Alkaline Thermo- and Oxidant-Stable Protease from Bacillus pumilus Strain TNP93: Laundry Detergent Formulations

Abstract

The study aims to produce a detergent-compatible and alkaline thermophilic protease from a Bacillus strain and to investigate its usability as a detergent bio-additive. The protease-producing bacterium was identified as Bacillus pumilus strain TNP93 according to the 16S rRNA sequence. The bacterium optimally synthesized the protease at 40 °C and pH 10 in 40 h. The raw protease displayed its optimum activity at pH 10 and 60 °C and its stability between pH 6–13 and 30–100 °C for 24 h. The molecular mass of the proteolytic band was estimated to be about 85 kDa. The protease was not inhibited by any of the metal ions used (Ba²⁺, Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Zn²⁺). 97 and 90% of its original activity with 5 mM PMSF and EDTA remained. The activity was measured as 84, 124, and 95%, respectively, in the presence of 1% concentrations of Tween 20, Tween 80, and Triton X-100. In addition, all of its activity was preserved when the enzyme was exposed to 5% H₂O₂. The end products of casein were detected as tyrosine, aspartic acid, glycine, and cysteine by thin-layer chromatography. Considering the wash performance analysis, the mix of 1% commercial detergent and enzyme almost removed all of the protein-based stains (blood and egg yolk albumin). These remarkable findings indicate that the alkaline, thermo-, and oxidant-stable TNP93 protease is a valuable candidate for usage as a biological additive in various laundry detergents.

Introduction

Enzymes, as non-toxic biocatalysts, facilitate reactions without necessitating high energy input. They exhibit substrate specificity and enantioselectivity, playing a vital role in mitigating the impact of environmental pollution and energy wastage [1, 2].

Proteases, one of the essential enzyme groups, are hydrolytic enzymes defined as proteinases or peptidases. By catalyzing the hydrolysis of peptide bonds in polypeptides or proteins, they release free amino acids and short peptides [3].

Due to their wide-ranging applications across various industries including food, pharmaceutical, cosmetics, medicine, textile, leather, photography, detergent industries, proteases, which constitute approximately 60% of the worldwide enzyme market, have a 40% share of the global enzyme sales [4, 5]. For further evidence of their industrial significance, one can refer to the website of the U.S. Patent and Trademark Office (USPTO, https://www.uspto.gov). According to the report of Future Market Insights (FMI), the market valuation of the protease enzyme as of 2023 is 2.1 billion USD. This value is expected to reach 4.6 billion USD by 2033. (https://www.futuremarketinsights.com/reports/protease-market).

Plants, animals, and microorganisms are utilized for the isolation of enzymes. Obtaining enzymes from animal sources presents challenges, including the necessity to slaughter a significant number of farm animals, which is a limiting factor in process. In addition, to isolate enzymes from plant sources, restrictive conditions such as providing and maintaining the climatic conditions necessary for plant growth should be considered. Because of such disadvantages, researchers prefer using microorganisms to isolate industrial enzymes [5]. Microorganisms are favored as sustainable resources in industrial applications thanks to their notable advantages. These include such as their rapid reproduction in short fermentation times, ability to grow easily in the presence of simple substrates, ability to synthesize enzymes with high catalytic activities, and broad substrate specificities.  Additionally, microorganisms can be produced in  substantial quantities with high purity and at low production costs [6, 7]. Bacillus species are among the most important bacterial genera from which microbial enzymes are obtained [7, 8]. Many advantages such as being able to reproduce under extreme conditions, being isolated easily, producing abundant extracellular enzymes, the ease of obtaining the enzymes they produce, and the enzyme’s long shelf life make them superior to other genera in commercial applications [9].

Among the bacterial enzymes produced, especially alkaline proteases (Subtilisins, EC 3.4.21.14), are active and stable in high pH buffers and in the presence of surfactants/oxidizing agents [10, 11], they facilitate the removal of protein-based stains in the laundry [12]. Due to these properties, alkaline proteases draw much attention as an additive compatible with detergents in the detergent industry [4].

The addition of enzymes in detergent formulations increases the efficiency of stain removal during laundering, reducing the required time, energy, and therefore, the cost. Moreover, this approach holds significant value in providing a more eco-friendly, alternative approach, unlike chemicals that create pressure on the environment. Protease used in detergents constitutes approximately 30% of the world enzyme market [5]. The usage of enzymes in detergent compositions is a widespread practice in advanced countries and is becoming popular in developing countries as well [13]. In this context, due to the increasing need for alkaline proteases in the detergent industry, it is important to isolate new proteases that can function at high capacity from natural strains.

For this reason, in our study, the isolation and identification of the natural Bacillus strain with high alkaline protease activity and production, partial purification, and characterization of the enzyme were performed. At the same time, the potential of using the characterized TNP93 protease in the laundry detergent was evaluated by performing a washing performance analysis.

Methods

Bacterial Isolation and Identification

For the isolation of strains, soil samples from the chicken farm were collected and one gram samples were transferred into the 50 mL Luria Bertani (LB) media (pH 9) containing (g/L): NaCl 10; yeast extract 5; tryptone, 10 [14]. To obtain endospore-forming bacteria, the soil specimens were heat-shocked at 80 °C for 10 min and incubated at 37˚C for 24 h. After the incubation period, the samples were serially diluted, spread on plates of LB agar (pH 9), and incubated for a night at 37 °C. In the isolations performed, a total of 300 bacteria were obtained. For screening of the proteolytic strains, morphologically different colonies growing on LB agar were plated onto skimmed milk agar plates (SMA) (pH 9) and incubated at 37 °C for 40 h. SMA (g/L): Peptone, 10; meat extract, 3; NaCI, 5; skimmed milk powder, 11; agar, 15 [15, 16]. Clear halo zone-forming colonies were indicated as protease-producing strains. The protease-producing TNP93 strain with the largest zone of clearance was selected for our study.

The preferred strain was identified first by morphological and biochemical tests (Gram staining, Endospore-forming, and Catalase test: according to Bergey’s Manual Systematic Bacteriology) [17]. The molecular tests (16S rRNA sequencing) were performed by BMLabosis Company (Ankara, Türkiye). For DNA isolation, “the EurX GeneMATRIX Tissue and Bacterial DNA isolation” kit (Gdansk, Poland) was preferred. To determine the species, the targeted region of the gene was amplified using the universal primers of 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) in PCR. In the PCR analysis, firstly, the denaturation process was performed at 95 °C for 5 min. Then, the cycle consisting of the following conditions was repeated 35 times; 45 s at 95 °C, 45 s at 57 °C, and 1 min at 72 °C. The final extension step was carried out at 72 °C for 5 min. The BLAST program was used to search for homologous sequences in the NCBI database. MEGA 6 software was used during the construction of the phylogenetic tree [18].

Production of Protease

The strain selected as the protease producer was cultivated on an SMA medium at between 30–60 °C and pH 6–13 for 40 h and thus the optimum growth conditions were determined. The production medium (skimmed milk broth, pH 10) was inoculated with 5% (v/v) inoculum of the 24 h old pre-grown strain TNP93. The cultivation was performed at 40 °C for 40 h [19]. The medium of culture was centrifuged at 7500 rpm and at 4 °C for 20 min. Thus, the supernatant, which is the source of the enzyme, was obtained [20].

Enzyme and Protein Assays

The standard proteolytic activity was determined according to the procedure of Takami et al. [21] with some modifications. 1 mL of the supernatant and 0.5 mL of 0.2 M Glycine–NaOH buffer (1% (w/v) casein added, pH 10) were mixed and incubated at 60 °C for 60 min. The reaction was terminated by the supplementation of 1.5 mL of 10% (w/v) trichloroacetic acid (TCA) and the mixture was incubated at room temperature for 30 min. Finally, it was centrifuged at 10.000 rpm for 10 min to eliminate the precipitated proteins. 0.5 mL of supernatant was mixed with 2.5 mL 0.5 M Na₂CO₃ and 0.5 mL Folin-Ciocalteu’s phenol reagent: distilled water (1:2, v/v). The mix was allowed to stand at room temperature for 30 min and measured at 660 nm [22, 23]. The assays were carried out in triplicates and the mean values of the results were stated. Tyrosine (0–200 μg/mL) calibration plot was used as a standard curve. The protein content was evaluated according to the procedure of Lowry et al. [24], in which bovine serum albumin (BSA) was used as the standard. One unit (U) of protease activity was determined as the amount of enzyme releasing the equivalent of 1 μg of tyrosine per minute under the determined experimental circumstances [25].

SDS-PAGE and Zymogram Analysis of Protease

The SDS-PAGE analysis was performed at 15 mA using 12% resolving gel according to the method of Laemmli [26]. The enzyme samples were diluted in a sample buffer (1:5) and denatured at 100 °C for 5 min. Following the electrophoresis, the gel was stained with 0.1% (w/v) Coomassie Brilliant Blue R-250 (CBB R-250). For determining the relative molecular mass of the enzyme, a standard prestained protein ladder kit was used (Broad range: 10–180 kDa, Thermo Fisher Scientific).

The zymography analysis was performed to the methodology of García-Carreño et al. [27] and Hammami et al. [28] with minor changes. It was carried out at 15 mA with the running gel (12%) containing 0.5% (w/v) of skimmed milk and with the non-heated samples. After the analysis, the gel was soaked in 200 mM glycine–NaOH buffer (pH 10) containing 2.5% (v/v) Triton X-100 at room temperature for 30 min with gentle shaking to eliminate SDS and renature the enzyme. The gel was rinsed with 200 mM glycine–NaOH buffer (pH 10) for three times to eliminate the Triton X-100. Afterward, the gel was incubated in 200 mM glycine–NaOH buffer (pH 10) including 1% (w/v) skimmed milk at 60 °C for 60 min. The gel was first soaked in a staining solution containing 0.25% CBB R-250, 45% ethanol, and 10% acetic acid for 2 h. It was then kept in the de-staining solution containing 5% ethanol and 7.5% acetic acid. Transparent bands formed on the blue-colored gel showed the existence of proteolytic activity.

Effects of pH and Temperature on Protease Activity and Stability

pH buffers (6–11) containing 1% (w/v) casein were used to determine the pH value at which the enzyme showed optimal activity [29]. In order to evaluate the pH stability, the enzyme was pre-incubated between pH 6–12 (without casein) at 25 °C for 24 h [22]. The remaining activities were evaluated by the standard method. The following buffer systems were used at 0.2 M with minor modifications: Sodium phosphate (pH 6–8) [30], Glycine–NaOH (pH 9–10) [22], and Borax-NaOH (pH 11–12) [31].

The optimal activity temperature of the enzyme was determined at 30–80 °C and pH 10 buffer containing 1% (w/v) casein for 30 min [32]. Based on the determination of the thermal stability, the enzyme was pre-incubated at 30–70 °C for 24 h [22, 33]. Then, the retained activities were measured as described previously. The unheated enzyme's activity was accepted as a control (100%). Each assay was carried out in triplicate.

Effects of various Chemicals on Protease Stability

The effects of metallic ions (Ba²⁺, Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, Zn²⁺) on the enzyme were determined by pre-incubating the TNP93 enzyme with each of the metal chloride salts (5 mM) at 60 °C for 1 h [30].

For the determination of the effect of inhibitory chemicals on the enzyme, TNP93 protease was pre-incubated with different inhibitors like phenylmethylsulfonyl fluoride (PMSF, 5 mM), N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK, 1 mM), ethylene glycol tetraacetic acid (EGTA, 5 mM), ethylenediaminetetraacetic acid (EDTA, 5 mM), 1,10-phenanthroline (1 mM), β-mercaptoethanol (0.05%, v/v), iodoacetamide (5 mM), urea (4 M), and Na-sulfide (5 mM) at 60 °C for 1 h [22, 30].

The effects of an anionic surfactant (SDS, 0.5%, w/v), various non-ionic surfactants (Tween 20, Tween80, Triton X-100, all at 1%, v/v), and an oxidizing agent (H₂O₂, 5%, v/v) on the enzyme were evaluated by pre-incubating the TNP93 protease with each of the chemicals at 60 °C for 1 h [25].

The enzyme was mixed with various organic solvents (acetone, butanol, ethanol, isopropanol, methanol (50%, v/v) and pre-incubated at 60 °C for 1 h [34].

NaCl stability was determined by pre-incubating the TNP93 protease with salt concentrations (1–15%, w/v) at 60 °C for 60 min [35].

All analyses were performed under the standard experiment conditions mentioned before. The residual activities were evaluated by comparing them with the control. Enzymatic activity measured without any chemicals was evaluated as a control (100%).

End Product Analysis of Protease

TLC F₂₅₄ plates (20 × 20 cm², Merck, Darmstadt, Germany) were used to analyze the release of end products of casein. Tyrosine, glycine, proline, cysteine, and histidine amino acids (Merck, Darmstadt, Germany; Sigma-Aldrich, St. Louis, USA) at 1% concentrations were used as markers. For the determination of the hydrolysis reaction products, enzyme and pH 7 buffer containing casein were reacted at 60 °C for 12 h. The reaction products (20 µL) and reagents (5 µL) were applied to the plate at a distance of 2.5 cm from the bottom of it. The chromatography was performed using a mobile phase containing acetic acid-n-butanol-distilled water (1:3:1, v/v/v) solvent system. The TLC plate was dried at room temperature for 1 h to prevent the formation of blurry images on the plate after the dye had been sprayed. To visualize the spots, the solution of ninhydrin (0.25% w/v, in acetone) was sprayed on the dried plate and heated at 90 °C for 20 min [36–38]. After the chromatography process was completed, visual alignment was made based on the locations of the spots of the markers and final products on the plate, and the final products were determined by comparing the spots.

Compatibility of Protease with Some Detergents

To assess the compatibility of the alkaline protease with commercially available laundry detergents, ABC (ABC Detergent Company, Türkiye) (solid) and Ariel (Procter and Gamble, India) (liquid) were used. 1% (w/v) concentrations of the commercial detergents were prepared and heated for 5 min to inactivate the endogenous proteases. The incubations of the enzyme and detergents were performed at 60 °C for 1 h. Following the incubation, the remaining enzyme activities were calculated according to the standard activity analysis. Enzymatic activity without any detergent additive was evaluated as a control (100%) [28, 39].

Stain Removal Activity of the Protease

The capability of the TNP93 enzyme in removing stains was evaluated according to the methodology of Lam et al. [34] with some modifications. White cotton fabrics (4 × 4 cm²) were stained with 500 μL of blood and egg yolk albumin and dried overnight at room temperature. The commercial liquid detergent was boiled for 5 min to inactivate the endogenous enzymes.

To clean the stains from the fabrics, the procedure described below was applied:

• A.Stained fabric + 50 ml tap water (control).
• B.Stained fabric + 0.5 mL liquid detergent + 49.5 mL tap water (1%).
• C.Stained fabric + 0.5 mL enzyme (500 U/mL) + 49.5 mL tap water.
• D.Stained fabric + 0.5 mL enzyme (500 U/mL) + 0.5 mL liquid detergent + 49 mL tap water.

These various washing solutions prepared in beakers were incubated at 60 °C for 1 h and 100 rpm. Then the fabrics were rinsed with tap water and air-dried. Removal of the blood and egg yolk albumin stains on the dried fabrics was visually evaluated. The untreated fabrics stained with blood and egg yolk albumin were taken as controls.

In addition, standard activity analysis was carried out with the washing water (containing the hydrolysis products of protein-based stains) remaining in the beaker after the fabrics were removed.

Results

Characterization of Protease-Producing TNP93 Strain

A bacterial collection was created with the bacteria samples isolated from chicken farm soils (Adana, Türkiye) and they were screened for their protease-producing abilities on skimmed milk agar at 37 °C and pH 9 (Fig. 1). The selected strain was named “TNP93”.

Fig. 1.

Proteolytic activity on SMA

The novel strain was an aerobic, catalase-positive, and endospore-forming rod-shaped gram-positive bacterium and its colony was circular, undulate, and dull-white. Besides, the 16S rRNA gene sequence of the strain showed significant homology with Bacillus pumilus by the BLAST program. Thus, it was identified as Bacillus pumilus strain TNP93. In the GenBank database, the accession number of the TNP93 strain's 16S rRNA gene sequence (1288 bp) is MH686032 (https://www.ncbi.nlm.nih.gov/nuccore/MH686032.1). The phylogenetic tree of the TNP93 strain constructed by MEGA6 is shown in Fig. 2.

Fig. 2.

Phylogenetic tree of strain TNP93

Production of Protease

Optimum growth and protease-producing conditions of the strain TNP93 were determined as pH 10 and 40 °C on SMA plates. After the enzyme production in SM broth for 40 h at 40 °C, centrifugation was performed to obtain the supernatant to be used as a raw enzyme source in the next experiments. The obtained enzyme had 7.60 U of total activity and 5.90 U/mg of the specific activity.

Characterization Parameters of TNP93 Protease

SDS-PAGE and Zymogram Analysis of TNP93 Protease

The molecular mass and homogeneity of TNP93 protease were detected by SDS-PAGE under reducing conditions. As seen in Fig. 3 Lane 2, nine protein bands with various molecular weights were observed. The zymography of B. pumilus strain TNP93 protease was done with 12% gel containing skimmed milk (0.5%, w/v) under non-reducing conditions (Fig. 3, Lane 3). The hydrolysis zone formed in the zymogram gel was compared with the protein band at the same level in the SDS-PAGE gel analyzed under the same conditions (Fig. 3, Lane 2). The formation of a clear hydrolytic zone of proteolysis against a dark blue background confirmed the presence of a single band of about 85 kDa.

Fig. 3.

SDS-PAGE and Zymogram analysis of TNP93 protease. 1. lane: Standard protein ladder; 2. lane: SDS-PAGE of TNP93 protease; 3. lane: Proteolysis zone of TNP93 protease

Effect of pH and Temperature on TNP93 Protease Activity and Stability

TNP93 enzyme displayed its optimal activity at pH 10 (Fig. 4a). The enzyme retained 96.25% activity in the pH range of 8–11 and this indicates that it is an alkaline protease. According to the pH stability evaluation of TNP93 protease, more than half of its initial activity in a range of pH 6–12 for 24 h remained (Fig. 4b).

Fig. 4.

Influence of pH on activity (a) and stability (b) of TNP93 protease. Each value represents the mean of three assays and standard error bars are shown

TNP93 protease was active at all the temperatures tested (30–80 °C), with an optimal activity at 60 °C (Fig. 5a). As seen in Fig. 5b, the enzyme showed high stability after 24 h of incubation at 30–70 °C, maintaining an average of 94% of its initial activity.

Fig. 5.

Influence of temperature on activity (a) and stability (b) of TNP93 protease. Results are the average of three assays and error bars are shown

Effects of Various Chemicals on Protease Stability

The influences of some metallic ions, inhibitors, organic solvents, surfactants, oxidizing agents, and NaCl on the stability of the enzyme were examined as described in the Methods section. All results are given in Table 1. While the TNP93 protease stability was almost completely preserved in the presence of Ba²⁺, Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, and Zn²⁺, Mn²⁺ increased the stability by 21%. TNP93 protease maintained almost all of its activity when exposed to the serine protease inhibitors PMSF (5 mM) and TLCK (1 mM) [40], indicating that it is not included in the serine protease family. A slight inhibition of enzyme stability of approximately 10% occurred in the presence of 5 mM EDTA and EGTA, while the enzyme remained stable when exposed to 1 mM 1,10-phenanthroline.

Table 1.

Effects of various chemicals on TNP93 protease stability

Chemicals	Concentrations	Residual activity (%) ± SD*
None (control)	–	100
Metal ions
BaCl2	5 mM	109 ± 0.02
CaCl2	5 mM	103 ± 0.04
CoCl2	5 mM	100 ± 0.02
CuCl2	5 mM	108 ± 0.04
MgCl2	5 mM	99 ± 0.01
MnCl2	5 mM	121 ± 0.01
ZnCl2	5 mM	103 ± 0.02
Inhibitors/chelators
PMSF	5 mM	97 ± 0.01
TLCK	1 mM	90 ± 0.02
EDTA	5 mM	90 ± 0.01
EGTA	5 mM	87.9 ± 0.01
1,10-Phenanthroline	1 mM	106 ± 0.04
β-mercaptoethanol	0.05%	39 ± 1.46
Iodoacetamide	5 mM	56 ± 1.71
Urea	4 M	69 ± 0
Na-Sulfide	5 mM	106 ± 0.02
Surfactants/oxidizing agent
SDS	0.5%	77 ± 1.34
TritonX-100	1%	95 ± 1.63
Tween 20	1%	84 ± 1.07
Tween 80	1%	124 ± 0.14
H2O2	5%	104 ± 0.01
Organic solvents
Acetone	50%	60 ± 0.01
n-Butanol	50%	52 ± 0.01
Ethanol	50%	49.1 ± 0
Isopropanol	50%	51 ± 0.01
Methanol	50%	53.33 ± 0.01
NaCl
	1%	94 ± 0.43
	5%	85 ± 0.13
	10%	59 ± 0.65
	15%	35 ± 0.22

^*Values represent the means of three replicates. The control as 100% was the protease activity without incubation with metal ions or chemical reagents and ± standard deviations (SD) are shown

The activity of TNP93 protease decreased sharply to 39% when pre-incubated with 0.05% β-mercaptoethanol. Similar to β-mercaptoethanol, 44% inhibition in enzyme activity with iodoacetamide (5 mM) supports that the TNP93 protease is a cysteine protease [41]. Na-sulphide had almost no influence on enzyme stability.

Urea, a hydrogen bond breaker [34], inhibited the TNP93 protease by 31%. In the presence of 0.5% SDS, 1% TritonX-100, and Tween20, TNP93 protease retained 77, 95, and 84% of its stability, respectively. Besides, Tween 80 increased the initial activity of the protease by 24%. Furthermore, TNP93 protease retained all its initial stability after treatment with 5% H₂O₂ (104%). Upon incubation with 50% of acetone, n-butanol, ethanol, isopropanol, and methanol for 1 h, TNP93 protease exhibited 60, 52, 49.1, 51, and 53.33% stability, respectively.

The stability of the TNP93 protease after the incubation with 1, 5, 10, and 15% (w/v) NaCl concentrations was calculated as 94, 85, 59, and 35%, respectively. At the same time, the enzyme stability was affected considerably at high NaCl concentrations (10 and 15%).

End Product Analysis by TLC

The final products of casein degradation were analyzed qualitatively by TLC. As shown in Fig. 6, alkaline protease TNP93 effectively hydrolyzed casein and produced tyrosine, aspartic acid, glycine, and cysteine.

Fig. 6.

TLC of enzymatic hydrolysis products of casein by TNP93 protease. Marker amino acids; (Arg) Arginine, (His) Histidine, (Cys) Cysteine, (Gly) Glycine, (Asp) Aspartic acid, and (T) Tyrosine. (P) Hydrolysis products of casein by TNP93 protease

Compatibility of Protease with Some Commercial Detergents

The compatibility and stability of the TNP93 protease were tested with enzyme-based commercial laundry detergents. As shown in Table 2, the enzyme maintained 72 and 84% of its original activity against ABC and Ariel, respectively.

Table 2.

Compatibility of TNP93 alkaline protease with commercial laundry detergents

Commercial detergent (1%)	Residual activity ± SD (%)
Control	100
ABC (powder)	72 ± 2.07
Ariel (liquid)	84 ± 1.95

The enzyme activity without detergent was considered as control (100%)

The Protein-Based Stain Removal Activity of the Protease

The capacity of the enzyme to remove protein-based stains was demonstrated in Fig. 7. Washing with commercial liquid detergent only showed better stain removal performance on both blood and egg yolk stains compared to washing with TNP93 enzyme alone. However, it has been observed that the combined use of the enzyme and detergent is more effective in removing stains than washing with detergent alone, as displayed in Fig. 7A (v) and B (v).

Fig. 7.

Protein-based stain removal activity of TNP93 protease A Blood stained fabrics; B Egg yolk stained fabrics; (i) untreated stained fabrics (control); (ii) stained fabrics washed with tap water; (iii) stained fabric washed with liquid detergent; (iv) stained fabric washed with TNP93 protease; (v) stained fabric washed with TNP93 protease and liquid detergent

According to the results of enzymatic activity analysis at 660 nm using washing water, the highest efficiency in removing stains was obtained in washing water in which liquid detergent and enzyme were used together (Fig. 8a and 8b).

Fig. 8.

a Residual hydrolysis products in the wash water of blood stain, b Residual hydrolysis products in the wash water of egg yolk stain

Discussion

This study aims to isolate and characterize an alkaline and a thermophilic protease from a bacterium for use in the laundry detergent industry. For this purpose, an alkaline Bacillus pumilus strain TNP93 was isolated and then a thermo-active alkaline protease obtained from the strain was produced and characterized. B. pumilus is one of the prominent Bacillus species involved in many studies about the production and characterization of alkaline and thermophilic protease [9, 42].

Proteases from B. pumilus were investigated for their use in washing and cleaning agents. Besides having optimal activity at high pH (> 10) and temperatures (> 65 °C), they also show significant stability and compatibility against oxidants, denaturing agents, and bleaches [35].

In this study, the protease produced by B. pumilus TNP93 at 40 °C and pH 9 was characterized as described in the Methods section. Only one of the nine protein bands determined in SDS-PAGE analysis exhibited proteolytic activity, and the molecular weight of this band was calculated as 85 kDa (Fig. 3, Lane 2 and 3). Our result is consistent with the proteases of B. pseudofirmus SVB1 (85 kDa) [41] and Bacillus sp. SM2014 (71 kDa) [43]. However, in previous works, alkaline proteases with different molecular weights from TNP93 such as B. halotolerans strain CT2 (250 kDa) [4], B. halodurans strain US193 (37 kDa) [44], and B.safensis strain RH12 (28 kDa) [39] were reported. The presence of a single proteolytic activity band of the enzyme on the zymogram gel indicates that TNP93 protease is a monomeric enzyme.

In the analysis in which the effects of pH on enzyme activity were determined, TNP93 protease showed its optimum activity at pH 10. Moreover, it retained about 96 and 95% activity at pH 9 and 11, respectively (Fig. 4a). It was stable at an average of 54% in a range of pH 6–12 for 24 h at 25 °C (Fig. 4b). Generally, the optimal pH range at which laundry detergents work is 9–11 [35]. Based on the pH stability and activity values of TNP93 enzyme, it is a quite suitable enzyme to be incorporated in laundry detergent formulations [4]. These results were found to be similar to the previous research [9, 25, 45]. Compared to the proteases in the formulations of some commercial detergents such as AlcalaseTM (Novozymes) (from B. licheniformis, max. activity pH 8–9) and SavinaseTM (Novozymes) (from B. clausii, max. activity pH 8–10), it can be said that TNP93 protease is a highly effective enzyme in alkaline conditions [35].

Considering the analysis in which the optimum temperature was determined, TNP93 protease was optimally active at 60 °C and exhibited 83% activity at 80 °C (Fig. 5a). According to these results, TNP93 enzyme can be classified as a moderately thermo-active protease [33]. Furthermore, the enzyme is highly stable at between 30–70 °C and it maintained 88% of its initial activity even at 70 °C (Fig. 5b). Due to its thermostable nature, TNP93 protease may be a preferable enzyme for high-temperature washing in the detergent industry. Similar data were obtained in previous studies in the literature. Shaikh et al. [25] reported the influence of the temperature (30–70 °C) on the activity of APP-07 alkaline protease. The outcomes indicated that the enzyme had been optimally active at 55 °C. Also, it was determined that while the APP-07 enzyme was stable up to 60 °C, there was a decrease in the stability of the enzyme above 60 °C. Bhatt and Singh [31] pointed out that protease from B. lehensis JO-26 was active at temperatures of 30–70 °C with optimum activity at 50 °C and stable in the range of 40–80 °C. The JO-26 protease was completely stable at 40 °C after 20 h of incubation, while retaining 62% of its activity at 50 °C. It completely lost its stability after 3 h of incubation at 80 °C.

Among the various metal ions analyzed, TNP93 protease activity was induced in the presence of Mn²⁺. The enzyme maintained all of its original activity when exposed to metal ions (Table 1). These data are compatible with the literature [25, 33, 46]. Ca²⁺, Mg²⁺, and Mn²⁺ ions play an important role in protecting the enzyme from thermal denaturation and maintaining its structure [47–49].

It is very important to determine the effects of inhibitors on the enzyme as it gives information about the nature of the enzyme's active site and whether the enzyme needs a cofactor [19]. In the presence of PMSF (5 mM), a chemical that inhibits enzymatic activity by blocking the serine residues in the enzyme's active site [34, 50], the TNP93 enzyme preserved its initial activity. 1 mM of TLCK, a chymotrypsin alkylating agent [51], did not exhibit a notable inhibitory effect on TNP93 protease. EDTA and EGTA, metalloprotease inhibitors, remove metal ion(s) required for the activity of enzymes by chelating divalent cations. Thus, it causes a decrease in enzymatic activity [52]. TNP93 protease preserved 90% and 88% of its activity when exposed to 5 mM EGTA and EDTA, respectively. Besides, TNP93 protease remained stable when treated with 1 mM 1,10-phenanthroline (metalloprotease inhibitor). Our data show that the TNP93 protease does not need metal cofactor(s) for its activity [53]. The enzyme's resistance to chelating agents, which are used as stain removers and water softeners in detergent formulations [53], is a valuable feature in terms of the enzyme's functionality as a detergent additive [43].

The 61% inhibition of the enzyme in the presence of 0.05% β-mercaptoethanol (cysteine protease inhibitor) indicates that the -SH groups and cysteine residues may be located in the enzyme’s active site and they are important for the enzyme stability [4]. Furthermore, the decrease in enzyme stability in the presence of β-mercaptoethanol indicates that TNP93 enzyme is a cysteine protease [19] and may be a thiol-dependent enzyme [54]. Compared with the findings in the literature, it was observed that TNP93 protease lost its activity strongly when treated with β-mercaptoethanol [29, 55, 56]. In addition, TNP93 alkaline protease had almost lost half of its stability in the presence of 5 mM iodoacetamide (cysteine protease inhibitor). This is another indicator that the enzyme is a cysteine protease.

A protease to be included in a composition of detergent must be compatible with oxidizing agents, bleaches, and surfactants, which are the components of the formulation [57]. As shown in Table 1, it was determined that TNP93 protease preserved its activity at good rates with anionic and non-ionic surfactants (SDS, TritonX-100, Tween 20, and Tween 80). In particular, SDS likely increases the access of the enzyme's active site to the substrate and improves the interaction between the substrate and enzyme [4].

Additionally, the initial activity of the TNP93 protease was preserved with H₂O₂, a strong oxidation reagent found in the composition of the detergent [19]. H₂O₂ inactivates the enzyme by oxidizing the methionine residue in the structure of the enzyme [58]. Therefore, most alkaline proteases show low activity against peroxides used in bleach-based detergents [19]. This indicates that TNP93 protease is an enzyme with a oxidant-stable nature. This feature highlights the usability of the enzyme in the detergent industry. In line with all these results, it is seen that our enzyme is a feasible laundry detergent additive candidate for the washing industry in which surfactants and oxidizing agents are used [42, 59].

The TNP93 protease was moderately stable when exposed to acetone, ethanol, n-butanol, isopropanol, and methanol (Table 1). When the water molecules in the enzyme's structure are replaced by the molecules of the organic solvent to which it is exposed, the enzyme's structure becomes stabilized [60]. Accordingly, it can be concluded that the structure of the solvent chemicals tested in our study is effective in the stability of the TNP93 protease.

NaCl is an important ingredient in detergent formulations [61] and is used in the granulation of the protease to be added to detergents [35]. As shown in Table 1, TNP93 protease showed a halotolerant feature. And also, the activity of the TNP93 enzyme when exposed to various concentrations of NaCl indicates that it can be an effective detergent additive for laundry detergents used in washing applications with high salt-content groundwater [52, 62].

It is expected that the protease to be added to detergents will have some properties such as ensuring the degradation of proteinaceous stains and being compatible with other components of the detergent [22]. The proteinaceous stain removal efficiency showed that TNP93 protease can enhance the washing performance of commercial laundry detergent (Fig. 7A (iii) and (v), 7B (iii) and (v)). Similar results have been obtained from some studies notifying the usage of alkaline proteases from various Bacillus species in facilitating the removal of blood and egg stains from cotton fabric [22, 34, 42, 63].

In addition, the data obtained by spectrophotometric measurement of the protein hydrolysis products transferred to the washing water (Fig. 8) support the results of our visually evaluated stain removal experiment (Fig. 7). Based on our research of the literature, we can say that the activity analysis from washing water was first performed in this study. We predict that this novel parameter will provide a different perspective for researchers.

In this regard, according to the results of the characterization experiments, the compatibility tests with the detergent components, and the washing performance analysis, TNP93 protease from natural strain was found to be suitable for use as a biological additive in various commercial laundry detergent formulations. TNP93 protease, as an eco-friendly biological agent, is an important potential candidate that can reduce the harmful effects of chemical agents used in commercial detergents on nature.

Author Contribution

The authors planned the research, performed all the assays, analyzed the results, wrote, and edited the article.

Funding

The Scientific Research Project Unit of Cukurova University supported this work (Grant number: FBA-2018–10883).

Data Availability Statement

All data belonging to this study are included in this article.

Declarations

Ethics approval There are no studies with humans or animals in this article.

Supplementary Information

No supplementary files.

Conflict of interest

There is no conflict of interest between the authors.