produced by various strains of Bacillus subtilis and other species.⁵⁰ Subtilisin exhibits broad substrate specificity, hydrolyzing proteins with a preference for large uncharged residues at the P1 position.³⁴ Specifically, subtilisin E from Bacillus subtilis (EC 3.4.21.62) exhibits kinetic parameters when acting on casein: a Michaelis constant (K_M) of 0.059 at pH 10 and 45 °C, a maximum reaction velocity (V_max) of 0.336 mg min^–1, and a catalytic efficiency (k_cat K_M-1) of 2,433 at 45 °C and pH 8.6.⁵¹,⁵²

Effect of pH value, temperature and NaCl concentration on the protease activity

The crude ProT27 showed excellent catalytic activity at 50 °C and pH 8 using azocasein as a substrate. Furthermore, the results showed that the enzyme is highly stable, maintaining its activity above 70% through all temperature values (20 to 80 °C) and pH values tested (6 to 11) (Figures 7a and 7b). The analysis of the stability of the enzyme at different concentrations of NaCl showed a residual activity of 56% even at the concentration of 2 M NaCl, which corresponds to 11.6 % (m/v) of salt (Figure 7c).

Figure 7
Effect of temperature (a), pH (b) and NaCl concentration (c) on the activity of the crude ProT27.

The Bacillus subtilis TIM27 strain was isolated from sediments from a semiarid mangrove, which are coastal ecosystems located on estuaries shorelines where harsh environmental conditions are found, such as periodic flooding by tides and variations in salinity, oxygen, temperature and low nutrient availability.⁵³,⁵⁴ Those extreme conditions are selective factors for the species of microorganisms that can inhabit these ecosystems, becoming valuable sources for the prospecting of new resistant biomolecules for industrial applications.⁵⁵,⁵⁶ The results of the crude ProT27 activity and stability support this statement, as ProT27 exhibited optimal activity at pH 8 and 50 °C. Moreover, it retained over 70% of its activity across all tested temperature and pH values.

Based on the optimal pH of ProT27 and its stability across a wide pH range, its potential for incorporation into commercial detergent formulations becomes clear. When compared to other proteases in commercial formulations, such as Alcalase™ (maximum activity at pH 8-10) from Bacillus licheniformis and Savinase™ (maximum activity at pH 8-10) from Bacillus clausii, ProT27 stands out for its high effectiveness under alkaline conditions.⁵⁷

From the study of the optimal temperature for enzymatic activity, we identified that ProT27 is a moderately thermophilic esterase.⁵⁸ Additionally, we observed its high stability within the tested temperature range (30-60 °C), maintaining over 70% of its relative activity. Due to this inherent stability, ProT27 shows strong potential for use in industrial washing processes at high temperatures.

NaCl is an important component in detergent formulations and is still used in the granulation of enzymes for inclusion in detergents.⁵⁷ As shown in Figure 3b, ProT97 demonstrated halotolerance, maintaining 80% of its activity even in the presence of high concentrations of NaCl. This characteristic is highly favorable for its use as an additive in detergents intended for washing applications with high-salinity water, such as groundwater.⁵⁹

Effect of metal ions and inhibitors in crude protease activity

The effect of metal ions K⁺, Na⁺, Ca²⁺, Mg²⁺, Mn²⁺, Cu²⁺ and Zn²⁺ on crude protease activity is shown in Table 4. The concentration of 5 mM of the monovalent ions K⁺, Na⁺, and the divalent ions Ca²⁺, Mg²⁺ had no effect on enzyme activity, however, the ions Mn²⁺, Cu²⁺ inhibited 15 and 50%, respectively, while Zn²⁺ inhibited protease activity 90%. The effect of common enzyme inhibitors was evaluated at two concentrations, 1.0 and 5.0 mM. The crude ProT27 was highly resistant to β-mer at both concentrations, with residual activity of 90%. PMSF inhibited about 30% at both concentrations and EDTA at a concentration of 5.0 mM inhibited approximately 40% protease activity (Table 4).

Table 4
Effects of metal ions and some enzyme inhibitors on the activity of the crude ProT27

Chemical	Concentration / mM	Residual activity / %
None	−	100 ± 0.2
K+	5	99 ± 1.3
Na+	5	100 ± 0.9
Ca2+	5	101 ± 0.2
Mg2+	5	103 ± 0.1
Mn2+	5	87 ± 1.3
Cu2+	5	49 ± 0.4
Zn2+	5	19 ± 0.5
PMSF	1	74 ± 0.3
	5	68 ± 0.2
EDTA	1	82 ± 0.03
	5	61 ± 0.03
β-mer	1	97 ± 0.09
	5	90 ± 1.0

• PMSF: phenylmethylsulfonyl fluoride; EDTA: ethylenediaminetetraacetic acid; β-mer: β-mercaptoethanol.

Alkaline proteases typically exhibit their best catalytic activity in the presence of divalent ions such as Ca²⁺, Mg²⁺, and Mn²⁺.⁶⁰ However, the results showed that Mg²⁺, Na⁺, and K⁺ ions did not interfere with the proteolytic activity, and unlike other studies in the literature, the addition of Ca²⁺ also had no effect on protease activity.⁶¹,⁶² The addition of Mn²⁺, Cu²⁺, and Zn²⁺ negatively impacted the performance of crude ProT27, reducing relative activity by 14, 50, and 80%, respectively. Inhibition of proteases by Cu²⁺ and Zn²⁺ has been similarly reported by Jeong et al.⁶³

PMSF is a specific inhibitor of serine proteases that completely inhibits the activity of subtilisins, though it does not inactivate all serine proteases. It reduced the activity of crude ProT27 by 26% at 1.0 mM and 32% at 5.0 mM, indicating that higher concentrations are needed for complete inhibition. The addition of EDTA reduced activity by 18% at 1.0 mM and 39% at 5.0 mM, suggesting that metal ions are necessary for enzyme activity and stability.⁶⁴ Metal ions can enhance the enzymatic activity of proteases by stabilizing their structure, protecting them from thermal denaturation, or acting as electron donors/acceptors.

Effect of detergents and oxidizing agents on the crude protease activity

The biosurfactant produced by the TIM27 (BiosT27) did not affect the protease activity of crude ProT27, whereas hydrogen peroxide reduced the protease activity by 4%. The detergents Tween 80, the commercial solid detergent, and Triton X-100 each reduced activity by around 20%. Additionally, enzymatic activity was fully inhibited in the presence of SDS (Table 5).

Table 5
Stability of crude ProT27 in the presence of different detergent components

Surfactant/oxidizing agent	Concentration	Residual activity / %
None	−	100 ± 1.4
BIOS27	300 ppm	102 ± 6.4
Commercial solid detergent	7 mg mL-1	79 ± 3.8
H2O2	1% (v/v)	96 ± 2.6
TritonX-100	1% (v/v)	74 ± 2.2
Tween 80	1% (v/v)	80 ± 1.0
SDS	1% (m/v)	2.9 ± 1.0

• SDS: sodium dodecyl sulfate.

Furthermore, crude protease has been shown to be quite stable in the presence of 2 M NaCl (56%) and in the different classes of additives of commercial detergents, such as non-anionic surfactants (Triton X-100 and Tween 80) and oxidizing agent (H₂O₂). However, in the presence of the anionic surfactant SDS, the crude ProT27 suffered a strong inhibition, with a 97% reduction in its activity after 30 min incubation. In fact, some alkaline proteases are known to be stable in the presence of non-ionic detergents but are strongly inhibited by anionic detergents such as SDS.⁶⁵,⁶⁶

The relative activity of ProT27 remained at 96% even in the presence of H₂O₂, a strong oxidizing agent commonly found in commercial detergents.⁶⁷ Many alkaline proteases lose activity or are completely inhibited by peroxides used in bleach-based detergents. H₂O₂ typically affects enzymes negatively by oxidizing methionine residues within the enzyme structure and/or at its active site.⁶⁸ This performance suggests that ProT27 has natural stability against oxidizing agents, making it suitable for inclusion in laundry detergents that contain surfactants and oxidizing agents.⁶⁹

Washing performance assay

The washing performance assay showed that the combination of crude ProT27 and BiosT27 provided better egg stain removal from cotton fabric compared to BiosT27 alone. However, it was less effective than both heated and unheated commercial solid detergent. Notably, heated commercial detergent combined with crude ProT27 performed better than unheated commercial detergent in its standard form (Figure 8), making it the most effective washing treatment.

Figure 8
Egg yolk stain before (a) and after washing performance assay by BiosT27 and ProT27 crude protease (b) compared to commercial detergent: (A) none; (B) commercial detergent (7 mg mL^-1); (C) heated commercial detergent at 70 °C; (D) BiosT27 (300 ppm); (E) BiosT27 (300 ppm) + Crude ProT27 (1000 U); (F) heated commercial detergent at 70 °C + Crude ProT27 (1000 U).

At the end of the washing performance assay, it was observed that the combination of crude ProT27 and BiosT27 resulted in more efficient egg stain removal from the cotton fabric compared to BiosT27 alone. In fact, biosurfactants are known to work synergistically with extracellular enzymes by enhancing solubilization, mobilization, and increasing the bioavailability of hydrophobic residues for their producing organisms.⁷⁰ Therefore, it can be inferred that in this experiment, the molecules worked together to improve egg stain removal from the cotton fabrics. The BiosT27 biosurfactant likely aided the catalytic activity by solubilizing hydrophobic proteins and facilitating the mobilization of these compounds, making them easier to remove during washing.⁷¹

Similarly, the addition of crude ProT27 to the commercial detergent improved washing performance compared to unheated and heated detergent (with its endogenous enzymes denatured). This result shows that the crude protease was effective in removing not only the egg stain with the commercial detergent but also a better performance in comparison to the endogenous enzymes of the detergent.

In detergency tests such as those conducted in this study, the results can vary significantly due to the chemical nature of the stains. Coffee stains, for example, are known to be difficult to remove due to the presence of phenolic acids. For this type of stain, bleaches or acids are commonly used in detergent formulations, which can result in fabric damage. In contrast, biosurfactants have shown similar potential to traditional detergents, as there is evidence that they are effective in removing most stains.⁷²

In the detergency assay, it was observed that the commercial detergent performed better when combined with ProT27. However, it is important to emphasize the excellent results obtained with the combination of ProT27 and BiosT27, even without any additives in its formulation. Several studies have demonstrated the effectiveness of biosurfactants in stain removal. For instance, Bouassida et al.⁷³ conducted a comparative study using isolated biosurfactant, biosurfactant combined with commercial detergent, and commercial detergent alone. The results showed that the biosurfactant, even without additives, was more effective in removing oil and tea stains compared to the commercial detergent. Furthermore, a 33 to 45% increase in the effectiveness of commercial detergents was observed when used together with the biosurfactant. Given its low toxicity, these findings highlight the significant potential of incorporating biosurfactants into commercial detergent formulations.

Conclusions

The Bacillus subtilis TIM27 strain, isolated from mangrove sediments in Northeast Brazil, proved to be an excellent source for producing subtilisin ProT27, which exhibits both esterase and protease activities, as well as lipopeptide biosurfactants (BiosT27). The combination of ProT27 and BiosT27 demonstrated stability under harsh conditions commonly encountered in laundry applications, such as extremely alkaline pH, high temperatures, and salinity. Additionally, it showed superior dirt removal performance compared to commercial detergents. Given their efficiency, environmental safety, and the fact that they can be derived from renewable resources, ProT27 and BiosT27 present promising potential as biodegradable additives for laundry detergents, helping to reduce the significant volume of synthetic waste generated in laundries while contributing to environmental conservation.

Supplementary Information

Supplementary information is available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

The authors thank the National Council for Scientific and Technological Development (CNPq) for grants and Foundation for Scientific and Technological Development of Ceará (FUNCAP) for the scholarship provided to FJA.

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Edited by

• Editor handled this article:
  Hector Henrique F. Koolen (Associate)

Publication Dates

• Publication in this collection
  06 June 2025
• Date of issue
  2025

History

• Received
  22 Jan 2025
• Accepted
  07 May 2025