Exploration of Compost Soil for the Production of Thermo-Stable Bacillus Protease to Synthesize Bioactive Compounds through Soy Protein Hydrolysis

Abstract

Application of bioactive peptides (BAPs) is promising due to their potential antimicrobial, antioxidant, agonistic, and ACE inhibition properties. To achieve a stable and active peptide at relatively high pH and temperatures by microbial fermentation, a wide variety of microorganisms need to be explored from diverse habitats, and compost is the excellent source. In an attempt to isolate potent protease-producing bacteria, gelatin-supplemented DM agar medium was used. Out of 140 pure cultures, initial protease production selects isolate D3L/1 (26 U/mL), and 16S rDNA sequencing confirmed it as Bacillus subtilis. Protease production was increased to 55.55 U/mL, with pH 7.5, 1% glucose, 1% casein, 1% ammonium sulfate, for 96 h of fermentation, at 37 °C under 140 rpm of shaking. Ion-exchange, and size-exclusion chromatography, 30 KDa protease was purified up to 4.1-fold (specific activity 3448.62 U/mL; 67.66% yield). The enzyme was active under broad temperatures (60 °C optimum), organic solvents, and pH variations. A total of 5% H₂O₂ can only reduce 40% of enzyme activity. However, 1 mM, Fe²⁺, and Cu²⁺ increased enzyme activity by five times. Soy hydrolysis (SPI) byD3L/1 protease produces bioactive compound (<3 KDa), which confirmed the peptide bond in the far UV region (205 nm, 215 nm, 225 nm, and 280 nm). The compound was ineffective towards Serratia marcescens but active against Escherechia coli (47%), Staphylococcus aureus (28%), and Pseudomonas aeruginosa (12%).

1. Introduction

Production of protease through microbial fermentation is highly demanding, which can be applied not only in detergent, food, medicine, and tannery but also in textile, leather processing, animal feed, and chemical industries [1]. Proteases are progressively limiting the use of inorganic chemicals and solvents [2]. Among their broad applications, the formation of bioactive peptides (BAPs) is promising both for food and medical aspects. Bioactive peptides from different protein sources and their potential antimicrobial role is established already. Measuring antioxidant activity, agonistic property, antagonistic activity, and angiotensin I converting enzyme (ACE) inhibitory activity are the additional characterizations for them. Under controlled conditions, the enzyme works on exposed polar groups of a protein by affecting the molecular size, hydrophobicity, and functional properties of proteins. As enzymatic hydrolysis can control the protein emulsification and foam formation process, it can affect their application as food ingredients [3]. Synthesis of a bioactive peptide from food was primarily performed by protein hydrolysis either by using digestive enzymes or by microbial fermentation [4]. For proteolytic enzyme production, a wide variety of microorganisms are being explored from diverse habitats, which indicate predominating genera of Bacillus due to their significant enzyme activity and stability at relatively high pH and temperatures [5,6]. Furthermore, extensive information about cell physiology, biochemistry, and genetics of Bacillus species facilitates these organisms for their wide range of industrial applications [1].

Compost is a reservoir of microorganisms with high degradability and physiological activity [7]. Cellulolytic, amylolytic, and proteolytic bacteria appear in high concentrations during the process [7]. Humification of high lignin waste increases the concentrations of ligninase, xylanase, protease, and urease. However, raw materials with low lignin content enhance cellulase, beta-glucosidase, and alkaline phosphomonoesterase concentrations [8].Hence, utilization of soil microbial resources is a crucial task, and compost is a unique niche of microbial habitat. It acts as self-heating, aerobic or anaerobic, biodegradation, to convert organic matter present in wastes [9,10]. For the degradation of native proteins into smaller subunits (amino acids) different types of proteases such as serine protease (EC 3.4.21), cysteine (thiol) protease (EC 3.4.22), aspartic protease (EC 3.4.23), and metalloprotease (EC 3:4.24) are used. Proteases are not a single enzyme but a mixture of endo-protease and exo-proteases, and amidases enzymes [11,12,13,14]. In early 1977 Priest et al. [15] demonstrated that during composting, bacteria at the late phase of their growth cycle can secrete proteases, esterases, and many other kinds of extracellular enzymes, and among the producers spore-forming Gram-positive B. Subtilis is predominating. It can produce both alkaline and neutral proteases [16]. Temperature-sensitive neutral proteases (metalloprotease group) can work actively in a narrow pH range (pH 5.0–8.0) [17,18]. A detailed analysis of composting revealed that environmental parameters, carbon, and nitrogen sources simultaneously affect the growth of bacteria (1). Apart from that, temperature, aeration, pH, the concentration of carbon, nitrogen sources, and enzyme/substrate (E/S) ratio are the crucial parameters to be monitored during bioactive peptide synthesis [3].

To date, milk proteins are considered the largest number of bioactive peptides source [19,20,21]. However, soybean meal is a second important source of these bioactive compounds [22,23,24]. The formation of bioactive peptides can entirely depend on the type of enzyme used. Those peptides can undergo physiological transformations as they are passing through the gastrointestinal tract which determines their bioavailability and activity [25]. Therefore, the complete understanding of the hydrolyzing enzyme is the prerequisite for the analysis of bioactive peptides’ effectiveness as the diet requires their active sequences which can resist gastrointestinal digestion. The present study focuses on compost soil exploration to screen a high-yielding, novel, extracellular protease-producer, and its growth parameters, purification, and characterization of protease to synthesize bioactive compounds from natural sources by cost-effective enzyme hydrolysis.

2. Materials and Methods

2.1. Sample Collection

Soil samples were collected from diverse compost piles of Shantiniketan and its surrounding areas such as Subhahspally (SUB), Ratanpally (RAT), Bolpur (B), and Dubrajpur (D), located in Birbhum District of West Bengal (23°39′46.01″ N, 87°41′49.02″ E) in India. A total of 20 gm of samples were collected separately from the upper layer (U), middle layer (M), and lower/bottom layer (L) of these piles in a plastic bag and were labeled according to their place and area of collection mentioned above. All of them were initially enriched with Carboxy Methyl Cellulose to obtainan overall population of soil isolates [2,26]. For isolating, protease producer organisms were selected based on their ability to utilize casein as a substrate.

2.2. Bacterial Growth and Medium Used

For the isolation of the proteolytic organism from enriched compost soil, Czapek Mineral Salt Agar medium containing (gL⁻¹) NaNO₃ 2, K₂HPO₄ 1, MgSO₄, 7H₂O 0.5, KCl 0.5, CMC 5.0, Peptone 2.0, Agar 15, Gelatin 10, and pH 7.0 was used as growth medium [26]. Inoculated plates were incubated at 37 °C for 2 days, and the selected screened isolates were maintained at 4 °C in the slants of the same medium. Inorganic broth of Davis et al. [27] containing (gL⁻¹) KH₂PO₄ 3.0, Sodium citrate 0.5, K₂HPO₄ 7.0, MgSO₄, 7H₂O 0.1, (NH₄)₂SO₄ 1.0, Glucose 10, pH 7.0, and Casein 10.0 as substrate was used as production medium. A total of 2% of 24 h old broth culture (10⁸ CFU/mL) was inoculated in the sterilized medium and incubated at 37 °C under shaking (140 rpm). Bacterial growth (OD) was measured at 600 nm and biomass was separated through centrifugation (at 5000 rpm for 30 min) [28]. The cell-free extract served as the crude source of enzyme and was stored for further assay.

2.3. Identification of Bacteria

2.3.1. Morphological and Biochemical Analysis

Morphological characters, such as cell shape and size (length and width), were measured through micrometry. Identification of bacteria was performed with Gram staining and endospore staining by following standard protocols [26]. To check biochemical characters, carbohydrate utilization by the selected isolate was determined through the HiCarboTm kit.

2.3.2. PCR Amplification and 16S rDNA Sequencing

The 16S rRNA genes from the bacterial cell were extracted through boiling and were amplified using bacterial specific primers f27 and r1492 [29]. The PCR amplification was performed using high fidelity PCR Master Kit (Roche Applied Science, Penzberg, Germany) by following the manufacturer’s instructions. To obtain 50 μL of final reaction volume, a 25 μL PCR master mix (provided by the manufacturer) was mixed with 300 nM concentration of each primer. The thermal cycles were as follows: 94 °C for 5 min; for 30 cycles each consisting of 30 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C; and finally 72 °C for 7 min to complete the process. Electrophoresis of the PCR products was performedon agarose gel prepared in 1× TAE (20 mM Tris-acetate, 0.5 mM EDTA, pH8), followed by excision and gelelution by using Qiagen gelelution kit. The 16S rDNA sequences from the PCR products were determined using ‘universal primers’ 27f, 357f, 530f, 704f, 926f, 1242f, 321r, 685r, 907r, 1069r, and 1220r [30], a terminator sequencing kit, and an automated DNA sequencer ABI 377 (Applied Biosystems, Waltham, MA, USA). The 16S rDNA sequence of the new isolate was compared against other available sequences in the EMBL, GenBank, and DDBJ databases using FASTA (version 3.4t) [31].

2.4. Optimization of Fermentation Condition

To identify the optimum fermentation period, the isolate D3L/1 was inoculated (10⁸ CFU/mL) in sterile production broth and incubated at 37 °C for 24 to 120 h with an agitation of 140 rpm. To acquire the optimal pH of the fermentation broth, the media pH was adjusted to a different range before sterilization. The effect of carbon source on bacterial growth and enzyme production was determined by inoculating sterile broth with variable percentages of glucose (1 to 6% w/v) [16]. To check the substrate effect on protease production, 1% casein, 1% gelatin, and 1% skim milk was supplemented separately in the sterile production medium and incubated with isolate D3L/1 for 96 h at 37 °C with an agitation of 140 rpm (Supplementary Figure S1). For analyzing the effect of nitrogen source, the production media was supplemented with 1% of different nitrogen sources such as Ammonium sulfate (AS), Ammonium persulfate (APS), and Ammonium ferrous sulfate (AFS) separately, and incubated for 96 h [32]. Regular production media with 0.1% Ammonium sulfate was the control for the experiment. The effect of the nitrogen source and its concentration optima was determined by inoculating working isolate D3L/1 in sterile broth medium with various nitrogen sources with concentrations differences (1–4 % w/v) and incubated for 96 h.

2.5. Purification and Characterization of Protease

In the purification procedure, the cell-free extract containing all the protease activity was used as the starting material. To obtain 80% supersaturation, varied amounts of ammonium sulfate were added slowly to the cell-free extract containing crude enzyme under stirring conditions at 4 °C to precipitate it [33]. The precipitate was then collected through centrifugation at 5000 rpm for 20 min and dissolved in 1 mM Sodium phosphate buffer pH 7.6. The semi-purified enzyme was dialyzed against the same buffer overnight at 4 °C under stirring conditions [34]. The dialysis bag was kept in a Petri dish containing PEG20000 at 4 °C to remove excess water to concentrate the protein sample up to 30 mL (desired volume) and stored at −20 °C [35]. To purify near homogeneity, the semi-purified protease loaded on the DEAE-Cellulose (DE52) anion exchange column, equilibrated with 100 mM sodium phosphate buffer (pH 7.6).The column was washed with 100 mM sodium phosphate buffer (pH 7.6) until the absorbance of the flow-through at 280 nm became constant. The bound protease was eluted by using the same buffer with an increasing concentration (0.1 to 1.0 M) of sodium chloride (NaCl) [35]. The fractions with protease (15 mL) were then pooled and concentrated by lyophilization at 4 °C. The pooled fraction of the anion-exchange column (DE52) was loaded onto Sephadex G-100 size-exclusion chromatographic column which was equilibrated before with 100 mM sodium phosphate buffer (pH 7.6). The protein was eluted with the same buffer having a flow rate of 0.25 mL/min. Enzyme activity of each fraction was determined and fractions that showed protease activity were pooled (5 mL) for further analysis. The amount of protein in each step was determined with BSA as a standard [36]. The specific activity of bacterial protease was expressed as unit/mg.

Molecular weight of the purified protease was determined by SDS-PAGE. The 12% gel was prepared by following the method of Laemmli [35] and stained with Coomassie Blue R250 prepared in methanol: acetic acid: water (4:1:5, v/v). The destaining solution used in this experiment has the same composition methanol:acetic acid:water (4:1:5, v/v) except Coomassie blue [34].

The protease was incubated in a water bath at different temperatures for 30 min to check the effect of temperature on its stability [37]. Total protease activity (100%) at a specific temperature indicated its optimum temperature. Before measuring the enzyme activity, the treated enzymes were placed immediately on ice. Using the modified method of Anson, described later, the enzyme activity was calculated for both time zero and after the respective time of incubation.

To check the effect of metal ions on extracellular protease activity the divalent and monovalent cations were added to the reaction mixture to achieve a 1 mM final concentration [34]. The enzyme assay was carried out following standard procedures with Ca²⁺, Mg²⁺, Ni²⁺, Fe²⁺, Cu²⁺, Mn²⁺, Na⁺, and Zn²⁺.

To check the pH effect on protease activity, the reaction mixture contained 1 mL of the enzyme with 2 mL of 100 mM buffers (acetate/sodium phosphate/Tris-HCl, as suggested) adjusted to pH of 3.4 to 9.0 having 0.5% of casein, and a standard assay was performed [37]. Under diverse pH ranges, the stability of protease was measured by incubating the enzyme at pH 3.4 to 9.0 for 24 h, and residual activity was estimated.

Checking the effect of organic solvents on enzyme stability was determined by mixing an equal volume of semi-purified protease (1 mL) with organic solvents for 24 h at room temperature (1); 50% (v/v) phenol, cyclohexane, methanol, propanol, dichloroethane acetone, and ethanol were solvents of choice;distilled water was the control. The next day, standard assay procedure was applied to measure residual enzyme activity.

To examine the effect of denaturing agents on enzyme activity an enzyme and various denaturing agents were mixed separately in equal (v/v). Both ionic and non-ionic surfactants such as5% Triton ×100, Tween 80, and 5% SDS, were used. To understand the consequences of oxidizing agents on protease activity,1 mL of hydrogen peroxide (5% H₂O₂) was mixed with 1 mL of cell-free extract and was allowed to stand for 24 h at room temperature, followed by the measurement of residual activities as per protocol [1,38].

2.6. Enzyme Assays

2.6.1. Gelatin Hydrolysis

The protease activity was determined by measuring the gelatin clearing zone (GCZ) on Davis Mingoli’s (DM) agar media [39]. In this assay, culture plates containing DM agar medium of pH 7.0 were supplemented with (1% w/v) soluble gelatin inoculated with test organisms and were incubated at 37 °C for 72 h [26]. After incubation, freshly prepared Mercuric chloride (HgCl₂) in hydrochloric acid (HCl) solution (HgCl₂ 15 g and 20 mL of 6N HCl to obtain a final volume of 100 mL) was added [40], and the mean diameters of the cleared zone were calculated and expressed as millimeters [32].

2.6.2. Quantitative Estimation of Protease Activity

The activity of protease was determined by the modified method of Anson [27]. One unit is defined as the e that hydrolyzes casein to develop a color equivalent to 1.0 μmole of tyrosine per minute in standard assay condition and represent as U/mL. Tyrosine was the standard in quantitative estimation.

2.6.3. Soy Protein Hydrolysis Assay

Soy protein was hydrolyzed following the standard assay proposed by Coscueta et al. [41]. The soy protein isolate (SPI) was obtained by treating defatted soybean meal at 70 °C. Before enzyme addition, 1% aqueous SPI (100 mL) was heated to solubilize. To continue proteolysis, 1% w/w D3L/1 protease was added (enzyme: substrate =10 mg enzyme/g SPI). The enzymatic hydrolysis continued for 3 h at 60 °C with the optimum pH of 7.6. After every hour, 2 mL aliquots were stored. The enzyme inactivation at 80 °C for 20 min stopped the reaction finally. The entire reaction mixture was centrifuged at 2370× g for 45 min to separate the supernatant. In the end, the soy protein hydrolysates (SPHs) were used for their antimicrobial property checking and stored at −80 °C. The non-hydrolyzed initial sample (before enzyme addition) was used as a control.

2.6.4. Bioactive Compound Synthesis from Casein, Skim Milk and Soybean Meal

To obtain bioactive compounds from casein and skim milk with B. subtilis D3L/1 protease, 5 gm of each sample was dissolved into 100 mL of distilled water separately. Then, 5% inoculum (D3L/1 protease) was added upon complete solubilization of casein and skim milk in water. With 0.1 N NaOH, the pH of the reaction mixture was adjusted to 9.5 and was kept for 6 h at 45 °C. Heating at 90 °C for 15 min stopped the reaction. Upon completion, the final temperature was 25 °C, and the final pH was adjusted to pH 7.0. The reaction mixture was centrifuged at 10,664× g for 15 min to separate bioactive compounds. The supernatant was collected for antimicrobial activity and kept at −80 °C for further analysis [37,42].

Aqueous soy protein extract (a defatted soybean meal in water) was treated at 70 °C for 1 h to obtain the bioactive compounds, designated as soy protein isolate (SPI). The active compound to water ratio was maintained at 1:10 with an initial pH of 8.5 following standard assay conditions [43]. The extract was centrifuged at 2370× g for 45 min, and the compound was precipitated by acidification with 2 N HCl of pH 4.5. The precipitate was collected by centrifugation at 2370× g for 30 min to perform the soy protein hydrolysis assay.

2.7. Partial Identification of Bioactive Compound

Ultrafiltration determined the size of the active compound in soy protein hydrolysates (SPHs) [44]. The 3 KDa, 10 KDa, and 30 KDa cutoff filters were used (Amicon Ultra-0.5 Centrifugal Filter Unit) following the manufacturer’s instructions. SPH fractions SPH-F1-SPH-F4 (>30 KDa, 10–30 KDa, 3–10 KDa, and <3 KDa) were represented based on the approximate size of the active compound. All the fractions were analyzed for antimicrobial properties and the type of active compound was verified through UV Vis spectroscopy by measuring absorbance at 205 nm, 215 nm, 225 nm, and 280 nm [45,46].

2.8. Determination of Antimicrobial Activity

The antimicrobial assay of bioactive compounds generated from all three sources was performed by checking both growth inhibition of E. coli, S. aureus, P. aeruginosa, and S. marcescens in Nutrient Broth medium supplemented with bioactive compound (1:50) and in nutrient agar (NA) media by measuring zone of clearance. To check the growth suppression (if any) by different bioactive compounds, OD₆₀₀ was measured after 48 h of incubation. In parallel, 100 μL of the S. aureus, P. aeruginosa, S. marcescens, and E. coli were added aseptically in 4 different nutrient agar plates. After solidification, wells were formed using a cork borer to add 20 μL bioactive compounds (final concentration 30 µg) from different time of hydrolysis and purification steps including control. Plate activity was monitored by placing a Kanamycin antibiotic disc (30 µg/disc) on one side of the experimental plate inoculated with each organism. The inoculated plates were incubated at 37 °C for 3 days to check the clear zone formation [47].

2.9. Statistical Analysis

All experiments were conducted in triplicate parallel sets. The values reported are means ± SD calculated as described by Snedecor et al. [48]. To check the statistical significance pairwise t-test was executed.

3. Results

3.1. Microbial Enzyme Production and Application

3.1.1. Isolation and Screening of Bacteria

In this study, 140 bacteria were isolated from 44 different compost samples. Depending on their enzyme production on DM agar media, 24 isolates were selected further. The preliminary selection depends on clear zone formation in the DM agar plates. Isolates utilized gelatin for protease production, and after 72 h of incubation, the maximum zone of clearance was formed by isolate D3L/1 (34 mm diameter) whereas the minimum (10 mm diameter) zone was created by isolate SUB3/2. The bacterial load of compost soil varied from 7 × 10² to 1.6 × 10⁴ CFU/g of dry wt. For next level of screening, 24 isolates were analyzed for growth rate and quantity of the enzyme produced. Based on their quantitative protease production (Figure 1A) and growth rate (Figure 1B), six isolates were selected for further analysis. Upon 96 h of incubation, compared with others, isolate D3L/1 produced a maximum of 26 U/mL of protease.

3.1.2. Identification of Isolate

Identification of the working isolate was performed by analyzing morphological, cultural biochemical (Table 1), and molecular characterizations (Figure 2). Isolate D3L/1 was a rod-shaped, Gram-positive, endospore-forming organism, and its length ranges between 5.3 and 5.5 μm; however, the width of the organism was 1.3–1.5 μm.

The complete 16S r DNA sequence of the selected compost organism isolate D3L/1 (1387 bp) was compared with those existing species available in the previously stated databases with an emphasis on plus/plus and plus/minus strains. Results showed that isolate D3L/1 had maximum homology (99–100%) with different strains of B. subtilis, Priestia megaterium, and Bacillus megaterium. Multiple alignments revealed that selected strain D3L/1 and B. subtilis were completely identical with 99% sequence similarity and differ from B. megaterium in only one nucleotide position. Priestia megaterium and Bacillus megaterium showed only 97–99% similarities with the D3L/1(Figure 2). Based on the Neighbor joining method of phylogenetic tree the working isolate D3L/1 belongs to the same phylogenetic group with B. subtilis (Gen Bank Accession No. GU723508).

3.1.3. Optimization of Physical Condition

• ➢. Effect of incubation period

The working isolate B. subtilis D3L/1 can grow well in the fermentation medium, and the amount of enzyme production relatively depends on time. The patterns of the enzyme as well as biomass production, were recorded with appropriate time intervals. Protease production maximized at the late exponential phase (Figure 3A). Maximum enzyme production (at pH 7.0) was noted at 96 h with the activity of 26 U/mL. The cell growth remained high up to 96 h and declined after.

• ➢. Effect of pH

The effect of pH on protease activity was measured by altering medium pH before sterilization. Here enzyme production was tested over a pH range of 6.5–8.0. For both enzyme production and bacterial growth, recorded optima were at 7.5 (pH 7.5), and the quantity of B. subtilis D3L/1 protease increased from 26 to 55.55 U/mL (Figure 3A). However, an acidic medium (pH 6.5) reduced bacterial growth and enzyme production.

• ➢. Effect of Carbon source concentration

As protease is an inducible enzyme, 1% of glucose maximized the enzyme activity (55.55 U/mL) under optimum conditions (pH 7.5, 96 h of incubation at 37 °C with an agitation of 140 rpm), and the enzyme production reduced drastically when the glucose concentration increased (Figure 3B).

• ➢. Effect of Substrate on Enzyme production

Compared withskim milk and gelatin, 1% casein maximized the enzyme production when supplemented in the medium. Almost 1.13-fold enhancement indicates its specificity towards casein as a substrate of choice. On the contrary, enzyme production suppressed up to 57% and 10%, respectively, when casein is replaced with 1% skim milk and gelatin. However, with skim milk supplementation, bacterial growth increased (Figure 4A).

• ➢. Effect of Nitrogen source and its concentrations

Protease production was increased up to 1.12-fold with 1% ammonium sulfate as a nitrogen source (Figure 4B). Further enhancement of ammonium sulfate concentration decreased enzyme production gradually. Ammonium persulfate follows a similar pattern to ammonium sulfate, and supplementation with more than 1% drastically reduced enzyme production up to two-fold. On the other hand, enzyme production increased progressively with Ammonium ferrous sulfate (Figure 4C). Ammonium ferrous sulfate did not make significant growth differences. However, with the increased concentration of ammonium sulfate and ammonium persulfate, the growth reduced.

3.1.4. Purification and Characterization of Protease

Initial protease activity was 55.55 U/mL in the cell-free extract of B. subtilis D3L/1. To precipitate the enzyme, ammonium sulfate concentrations were increased from 20% to 90% with an enhancement of 10%. The optimum saturation for bacillary protease was 80%. A total of 100 mM Sodium phosphate buffer of pH 7.6 [33] dissolves the resulting precipitate after centrifugation. The enzyme solution was further purified through dialysis using the same buffer. The dialyzed and partially purified enzyme solution was transferred to an ion-exchange column and Sephadex size-exclusion column for additional purification to achieve a 4.1-fold purified protease at the end with 67.66% yield, having a specific activity of 3448.62 U/mg and total protein 1.09 mg (Supplementary Table S1). SDS-PAGE analyses of the protease showed a single band with a molecular weight estimated to be 30 KDa (Figure 5).

• ➢. Thermostability of protease

The thermostability of extracellular protease was determined by treating the enzyme solution for 30 min at different temperatures ranging from 30 to 90 °C. After that, the assay was performed under standard conditions (constant pH of 7.5 and 1% casein concentration) (Figure 6A). While checking the optimum temperature for enzyme activity, the D3L/1 protease was fully functional at 60 °C. However, treatment at 90 °C reduced only 6% of enzyme activity. Thus, the optimum temperature for thermotolerant D3L/1 protease was 60 °C. Compared withexisting enzymes, this D3L/1 protease was temperature-resistant and suitable for industrial, medical, and biotechnological applications.

• ➢. Effect of Metal Ions

The effects of 1 mM monovalent and divalent metal ions in protease activity were estimated. Activities increased approximately five times with Fe²⁺ and Cu²⁺.Zn²⁺ reduced enzyme activity drastically less than one-fold. (Figure 6B).

• ➢. pH effect on Protease Activity and Stability

To check the pH effect on protease stability (Figure 6C), 1 mL of the enzyme and 2 mL of buffer with different pH ranges were mixed for 24 h at 4 °C. The D3L/1 protease was found active significantly from pH 3.6 to pH 9.0, and the optimum pH was 7.6. Under both acidic and alkaline pH, stability was more than 90%. Between pH 6.0–7.6, the enzyme activity increased. The optimum pH for 100% activity of D3L/1 protease was pH 7.6 (Figure 6D). Thus, B. subtilis D3L/1 protease appeared extensively active under wide ranges of pH.

• ➢. Effect of various Inorganic and organic compounds on the Protease Activity

Figure 7A demonstrated the effect of organic solvent on the relative activity of the protease. Methanol, Dichloroethane, Acetone, and Ethanol treatment did not create significant differences. However, enzyme activity increased up to 1% with Cyclohexane and Phenol treatment. Propanol reduced 3% of the relative activity of the protease. Figure 7B represented both ionic and non-ionic surfactants’ effect on D3L/1 protease activity. The relative activity of the D3L/1 enzyme was not changed while treating either with 5% triton ×100 or tween 80. However, anionic surfactant 5% SDS reduced the activity up to 10%. In addition, oxidizing agents (5% H₂O₂) reduced the enzyme activity up to 40%.

3.1.5. Antimicrobial Activity of Bioactive Compound Synthesized from Casein, Soy Protein, and Skim Milk

D3L/1 protease isolated from B. subtilis unable to produce bioactive compound (BAP) from casein and skim milk upon hydrolysis. The promising result was obtained only in soy protein after SPI hydrolysis with D3L/1 protease (1–3 h BAP samples). The bioactive compound upon SPI hydrolysis was effective against all the pathogens except S.marcescens. After 48 h of incubation, E. coli, S. aureus, and P. aeruginosa growth were suppressed, by almost 47%, 28%, and 12% (Figure 8A), respectively. No suppression of growth was observed in casein and skim milk hydrolysates. While analyzing the hydrolysates of different periods, in P. aeruginosa with 1 h BAP, the maximum zone of clearance was recorded. For E. coli and S. aureus, 3 h BAP provided the best result, though 1 hand 2 h BAP both can kill the pathogen (Figure 8B). Ultrafiltration confirmed that SPH-F4 had a bioactive compound with a similar pattern of antimicrobial activity present in 3 h BAP sample, and the approximate compound size was <3 KDa (Figure 8C). Positive biuret test and absorbance at 205 nm indicated the presence of the peptide bond [46]. Absorbance at 215 nm and 225 nm confirmed the concentration of bioactive protein or peptide (based on size) (Supplementary Table S2) [49].

4. Discussion

4.1. Isolation, Screening, and Identification of Bacteria

This report demonstrated the synthesis, purification, and characterization of compost soil-derived extracellular protease and its application for bioactive peptides production from soybean meal. Compost piles are an excellent source of extracellular enzymes of microbial origin, and their stability allows them to be active under different environmental conditions [34]. Bacillus is the most abundant genus. However, Gram-negative genera such as Escherichia, Klebsiella, Aeromonas, Alcaligenes, and Gram-positive Enterococcus [7] also dominate. The 16S rDNA sequencing serves as a molecular chronometer for bacterial identification with morphological and biochemical analysis [50,51].

4.2. Optimization of Enzyme Production

For the successful production of enzymes, cultivation conditions such as incubation period, pH, and temperature are the parameters to be optimized [39]. Malathu et al. [2] reported maximum activity of bacterial protease at pH 7.5, and as the pH shifted either to acidity or alkalinity, activity reduced. On the contrary, production increased when the optimum pH was 6.0–6.5 for B. subtilis [16]. B. subtilis can produce protease maximally between 48 and 96 h [52,53]. The concentrations of carbon and nitrogen sources are present in the basal medium, the pH-dependent solute, and solvent transportation across the cell membrane regulate protease production [39]. Andrade et al. [54] found that protease production reached its maxima with a low concentration of D-glucose (40 g/L). In Mucor circinelloides, glucose was an alternative source for protease production, and the production level dropped suddenly as glucose depleted from the media [16]. Amid et al. [37] checked proteolytic activities using azocasein, casein, gelatin, hemoglobin, and BSA as the substrates of choice. Relative activity maximized with azocasein and minimized with gelatin. On the contrary, Prakash et al. [55] isolated bacterial strains showed maximum activity with gelatin as a substrate.

Not only nitrogen sources’ production of some amino acids can inhibit the growth of proteolytic bacteria, which may also be the reason for reduced enzyme synthesis [56]. Enzyme production is restored only by the removal of amino acids. Among the various ammonium and nitrate salts, ammonium nitrate provided excellent yield for enhancing enzyme activity [57]. Safey and Abdul-Raouf [32] found good bacterial growth and enzyme yield with (NH₄)₂SO₄, whereas Gul et al. [56] found that sodium nitrate is optimum for both alkaline and neutral proteases. For Pseudomonas sp. sodium nitrate increased protease production, and ammonium nitrate suppressed it [58].

4.3. Purification and Characterization of Protease

The residual activity of Bacillus cereus BG1 protease was 89.5% after treating at 55 °C for 15 min [59]. For metalloprotease TKU004, only 10% residual activity remains after 60 °C treatment [60]. Their structural integrity and extraordinary operational stability at a high temperature make them applicable for industrial processes [61]. Metal ions also maintained the active conformation of the enzyme at a relatively high temperature to prevent thermal denaturation [62]. Treatment with Ca²⁺, Mg²⁺, and Mn²⁺ enhanced protease stabilization [16,38]. Though Ca²⁺ and Mn²⁺ had similar effects on B. subtilis D3L/1 protease, the enzyme activity reduced with Mg²⁺. Only serine proteases are not affected by chelating ions [16,61,63]. Synergistes sp. produces a protease that was active under acidic conditions [33], but Bacillus subtilis megatherium worked betterunder alkaline conditions (pH 8.0) [64].

Non-ionic surfactants, such as Tween 20 and Triton X-100, stimulated alkaline proteases activities isolated from Bacillus mojavensis, Bacillus clausii I-52, Bacillus circulans, and Bacillus licheniformis [1]. Both non-ionic (Triton X-100, Tween-80) and ionic surfactant (SDS) maximized the stability of Hylocereus polyrhizus protease [37]. The 5% SDS reduced 27%, and 1 h treatment with 2 M (v/v) hydrogen peroxide reduced 38% of the enzyme activity. Enzyme resistance to those reagents indicates its well-packed structure and native conformation rigidity [65]. On the contrary, 5% SDS reduced only 25% activity of Bacillus sp. KSMK16 protease [66]. However, Aspergillus parasiticus protease retained almost 97% of its residual activity when treated with 2% SDS [67]. Though the residual activity of B. clausii I-52 protease increased approximately between 11 and 17% after 1–5% H₂O₂ treatment [1], activity of Bacillus sp. KSM-KP43 protease was reduced slightly after 10% H₂O₂ treatment for 30 min [68].

4.4. Antimicrobial Activity of Bioactive Compound Synthesized from Soy Protein

The bioactive compound can be isolated from different sources through time and temperature-dependent proteolysis. Heating soy or milk proteins above 70 °C caused quaternary structures dissociation, enzyme subunit degradation, protein aggregation by disulfide interchange mechanisms, and electrostatic or hydrophobic interaction [41,69]. Hydrolysis of soy protein needed control parameters as higher temperature releases bitter peptides [70]. Bioactive peptides with membrane-lytic potential made them significant biologically as bacterial membrane permeability disrupted [21]. These peptides are effective against various pathogens and illnesses [21]. However, their activity depends on the area of application and the type of hydrolyzing enzyme used [69]. On the contrary, upon hydrolysis, negative bioactivity indicated proteolysis. Due to the sudden drop of pH destabilization of protein and exposure of hydrophobic peptides, protein aggregation is initiated, followed by less soluble and thermally unstable biologically inactive peptides [47].

5. Conclusions

The experimental data demonstrated that compost piles are an excellent source of hydrolytic enzyme producers. Among them, B. subtilis D3L/1, a potent thermostable protease producer, was screened for synthesizing bioactive compounds through soy hydrolysis. With 96 h of controlled fermentation (pH-7.5, 10⁸ CFU/mL of inoculum size, carbon and nitrogen source supplementation, and 140 rpm of agitation at 37 °C), recovered protease was 55.55 U/mL. The 4.1-fold purified 30 KDa enzyme was highly stable for creating bioactive compounds. Except for Serratiamarcescens, 3 h BAP efficiently reduced the growth of E. coli (47%), S. aureus (28%), and P. aeruginosa (12%). Absorbance at the far UV region confirmed that lesser than 3 KDa-sized proteins present in SPH-F4 had a similar antimicrobial pattern to 3 h BAP. The screening is inexpensive, and production does notreally need much controlled condition as these isolates are already resistant to extreme conditions. Thus, it canbe an excellent source of eco-friendly production of proteases which can be used for BAP production.

In the food manufacturing unit, the processing environments allow microbial growth resulting in surface biofilms. For more than 20 years, scientists have worked on various strategies, and now they are focusing on natural compounds to eradicate them. B. subtilis D3L/1 protease can tolerate extreme conditions with more than 90% residual activities. Moreover, the BAP synthesis process by using this organism is rapid, consistent, and highly economical with maximum yield. Exploration of efficient protease producer is in progress to obtainhigh-quality industrial grade protease for the production of various products including BAP. However, the production cost and stability of this enzyme are the main problem. In all cases, expensive substrate and growth parameters are required for the high-quality yield. It is estimated that almost 40% of the total production cost of enzymes is due to optimized parameters.Hence, significant interest is recorded to produce cost effective enzymes with easily available substrates. The production of protease from compost soil isolate is undoubtedly promising as compost are the rich source of hydrolytic enzymes. Eliminating surface biofilm and protecting food packaging surfaces with bioactive peptides can be addressed with that enzyme in future. Successful attachment of bioactive peptides on stainless steel, polystyrene, titanium, and polydopamine surfaces may solve the biggest problem of food andwater-borne biofilm diseases.

Author Contributions

Conceptualization, I.P., P.K.I. and B.R.; methodology, I.P., R.R., B.R. and P.K.I.; software, I.P. and K.N.; validation, I.P. and P.K.I.; formal analysis, I.P. and P.K.I.; investigation, I.P. and P.K.I.; resources, data curation; writing—original draft preparation, I.P., P.K.I. and B.R.; writing—Review and editing, I.P., P.K.I., B.R., R.R., S.A.B., A.A. and S.W.C.; project administration, P.K.I. and B.R.; funding acquisition, P.K.I. and B.R. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The 16s r DNA sequencing datasets for this study can be found in the NCBI having Gen Bank Accession No. GU723508.

Acknowledgments

I express my gratitude to the Saveetha School of Engineering, Saveetha Institute of Medical and Technical Sciences, Saveetha University, for providing the necessary infrastructure to carry out this work successfully. I am thankful to Sukanta Kumar Sen, Department of Botany, Visva-Bharati, West Bengal, India, for his valuable suggestions and the initial characterization of the working isolate.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Table

Figure 1. Effect of incubation period for extracellular protease production by selected screened isolates from compost soil. (A) Bar represents enzyme concentration (U/mL) and (B) line graph for bacterial growth (O.D.). The statistical significance was p < 0.05.

Figure 2. A neighbor joining phylogenetic tree of representative B. subtilis and B. megaterium. The optimal tree is shown (next to the branches). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. This analysis involved 26 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1387 positions in the final dataset. Evolutionary analyses were conducted in MEGA11.

Figure 3. Extracellular enzyme production by selected isolate Bacillus subtilis D3L/1. Bar represents enzyme concentration (U/mL),line graph represents bacterial growth; (A): effect of pH and incubation period. (B): effect of Carbon source concentration. The statistical significance was p < 0.05.

Figure 4. Effect of substrate concentration on extracellular protease production by selected isolate Bacillus subtilis D3L/1. Bar represents enzyme concentration (U/mL),line graph for bacterial growth; (A): effect of different substrate on protease production; (B): effect of inorganic nitrogen sources on protease production; (C): effect of inorganic nitrogen sources concentration on protease production. AS: ammonium sulfate; APS: ammonium persulfate; and AFS: ammonium ferrous sulfate. The statistical significance was p < 0.05.

Figure 5. SDS-PAGE analysis of protease. Lane 1: molecular weight marker (BSM0441, pre-stained 20–120 kDa mid-range protein molecular weight marker); Lane 2: sample (purified protease).

Figure 6. Characterization of semi-purified enzyme extracted from compost soil isolate Bacillus subtilis D3L/1: Bar represents enzyme activity; (A) thermostability of extracellular protease; (B) effect of heavy metal on protease activity; (C) protease stability under different pH range (D): relative activity of protease under different pH range. The statistical significance was p < 0.05.

Figure 7. Characterization of semi-purified enzyme extracted from compost soil isolate Bacillus subtilis D3L/1: Bar represents relative activity of protease (%); (A) effect of organic solvents on protease activity; (B) effect of ionic and non-ionic surfactants, inhibitors, and oxidizing agents on enzyme activity. The statistical significance was p < 0.05.

Figure 8. Determination of antimicrobial activity of bioactive peptides using Bacillus subtilis D3L/1 protease as hydrolyzing enzyme. (A) Enzyme hydrolysis effect on different substrates to check suppression of bacterial growth in order to confirm bioactive peptide formation. Bar represents bacterial growth; (B) determination of zone of clearance in soy protein-derived hydrolyzed bioactive compound samples of different time point to check antimicrobial efficiency. Bar represents the zone of clearance (in cm); K30: kanamycin antibiotic disc (30 µg/disc); UN: untreated; BAP: bioactive peptide.(C) Antimicrobial activity of partially purified bioactive sample to determine the possible size of the active compound through ultrafiltration. Bar represents the zone of clearance (in cm) and Fraction 1–4 (SPH-F1-SPH-F4) had the size approximately >30 KDa, 10–30 KDa, 3–10 KDa, and <3 KDa. K30: kanamycin antibiotic disc (30 µg/disc); UN: untreated; SPH F1: soy protein hydrolysate fraction 1; SPH F2: soy protein hydrolysate fraction 2; SPH F3: soy protein hydrolysate fraction 3; SPH F4: soy protein hydrolysate fraction 4. The statistical significance was p < 0.05.

Table 1

Biochemical characterization of selected screened isolate B. subtilis D3L/1.

Carbohydrates	Use (+/−)
Maltose	+
Fructose	+
Dextrose	+
Raffinose	+
Sucrose	+
L-Arabinose	+
Lactose	−
Inulin	+
Sodium gluconate	+
Glycerol	+
Salicin	+
Glucosamine	+
Mannitol	+
Ribose	+
Dulcitol	−
Xylose	−
Galactose	−
Melibiose	−
Mannose	−

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13041019/s1, Supplementary Table S1: Purification of protease from compost soil isolate Bacillus subtilis D3L/1. The statistical significance was p < 0.05, Supplementary Table S2: Detection of the type of active compound present in soy protein hydrolyzates through qualitative and quantitative analysis and compared with standard BSA solutions. The statistical significance was p < 0.05 and Supplementary Figure S1: Schematic representation of potent protease producer B. subtilis D3L/1 screening from compost pile, production optimization of B. subtilis D3L/1 protease, characterization and application of B. subtilis D3L/1 protease for bioactive peptide synthesis. Abbreviations: CMC: Carboxy Methyl Cellulose, RPM: Rotation per minute, AS: Ammonium sulfate, APS: Ammonium persulfate, AFS: Ammonium ferrous sulfate, BAP: Bioactive Peptide, are present in this file.

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