Immobilization and docking studies of Carlsberg subtilisin for application in poultry industry

Anum Munir Rana; Bart Devreese; Stijn De Waele; Asma Rabbani Sodhozai; Maryam Rozi; Sajid Rashid; Abdul Hameed; Naeem Ali

doi:10.1371/journal.pone.0269717

. 2023 Aug 16;18(8):e0269717. doi: 10.1371/journal.pone.0269717

Immobilization and docking studies of Carlsberg subtilisin for application in poultry industry

Anum Munir Rana ^1,^#, Bart Devreese ^2,^‡, Stijn De Waele ^2,^‡, Asma Rabbani Sodhozai ^1,^#, Maryam Rozi ³, Sajid Rashid ³, Abdul Hameed ¹, Naeem Ali ^1,^*

Editor: Afsheen Aman⁴

PMCID: PMC10431679 PMID: 37585472

Abstract

Carlsberg subtilisin from Bacillus licheniformis PB1 was investigated as a potential feed supplement, through immobilizing on bentonite for improving the growth rate of broilers. Initially, the pre-optimized and partially-purified protease was extracted and characterized using SDS-PAGE with MW 27.0 KDa. The MALDI-TOF-MS/MS spectrum confirmed a tryptic peptide peak with m/z 1108.496 referring to the Carlsberg subtilisin as a protein-digesting enzyme with alkaline nature. The highest free enzyme activity (30 U/mg) was observed at 50°C, 1 M potassium phosphate, and pH 8.0. the enhanced stability was observed when the enzyme was adsorbed to an inert solid support with 86.39 ± 4.36% activity retention under 20 optimized conditions. Additionally, the dried immobilized enzyme exhibited only a 5% activity loss after two-week storage at room temperature. Structural modeling (Docking) revealed that hydrophobic interactions between bentonite and amino acids surrounding the catalytic triad keep the enzyme structure intact upon drying at RT. The prominent hygroscopic nature of bentonite facilitated protein structure retention upon drying. During a 46-days study, supplementation of boilers’ feed with the subtilisin–bentonite complex promoted significant weight gain i.e. 15.03% in contrast to positive control (p = 0.001).

Introduction

Proteases (EC 3.4) have versatile applications in various fields and are active from acidic, neutral to alkaline pH herewith possessing maximal industrial interest. The proteases own a proven significant role in wastewater treatment, pharmaceutical, food, detergent, leather, poultry, and textile industry [1]. The demand for protease use in poultry has markedly increased, promising improved body weight and health status [2]. With ever-increasing demand, protease storage and its activity under room temperature needs to be improved. So far, several techniques have opted for the storage of enzymes such as freeze-drying, spray drying, and lyophilization. Likewise, immobilization of proteins on support material is one of the most important and efficacious methods to store proteins (at room temperature) with reusability [3]. Studies have shown that an extended shelf life and enzyme stability under storage conditions is vital to retain the efficacy of enzymes [4]. Up till now, several enzymes such as protease, laccase [5], peroxidase, catalase, pectinesterase have been successfully adsorbed on a variety of solid support materials [6–10]. Carlsberg subtilisin is a highly potent class of protease with noticeable stability at extreme temperature and pH. The immobilization of carlsberg subtilisin to bentonite has not been explored in past with an aim of poultry feed supplementation. The immobilization is classfied into two types on the basis of interaction among the enzyme and support material such as physical and chemical method. The monocovelent and weaker interactions among the enzyme and support material is known as physical immobilization including the hydrophobic interactions, van der waals forces, affinity binding, ionic binding, and hydrogen bonding. Moreover, thre are four principle techniques to immobilize the enzyme to solid support material namely, entrapment, adsorption, covelant bonding and cross linking. Organic supports are generally poor with a humid and temperature environment as the shelf life is greatly reduced increasing the need of preservative addition is required to avoid microbial growth. Moreover, the inorganic supports are less intervening with the feed and enzyme to be fed eventually increasing the potential for being used in poultry as feed supplement.

One of the crucial requirements for enzyme immobilization is the selection of appropriate solid support material. The support material, either organic or inorganic by nature, is selected based on limitations, usage method, and desired application [11]. In some studies, organic supports such as carbon SKN-2P, lignocellulosic substrates, graphene oxide particles [12] and lignin have been used as solid support for the immobilization of protease and cellulase, respectively [13,14]. Ideally a support that is inert can work best for use in poultry due to its nonreactive ability, since the shelf life and environmental factors can contaminate the supports and require addition of preapproved preservative for poultry feed. Besides organic, inorganic supports serve as a good option for protein immobilization [15]. Among inorganic supports clay, due to its diverse characteristics (surface chemistry, particle size, surface area, and particle shape) is extremely suitable for enzyme immobilization. The hydrated sodium calcium alumino-silicate, zeolites, activated charcoal, and bentonite have been widely used for detoxification of feed [16]. Bentonite is a low-cost inorganic matrix that lack toxicity and chemical reactivity, making it suitable most solid support for enzyme immobilization. Since the bentonite carries high swelling capacity it is being used as sorbent to reduces wet droppings, studies reported that it also contributes toward the increase chickens’ body weight, life expectancy, and egg size [17]. Additionally, an absorbed Carlsberg subtilisin to bentonite has never been exploited for its potential use in poultry as a feed supplement.

The protein-ligand docking gives an insight into possible binding regions among protein and support material that are the major contributors of interactions. Protein and ligand docking can be used as the source to predict patterns of binding proteins to specific solid support material after immobilization and also gives insight to predict reasons of attachment [18]. The docking studies is a newer approach that help find out the clues to possible stability retention after drying and poultry experiments confirms the active participation of enzyme in digesting proteins while being in the gut.

The current study was designed to improve the dietary protein assimilation among broilers. Carlsberg subtilisin isolated from Bacillus licheniformis PB1 was identified, characterized, and optimized for immobilization on the suitable solid support. To achieve maximum activity after adsorption, the enzyme immobilization was performed by using optimized parameters (OVAAT) viz., selection of the suitable solid support, molarity, temperature, and buffer pH was studied. To predict the possible reason for enzyme stability under drying conditions, molecular docking interactions of two subject proteins (serine protease (3QTL) and Carlsberg subtilisin (PDB ID:4GI3) having a high resemblance to the enzyme under study) with Bentonite were studied. Furthermore, the immobilized product was fed to the Broilers (Arbor acres) to prospect the impact of dried-enzyme-bentonite product on the overall weight improvement of birds and compared with the commercially available protease supplement.

Materials and methods

The work was approved by Bio-Ethical Committee (BEC-FBS-QAU2021-313) of Department of Microbiology, Quaid-i-Azam University, Islamabad.

Chemicals

The chemicals and reagents used in this research work are of analytical grade. Chemicals such as NaCl, yeast extract, casein, peptone, gelatin, ammonium sulfate, Magnesium sulphate (MgSO₄), Calcium chloride dehydrate (CaCl₂ 2H₂O, Dipotassium hydrogen phosphate (K₂HPO₄)_, azocasein, α-hydroxycinnaminic acid, acetonitrile (ACN), ammonium citrate, Trifluoroacetic Acid (TFA), and KCl were purchased from Sigma-Aldrich (Merck Group, Germany). Ultracell centrifugal filter tubes purchased from Millex Amicon Ultra filter (Merck-Millipore, Darmstadt, Germany). For the SDS-PAGE protein standard Precision Plus Protein All Blue Standards was purchased from BioRad (California, United States). Woogene B & G enzyme supplement (South Korea), All reagents have been prepared in sterilized Milli Q water.

Protease production, purification, and extraction

Bacterial cultivation

The alkaline protease was produced by Bacillus licheniformis PB1, previously isolated from desert soil of Sindh, Pakistan [19]. The stock culture was maintained on nutrient agar plates and stored in 30% glycerol at −80°C. The culture was grown in inoculum medium (w/v) composed of NaCl 0.5%, yeast extract 0.5%, casein 0.5%, peptone 0.3%, gelatin 0.3% with pH 8.5 and incubated at 60°C, 160 rpm for 24 hours. The culture was used to inoculate production flasks at optimal growth inoculum (69x10⁵ CFU/ml).

Enzyme production

The alkaline production medium (w/v) was prepared by adding soybean meal 1%, NaCl 0.01%, MgSO₄ 0.05%, CaCl₂. 2H₂O 0.05%, K₂HPO₄ 0.02% and KCl 0.2% with pH maintained at 8.0. This production medium (3 L) was inoculated with 2% (v/v) B. licheniformis and incubated at 60°C, 160 rpm for 7 days. Later, the extracellular enzyme was separated from the cell content by centrifugation at 10,000 rpm for 20 mins at 4°C.

Enzyme extraction and purification

Ammonium sulphate (w/v) at 80% saturation was added to the supernatant medium protein precipitation. Further in next line precipitated enzyme pellets were suspended in 0.05 M Tris-HCl buffer, pH 7.5. The precipitated enzyme was dialyzed using vivaspin utlraspin centrifugal dialysis tubes with cut off 10 kDa to remove excessive salts and assayed for protein content and enzyme activity.

Protease activity assay

The protease activity was determined by an azocasein (Sigma) assay at 440 nm [20]. Negative (without protease) and positive controls were run along to find out the activity. The total protein was estimated by Lowry assay using bovine serum albumin as a standard protein at 280 nm [21]. The enzyme activity, expressed in units, was calculated by Goose’s Formula and specific activity was determined [22]. One unit of enzyme activity is defined as amount of enzyme required to liberate 1 ug of tyrosine /ml at 50°C and pH 8.

Electrophoretic analysis

The enzyme was concentrated by ultrafiltration using an Ultracell centrifugal filter with a molecular weight cut-off of 10 kDa. Protein size was determined using SDS-PAGE on a 12% polyacrylamide resolving gel as reported by Laemmli et al [23]. Protein bands were observed by staining the gel with Coomassie brilliant blue G-250 and apparent molecular weight was estimated with Precision Plus Protein All Blue Standards. The 1% casein (copolymerized with 12% resolving gel) zymography was carried out according to the modified method of Garcia-Carreno [24].

Identification of proteins by MALDI-TOF/TOF mass spectrometry

Selected protein bands were excised from SDS-PAGE followed by trypsin digestion (0.1 ug/ul) [25]. These digests were subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Measurements were carried out on a 4800+ MALDI TOF/TOF Proteomics Analyzer (AB-SCIEX, Darmstadt, Germany). An equal amount (1:1) of tryptic digest mixtures from each band and matrix solution (5 mg/mL α-hydroxycinnaminic acid dissolved in 60% ACN, 10 mM ammonium citrate, 0.1% TFA) was spotted on a stainless-steel MALDI target plate. The MS spectra were obtained in positive ion mode. Spectra were acquired from 5000 laser shots with a 200 Hz laser (Nd: YAG laser; 355 nm), the laser intensity was 4200 reflector measurement. The proteins were identified using the Mascot (Matrix Science) search engine against the SwissProt database (2017 version). Database search was conducted with the following parameters: three missed cleavages, carbamidomethylation of Cys, and oxidation of methionine as fixed and variable amino acid modifications, respectively, allowing an MS/MS error of 0.6 Da to fragment ions and maximum accuracy of 200 ppm for parent ions were used to identify the specific protease produced from Bacillus licheniformis PB1. To identify a protein, the confidence threshold was adjusted to 95%.

Enzyme immobilization

Free enzyme optimization

To determine the physical parameters required for adsorption of protease to a solid support, we first assessed the effect of pH, temperature, and buffer molarity on enzyme activity. The varying molarity of potassium phosphate buffers (0.1 to 2.2 M with an interval of 0.1 M) was used to study the effect of electrolytes on enzyme activity. Likewise, a range of pH (2 to 9 with an interval of +1) was used to study its effect on enzyme activity. Furthermore, enzyme activity was determined at different temperatures (20 to 70°C with an interval of 10°C).

Enzyme adsorption on selected solid supports

The Subtilisin was adsorbed to a set of organic and inorganic support materials. The inorganic support was inert material, i.e. an active component of clay, bentonite (provided by SJ Enterprises, Pakistan). The enzyme was immobilized on bentonite by simple adsorption. Therefore, bentonite was pretreated with 1% HCl, washed several times, and dried overnight in a hot air oven at 100°C. The dried bentonite (1 gm) was ground and equilibrated with potassium phosphate buffer (1 M, pH 8). Next, it was incubated with 2 ml of protease solution containing 15.32 U/mg (SD ± 2.5%) in the same buffer at 4°C for 12 h at 50 rpm. Control experiments were performed without subtilisin. The unbound enzyme was removed by washing with the same buffer. Enzyme adsorbed product was further quantified to determine its specific activity. The activity yield was determined by dividing the total activity of immobilized enzyme with the total activity of free enzyme.

Wheat bran and soybean meal were selected as carbon-based solid support material. Further, optimized enzyme buffer suspension (18.34 U/mg) was adsorbed on soybean meal and wheat bran in 1:1 proportion, thin layers were spread out with the help of a spatula on Petri plates and left for 12 hours to dry at room temperature (25 ± 3°C) via air drying. Later, dried material was suspended in 0.05 M Tris-HCl (pH 7.5) and specific activity of protease was determined after 14 days of storage at room temperature.

Enzyme desorption

The total enzyme release was measured by suspending the Subtilisin bentonite dried product in Tris-HCl buffer with pH 7.5 kept at 37°C for 10 mins under shaking conditions. After incubation, the product was centrifuged (6000 rpm for 10 mins) and supernatant was assayed to study the activity of the released enzyme in each wash was evaluated. The process was repeated four times and total adsorbed protein activity was calculated.

Structural modeling

The X-ray crystal structure of Serine protease (3QTL) and Subtilisin (PDB ID: 4GI3) from Bacillus licheniformis was retrieved from Protein Data Bank [26]. The 2D structure (SDF format) of Bentonite (PubChem ID: 72941614), (Fig 4) was obtained via PubChem database, and Nanotube was constructed by combining four monomers (with a diameter of 9nm) subunits of sodium bentonite in Avogadro [27]. PDB format was generated through PyMol. The 3D structure of the nanotube was then subjected to energy minimization using GAFF force field [28].

Fig 4 — Surface view of (A) Serine protease (3QTL) (cyan) and (B) Subtilisin (PDB ID: 4GI3) -specific bentonite binding cavity. The binding pocket is indicated by a light purple colored surface onto Subtilisin (light green) while interacting residues are labeled in black. Catalytic binding pocket residues in Subtilisin are indicated by yellow color and the residue implicated in the hydrogen bonding is indicated by light green color. The bentonite nanotube is indicated by the red colored stick (UCSF Chimera).

Dockings of solid support material and its nanotube with Bacterial Serine Protease (BSP) and Subtilisin (PDB ID:4GI3) was accomplished using PatchDock server [29]. The refinement and rescoring tool FireDock was used to check the specificity of the respective interacting protein [30]. The input parameters in PatchDock were coordinate files of Serine Protease and sodium bentonite. Each enzyme transformation was calculated by scoring function to observe the geometric fit and atomic desolvation energy [31]. Root Mean Square Deviation (RMSD) clustering was engaged to the candidate solution to exclude the redundant one. The best scoring complex was subjected to UCSF Chimera ver. 1.11.2 and LigPlot for interaction analysis [32,33].

Supplementation of Carlsberg subtilisin-bentonite product to poultry as a growth enhancer

Aiming to evaluate the use of adsorbed-dried-product on weight gain, a one-day-old commercial broiler named Arbor acres (n = 150) was selected and raised on a poultry farm (near Attock). The work was approved by Bio-Ethical Committee (BEC-FBS-QAU2021-313) of Department of Microbiology, Quaid-i-Azam University, Islamabad. The birds were divided into three groups consisting of 1 day-old mixed-sex broiler chickens (Cobbs), 50 birds in each. During the first 3 days, chickens were fed on a diet without enzyme supplementation. From the day onwards, for 42 days their feed was supplemented differently. Group A feed was supplemented with adsorbed protease product with the activity of 27.04 U/mg (SD ± 0.0035) on 1 gm of bentonite added to 1 kg bag of basal feed. Group B was supplemented with B & G commercially available enzyme product as a positive control 1 gm/Kg, as advised by the manufacturer’s feeding chart. Group C was not supplemented with enzyme product and was run as a negative control. Both positive and negative controls have bentonite in them since it acts as a sorbent of excessive water and reduces wet litter from bird’s feces. Birds were weighed at a regular interval of 6 days from 4^th to 46^th day.

Statistical analysis

The experimental optimization results of three parallel measurements were assigned mean value and analysis was conducted. The results were statistically analyzed by ANOVA and Duncan’s test using SPSS version 2017 software, Chicago, SPSS Inc.

Results and discussion

Protease production, purification, and extraction

The protease was extracted from B. licheniformis PB1 under batch mode agitated condition (2% inoculum, 8.5 pH, and 160 rpm at 60°C). The highest enzyme activity (231.1 U/ml) was observed on the 7^th day of production [34]. Likewise, proteases were produced by B. licheniformis PB1 [35] under varying physiochemical production parameters. These parameters were key determinant of varying production of protease in quantity (activity) from different species. After partial purification through ammonium sulphate precipitation (80%) an about 8.21-fold increase in enzyme activity was observed with an overall yield of 59% from B. licheniformis PB1.

SDS PAGE, zymography and MALDI TOF-MS/MS

SDS PAGE gel analysis of partially purified B. licheniformis PB1 protease revealed five bands. The band activity of Carlsberg subtilisin has been observed through zymogram analysis and a clear casein hydrolysis zone confirmed proteolytic characteristics of the enzyme (Fig 1A & 1B, S1 Fig). The protein bands from SDS-PAGE were extracted from the gel, digested with trypsin, and analyzed through MALDI-TOF MS/MS. The MASCOT database search revealed that the protein band had an apparent molecular weight of 27 kDa and is most likely a Carlsberg-subtilisin from B. licheniformis as reported by Pannkuk [36]. The peptide confirmation was obtained by MS/MS analysis of a tryptic peptide peak with m/z 1108.496. MS/MS showed that this peptide is identical to the peptide covering amino acid residue 342–351 (KHPNLSASQVRN) of the apr gene product (Mascot score 80) (Fig 1C). The Unipept analysis revealed that peptide was hitherto solely found in B. licheniformis Subtilisin [37]. Further the peptide sequence was found to have 40 score on NCBI protein Blast as Carlsberg subtilisin of Bacillus licheniformis.

Fig 1 — Carlsberg Subtilisin produced by B. *licheniformis* PB1 (A) SDS-PAGE Excised Gel bands with protein marker for 80% precipitated enzyme extract (B) Excised zymogram (1% Casein) of potentially purified enzyme (C) MALDI -TOF MS/MS spectrum of a tryptic peptide with M/z 1108.5 (S1 Fig). *^A The SDS-PAGE was excised to visualize the thickest bands. *^B The excised Zymogram of purified Carlsberg subtilisin.

Further the peptide sequence was found to have 40 score on NCBI Protein Blast as Carlsberg subtilisin of Bacillus licheniformis.

>sp|P00780|SUBT_BACLI Subtilisin Carlsberg OS = Bacillus licheniformis OX = 1402 GN = apr PE = 1 SV = 1

MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKE

SGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADK

VQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALD

NTTGVLGVAPSVSLYAVKVLNSSGSGTYSGIVSGIEWATTNGMDVINMSLGGPSGSTAMK

QAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAEL

EVMAPGAGVYSTYPTSTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATY

LGSSFYYGKGLINVEAAAQ

Solid support selection for enzyme adsorption

To select appropriate solid support for adsorption of Carlsberg subtilisin, different organic and inorganic solid supports were tested for better activity after adsorption. Carlsberg subtilisin was successfully immobilized to both types of supports but overall activity was different. The adsorption on carbon-based solid support viz., soybean meal exhibited 27% greater activity (0.33 ± 0.007 U/mg) than that of wheat bran (0.25 ± 0.0014 U/mg). Moreover, bentonite immobilized Carlsberg subtilisin (0.45 ± 0.0071 U/mg) retained better residual activity in contrast to carbon-based solid supports (98%) (Fig 2A). This indicated bentonite has better immobilization ability as compared to carbon-based support material and the overall activity of enzyme was improved markedly. Similar results were previously obtained for α-amylase bound to chitosan and Na-bentonite composites with an overall increase in the activity (87%) of enzyme [38]. For prolonged storage of proteins, carbon-based immobilization support materials are not advisable for regions that have a wider range of temperature and humidity, increases the chance of fungal contamination. The storage capacity on wheat bran and soybean meal as solid support appeared to be very low and changing environmental conditions may contaminate the product and lead towards harmful aflatoxins production by fungal strain [39]. As the poultry is a very sensitive organism and mild fungal contamination in feed can possibly lead to loss of organism.

Optimization of conditions before adsorption

Effect of change in buffer molarity

The overall enhanced activity was observed with an increase in molarity of buffer until the optimum (1M potassium phosphate, pH 8) and it was dropped on further increase. Inclusion of potassium phosphate buffer (1 M) leads to 10-fold enhanced specific activity 14.805 ± 0.57 U/mg with respect to the crude enzyme, elucidating the marked impact of electrolyte strength on enzyme activity (Fig 2B). Likewise, McComb et al., revealed activity also relied on electrolyte molarity under consideration [40]. Therefore, the results strongly support that Carlsberg subtilisin exhibited a stable activity around higher electrolyte strength.

Free enzyme temperature optimization

In general, the temperature has a remarkable impact on Subtilisin activity. The specific activity of Subtilisin was found to be 33.667 ± 0.94 U/mg at 50 ˚C (Fig 2C). The enzyme is still active at a range of higher temperature up to 70°C after 30 min of incubation. Our findings coincide with a study reporting 50 ˚C as optimal temperature for Subtilisin. Although, the latter enzyme had a wider temperature range (30–60 ˚C) for its pronounced activity [41]. In terms of thermal stability, the activity of Subtilisin extracted from B. licheniformis PB1 was better as compared to other Bacillus species such as B. subtilis [42].

Influence of pH for enzyme activity

A wide range of Subtilisin has been produced from different Bacillus species with a relatively high optimal pH [43]. The specific activity of Subtilisin at optimum pH (8.0) was recorded 30.2 ± 0.26 U/mg indicating enhanced activity as compared to crude enzyme due to protein enrichment (Fig 2D). This specific activity could be due to an overall charge at the interfacial enzymatic surface. This charge distribution on the enzyme surface facilitates substrate binding and catalytic efficiency, increasing overall enzyme-substrate complex formation. In other words, there is an optimum pH value that favors the maximum concentration of the enzyme-substrate intermediate [44].

Free enzyme adsorption on a selected solid support

Successful immobilization of Carlsberg-Subtilisin could be a consequence of adsorption through hydrophobic interactions among enzymes and supporting material [45]. It can be said that involvement of possible strong interactions among enzymatic amino acids (essential for bonding or catalysis) and the surface of the solid support (clay mineral) are associated with successful immobilization [46]. This could affect the structure of active center surroundings and subsequently the final catalytic features of lipase [3].

Enzyme desorption

The extent of enzyme desorption from adsorbed carriers could be evaluated by suspending the product in the buffer. The Subtilisin adsorbed to bentonite in dried form was suspended in 1 M Potassium phosphate buffer pH 8 and placed for 10 mins at room temperature, later centrifuged at 6000 rpm. The overall desorption of Subtilisin from the product was 28.89 ± 3.24% in 40 mins after four washes. Consequently, the protein desorption was achieved with approximately 71.1 ± 4.1% of active protein attached to the solid support after concomitant four washes (Fig 2F).

Molecular docking analysis

Despite the difference, in structure, both enzymes were evaluated to validate the adsorption of the protease. Molecular docking analysis of Serine protease (3QTL) and Carlsberg-Subtilisin (PDB ID:4GI3) against bentonite was accomplished. To further characterize the serine protease and bentonite interaction, we mapped the bentonite-specific probable regions required for Serine protease and Carlsberg-Subtilisin binding. The 10 best docking solutions were designated for additional enhancement and rescoring scrutiny by the FireDock algorithm. The binding energy of the serine protease and bentonite complex was -73.94 kcal/mol, while the binding energy of Carlsberg subtilisin and bentonite was -47.58 kcal/mol. In both Serine protease-sodium bentonite and Carlsberg subtilisin-bentonite complexes, catalytic triad (Ser220, His63, and Asp32) and (Ser221, His64, and Asp32) residues were involved in the formation of substrate-binding clefts, respectively (Fig 3A and 3B).

Fig 3 — Detailed interaction of best-docked complexes with (A) Serine protease (3QTL) (B) Subtilisin (PDB ID: 4GI3). Protein is styled in ribbon representation while interacting residues are depicted in wire form with labeled residues in black color. Bentonite nanotube is illustrated in stick representation with red-colored (using UCSF Chimera).

Positioning of bentonite onto the surface of a serine protease and Subtilisin was keenly monitored to explore the binding pocket dynamics and residual contributions of protein in association with docked ligand (Fig 4A and 4B). revealed the predominant contributions of hydrophobic residues lying in the periphery of active sites. Noticeably, His63, Leu95, Gly99, Ser100, Gly101, Ser102, Tyr103, Ile106, Gly126, Gly127, Asn154, Leu216, and Asn217 residues of serine protease were involved in hydrophobic interactions with sodium bentonite. In contrast, Subtilisin-specific His64, Leu96, Gly100, Ser101, Gly102, Ser103, Tyr104, Ile107, Ser125, Leu126, Gly128, Asn155, Leu217 and Asn218, Ser221, Met222 residues were involved in bentonite binding (Fig 5A and 5B). In the Serine Protease-bentonite complex, Gly127 residue was involved in hydrogen bonding. Moreover, Ile107 and Gly127 residues of Subtilisin were also found in hydrogen bonding.

Fig 5 — Inter-molecular interaction pattern of the **(A)** Serine protease (3QTL) and (B) Subtilisin (PDB ID:4GI3) to Sodium bentonite. 2D structure of bentonite is indicated in the middle while interacting residues are illustrated by green balls. The water molecules are indicated by cyan-colored balls.

The docking analysis revealed significant involvement of van der Waals, Water, and desolvation energies in the binding of bentonite to Ile107, Gly127 residues of subtilisin exhibited more conformational changes as compared to Serine Protease due to binding. Likewise, Gilli et al., reported that acidic or basic medium plays a vital role in the establishment of a strong hydrogen bond or strengthens it to 6-folds followed by a 15% shorter bond [47]. This could be the reason for the establishment of stronger hydrogen bonds among bentonite and protease keeping the catalytic triad intact. Despite drying protease and bentonite, enzyme activity was sustained, and only a limited amount of activity was lost (how much). Additionally, the pH of Cobb’s gut varies greatly from acidic to mild basic (pH 3–7.5) that supported meliorate in desorption rate [48]. This could be a reason for increased weight gain when the dried product was subjected to poultry trials.

Poultry application

The Carlsberg-subtilisin-bentonite product prepared as a result of immobilization, subjected to poultry feed with aim of finding impact on chicken weight, when fed in comparison to a commercially available product. A gradual increasing trend was observed among treatment groups (A, B, and C) to the total number of days, product was fed to chickens. A significant weight gain was observed on day 10, 28, 34, 40, and 46. Therefore, treatment group A has a significant difference in weight gain as compared to group B (positive control) and C (negative control) showed in Table 1. This is in line with a previous study that proved visibly increased body weight of chicken vs. time (days) when fed on an enzyme-based diet. In contrast to 129 gm weight on the 7^th day, our results explicitly showed an efficient increase in weight gain from day 4 to day 10^th (168.06 gm) [49]. Treatment group A retained significantly higher weight gain (1821.8 gm) compared to the treatment group B and C. In this experiment, the enzyme continued to demonstrate catalytic activity in terms of weight gain. This could be an advantage for broiler feeding, since enzyme supplementation typically reduces overall digesta’s viscosity, which influences feed retention, lowering it from 14–11 hour (Table 2) [50]. Therefore, the addition of protease inorganic sorbent (bentonite) products slowed down the feed passage to an acceptable level. It can be inferred that approximately 11 hours of feed retention time, results in a slower release of enzymes from harnessing sorbent thus facilitating the hydrolysis of peptides throughout the gastrointestinal tract. Eventually, increased peptide assimilation was estimated in terms of prominent weight gain. The adsorbed subtilisin efficiently hydrolyzed protein-rich basal medium and enhanced peptide assimilation despite the bound state of the enzyme. Therefore, an overall ameliorated weight gain in chicken till 46 days was observed to be 15.03% as compared to the positive control. The adsorption of subtilisin to bentonite participates in securing the activity of the enzyme under drying conditions by keeping the active site intact.

Table 1. Comparison of enzyme immobilization to organic and inorganic solid supports.

Support Material	Enzyme	Immobilization Technique		Ref.
Inorganic Support
bentonite-derived mesoporous materials	Laccase	Physical adsorption	60% removal efficiency	[51]
Na-bentonite and modified bentonite	Alkaline phosphatase	Cation exchange and adsorption by van-der-Waals interaction		[46]
Bentonite	lipase		(30%)	[52]
Na-bentonite and modified bentonite	α-amylase	Adsorption		[53]
Organobentonite	Lipase	Adsorption	BC₁₀₀-lipase kept 82.5% of initial activity	[54]
Organic Support
Nanoporous rice husk silica	Soybean lipoxygenase	Adsorption	50%	[55]
DEAE-cellulose	α-amylase	Adsorption	80%	[56]

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Table 2. Weight gain in Broilers (46 Days) after addition to feed Carlsberg-subtilisin-bentonite complex, B&G enzyme product, and without enzyme.

Treatment groups
Age (days)	A	B	C	SDM^*	SEM^**
4	46.86^b	47.2^a	47.3^a	1.09	.08980
10	168.06^a	151.64^a^,^b	155.22^a^,^b	12.825	1.05066
16	343.92^a	333.54^b	318.42^c	22.21	3.14040
22	522.9^a	466.77^c	449.14^c	26.96	3.81308
28	863.8^a	754.54^b^,^c	735.92^b^,^c	41.98	7.61214
34	1280.2^a	1113.44^b^,^c	1072.73^b^,^c	62.9	16.30792
40	1613.2^a	1387.46^b	1351.5833^b	84.84	26.67894
46	1821.8^a	1583.76^b	1554.3958^b	86.95	26.98344

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^a-c Means in a row lacking common superscript letters differ significantly (P <0.05), Duncan’s test.

Note

Treatment group A: Protease product.

Treatment group B: Phytezyme (commercial product as positive control).

Treatment group C: Control (no enzyme product).

*Standard Deviation Mean.

**Standard Error Mean.

Conclusions

Protein catalyzing enzymes have diverse application in industry, specifically in poultry broilers, proteases are used as the feed supplement. For which proteases like enzyme was produced from Bacillus licheniformis PB1 and extracted, partially purified, dialyzed, and characterized using MALDI-TOF MS/MS to identify the specific protease involved in the immobilization is an efficient way to enhance protein stability (S1 & S2 Figs in S1 Raw images). With this property, numerous solid supports (organic and inorganic) have been reported as a promising solution to increase the shelf life of biological catalysts and contribute towards commercial utility. Where clay components’ enduring hydrophobic and adsorptive characteristics can be a good choice for protease immobilization. However, immobilization of Carlsberg-subtilisin to solid support required optimization of parameters to provide cushioning of the enzyme along with better adsorption on bentonite. Based on the results of present study, adsorbed-product contributed better stability at RT, and docking experiments gave a possible explanation. To be more specific, docking results confirmed that the presence of specific hydrophobic interactions among catalytic triad and bentonite is responsible for pronounced stability and activity of protein after adsorption. According to the present study, the active center of subtilisin is in close vicinity to the bentonite surface that created a hydrophobic environment around the active center compared to the aqueous medium surrounding dissolved enzyme.

Supporting information

S1 Fig. Carlsberg subtilisin produced by B. licheniformis PB1.

(A) SDS-PAGE Excised Gel bands with protein marker for 80% precipitated enzyme extract (B) Excised zymogram (1% Casein) of potentially purified enzyme (C) MALDI -TOF MS/MS spectrum of a tryptic peptide with M/z 1108.5.

(PDF)

Click here for additional data file.^{(253.8KB, pdf)}

S1 Raw images

(PDF)

Click here for additional data file.^{(277.3KB, pdf)}

S1 File

(DOCX)

Click here for additional data file.^{(86.5KB, docx)}

Data Availability

All relevant data are within the paper and its supporting information files.

Funding Statement

The authors received no special funding for this work.

References

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PLoS One. doi: 10.1371/journal.pone.0269717.r001

Decision Letter 0

Sanjay Kumar Singh Patel

1 Jun 2021

PONE-D-21-14945

Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite as a poultry feed supplement

PLOS ONE

Dear Dr. ali,

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Reviewer #1: This manuscript report about Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite as a poultry feed supplement. Manuscript needs following changes for further improvement.

Comments:

Introduction: Improve this section by adding more information about properties of matrix required for enzyme immobilization and various supports reported for protease immobilization.

Line 31: Add suitable reference in support: Molecular biology interventions for activity improvement and production of industrial enzymes.

Line 51: What are drawbacks of organic support used for immobilization? Why we need inorganic support. Need explanation.

Line 54: What are the advantages of clay over other inorganic supports reported?

Check the manuscript for typo errors, there should be space between value and units.

Line 165: Check and correct… pernatant was assayed

Protease was partially purified and used in experiments. There may be several other protein which have positive or negative effect on chicken growth improvement. Need justification.

What about the Native-PAGE? Is the reported protease in its monomeric form or multimeric? Add information.

Why there is no data about the effect of pH, buffer strength and temperature effect on immobilized enzyme. This information is important for comparison. Add data and discussion.

Reviewer #2: Manuscript title: Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite as a poultry feed supplement

The manuscript describes very simple method for the preparation of protease immobilized bentonite. The resultant material used for poultry feedstock to achieve healthy and productive chicken. Additionally docking study was also performed to find optimal binding between protein and ligand. Overall, the manuscript has been written well. In my opinion, the work may be accepted for publication in PLOS ONE after Major revision.

My specific comments are as follows:

1. Reduce some words in title and change the keywords which areal ready present in title

2. Line 17: The highest free enzyme activity (a maximum of 19.02-fold) was observed at 50 °C, 1M potassium phosphate, and pH 8.0. the enhanced stability was observed when 19 the enzyme was adsorbed to an inert solid support with 86.39±4.36% activity retention under 20 optimized conditions.

Mention the maximum enzyme activity in term of unit and rewrite the sentences.

3. Abstract: Line 14. Significance of partially-purified enzyme?

4. The introduction section is lack of focus. Please be selective on the background content and rewrite the introduction part after a wider literature investment.

5. Line 36 Mention how the application of protease in poultry feed is promising? Kindly highlight productivity and improved health via control on poultry zoonotic pathogens.

6. Author must mention the types of immobilization methods in brief and advantage of the method by which immobilization has been reported in this study.

Line 43 Consider the relevant works published within 5 year for possible citations such as

Suman, Sunil Kumar, Padma Lata Patnam, Sanjoy Ghosh, and Suman Lata Jain. "Chicken feather derived novel support material for immobilization of laccase and its application in oxidation of veratryl alcohol." ACS Sustainable Chemistry & Engineering 7, no. 3 (2018): 3464-3474. https://doi.org/10.1021/acssuschemeng.8b05679

Imam, Arfin, Sunil Kumar Suman, Raghuvir Singh, Bhanu Prasad Vempatapu, Anjan Ray, and Pankaj K. Kanaujia. "Application of laccase immobilized rice straw biochar for anthracene degradation." Environmental Pollution 268 (2021): 115827. https://doi.org/10.1016/j.envpol.2020.115827.

Sanjay KS PATEL, Raviteja PAGOLU, DIBYA BHOL, LEE Jung-Kul

Eco-Friendly Composite of Fe₃O₄-Reduced Graphene Oxide Particles for Efficient Enzyme Immobilization. ACS applied materials & interfaces, 9(3), 2213-2222.

Sanjay KS Patel, Rahul K Gupta, Sang-Yong Kim, In-Won Kim, Vipin C Kalia, Jung-Kul Lee, Rhus vernicifera Laccase Immobilization on Magnetic Nanoparticles to Improve Stability and Its Potential Application in Bisphenol A Degradation Indian Journal of Microbiology 61, no. 1 (2021): 45-54.

More relevant study may be cited related to immobilization support.

7. Recheck entire manuscript for additional space, for instance line 45.

8. If the protease producing strain has deposited in any microbial culture collection repository mention the ID number (section bacterial cultivation)

9. This production medium (3L) was inoculated with 2% (v/v) B. licheniformis and incubated at

99 60°C for 7 days. Author must mention other production parameter such as RPM and pH for protease production.

10. Correct the sentence, line 102, further in next line precipitated enzyme pellets were suspended in 0.05M Tris-HCl 104 buffer, pH 7.5, and assayed for protein content and enzyme activity. The precipitated enzyme was dialyzed using vivaspin utlraspin centrifugal dialysis tubes with cut off 10 kMW to remove excessive salts. Salts interfere with enzyme activity so it is necessary to remove the excess salt from the suspended buffer before taking the enzyme activity. The sentence mentioned (line 103) indicate that, enzymatic activity was assayed first then dialysis was performed. Change the sequences of experiment and rewrite the section.

11. Protease activity method and calculation should be explained in brief for readers,

Line 110 Please add space between numbers and unit, for instance 280 nm (space between 280 and nm, please make further corrections in all the text.)

12. Line 121 Digestion protocol not provided

13. Section 2.4 what was the peptide concentration for MALDI-TOF MS/MS analysis?

14. Line 130 how the database search was performed? Any specific genera or generic search kindly mention?

15. Line 132 Mention the peptide charge used in MASCOT search

16. Kindly provide peptide sequence showing identity with protease of Bacillus licheniformis along with score obtained in MASCOT search.

17. Reason for selection of clay as support material?

18. Line 148 significance of HCl pretreatment?

19. Line 168 correct as supernatant

20. Line 208 -211” Likewise, proteases were produced by B. licheniformis 209 PB1 (33) as well but under varying production parameters. Among them, physicochemical 210 parameters were a key determinant of varying production of protease in quantity and activity from 211 different species:. Rewrite the sentences for better clarity

21. Section 3.1 before mentioning the fold activity, author should mention the actual enzyme activity in terms of IU/ml

22. Section 3.1 line 207 “highest enzyme activity observed on seventh day” is the activity reduced or stable ,what was the reason for the reduction of activity explain in brief (1-2)lines.

23. Line 221“The peptide confirmation was obtained by MS/MS analysis of a tryptic peptide

peak with m/z 1108.496. MS/MS showed that this peptide is identical to the peptide covering amino acid residue 342-351 (K.HPNLSASQVR.N) of the apr gene product (Mascot score 80)” kindly justify the statement.

24. Section 3.3 line 235 Reason for better residual activity in bentonite immobilized subtilisin compared to other support used in the study?

25. Author studied the effect of change in buffer Molarity, and found at 1M strength the activity was maximum, at what temperature this activity was measured. Include this information.

26. Line 257, It has mentioned the enzyme is active at higher temperature range up to 70°C after 30 min of incubation. The loss in activity must be compared with the maximum activity at temperature 50 0 C.

27. Section 3.4.4, is not explained adequately. The adsorption of enzyme on a selected solid support should be explain properly.

28. Line 331“only a limited amount of activity was lost (how much).” check the statement and rewrite.

29. Section 3.6 I strongly suggest authors to compare the data of poultry trials with previously reported results. This may add merit to the present study and would help readers to understand the advantage of the work.

30. Section 3.5 This section should be more descriptive rather than straight forward observation of docking results. Mention about the ligand internal coordinates in grid for protein pocket calculation, score and binding energy with comparing where applicable.

31. Spell check the entire manuscript.

32. Check the units properly and follow standard units.

33. Check your reference format. It should be uniform.

Reviewer #3: Manuscript number: PONE-D-21-14945

Title: Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite

as a poultry feed supplement

The manuscript entitled ‘Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite as a poultry feed supplement' is an interesting piece of work that gives details about immobilized thermophilic Carlsberg Subtilisin on bentonite and its application to poultry feed supplement'. They prepared the Carlsberg Subtilisin enzyme, and then immobilized on to bentonite surface by physical adsorption, and then feed to poultry feeding. The results are deserved to be published in enzyme technology area. However, the level of results and discussion is not considered enough to merit its publishing in PLOS ONE. Some of the suggested changes to be made are given below.

Major

Q1. The authors should describe advantage of bentonite relevant with enzyme immobilization. This discussion would be very important to address originality of the manuscript.

Q2. Physico-chemical characterization of bentonite should be discussed.

Q3. The author conclude that bentonite is better than carbon materials for protease immobilization. However, depending on experimental condition, the result might be changed. This information must be clear in the manuscript.

Q4. Provide the supporting literature information about bentonite for enzyme immobilization (additional comparison Table) to justify the significance of this study.

Q5. Was determined the full loading of immobilized enzyme prepared under the optimized immobilization conditions? This information must be clear in the manuscript.

Q6. The loading capacity of enzyme immobilizaed prepared and the amount of attached enzyme: What were the optimum conditions? Please use numbers, no qualitative statements (high, good, higher, etc).

Q7. The kinetics and specific activity of immobilized protease on the bentonite based on the enzyme loading need to be more quantitative and should be given and discussed in the manuscript: Authors need to compare these results with other results in the literature.

Q8. Comparison of the free and immobilized enzyme in terms of kinetic parameters: Authors need to compare these results with other results in the literature.

Q9. The activity and stability of immobilized enzymes in bentonite carrier: The new biocatalysts presented were compared with a commercial material?? This information must be clear in the introduction.

Q10. The influence of metal ions and some inhibitors on the enzyme activity were examined?

Q11. The effect of the detergents and organic solvents was studied?

Q12. All discussion sections relevant with enzyme immobilization are very week, authors can improve it by suggesting the above citations such as how various in loading? why high activity? kinetic changes? And low reusability (may be leaching), what benefits of this system? etc.

Q13. Bentonite is mineral materials which might affect weight gain to chicken. However, the author didn’t include any proper experimental condition to discuss with potential synergetic effect of them. This information must be clear in the manuscript.

**********

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Reviewer #1: Yes: Shashi Kant Bhatia

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2023 Aug 16;18(8):e0269717. doi: 10.1371/journal.pone.0269717.r002

Author response to Decision Letter 0

10 Jan 2022

Comments:

Introduction: Improve this section by adding more information about properties of matrix required for enzyme immobilization and various supports reported for protease immobilization.

Thank you for highlighting the weak ends in the manuscript. The introduction is modified and additional information is added to the manuscript as per suggestion.

Line 31: Add suitable reference in support: Molecular biology interventions for activity improvement and production of industrial enzymes.

I will be publishing a whole study on this aspect. As it is more intricate and requires a lot of attention so a separate study is designed to address this aspect.

Line 51: What are drawbacks of organic support used for immobilization? Why we need inorganic support. Need explanation.

Organic supports are generally poor with a humid and temperature varying environments as the shelf life is greatly reduced and a need of addition of preservative are required to avoid microbial growth. For which an additional approval of poultry friendly preservatives is required to be tested and approved by the DRAP. – shelf life issue, microbial growth etc.

The inorganic supports are less intervening with the feed and enzyme to be fed eventually increasing the potential for being used in poultry as feed supplement. Also the bentonite is already a frequently used product in poultry industry to control wet liter.

Line 54: What are the advantages of clay over other inorganic supports reported?

Check the manuscript for typo errors, there should be space between value and units.

Majorly clay and its component is already being used poultry feed with an aim to control wet liter and adsorb trace amounts of aflatoxins present in feed. Additionally, the suggested spaces has been added between the values and units.

Line 165: Check and correct… pernatant was assayed

The typo errors has been removed after carefully review of paper.

Protease was partially purified and used in experiments. There may be several other proteins which have positive or negative effect on chicken growth improvement. Need justification.

We have designed another project and currently working on the comparative studies of the highly purified respective enzyme and partially purified. The formulations are being fed to the same bird types to test th impact of variation among the reported results.

What about the Native-PAGE? Is the reported protease in its monomeric form or multimeric? Add information.

Yes, true this is a very valid point. That experiment was not scope of the studies. We have mentioned above that we are working on the next part of this study where we are going to run comparison between purified and partially purified proteins to check if there are any other protein is participating and causing the effect. so we have included this experiment in our work flow to explore the potential participation of other proteins causing the net effect on the bird.

Why there is no data about the effect of pH, buffer strength and temperature effect on immobilized enzyme. This information is important for comparison. Add data and discussion.

Though the data was expected to be published in another manuscript, yet I have added the data in figures to highlight the successful immobilization impact. The discussion of immobilization variations and results will change the gist of the current aspects.

Reviewer #2: Manuscript title: Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite as a poultry feed supplement

The manuscript describes very simple method for the preparation of protease immobilized bentonite. The resultant material used for poultry feedstock to achieve healthy and productive chicken. Additionally, docking study was also performed to find optimal binding between protein and ligand. Overall, the manuscript has been written well. In my opinion, the work may be accepted for publication in PLOS ONE after Major revision.

My specific comments are as follows:

1. Reduce some words in title and change the keywords which areal ready present in title

We have changed the title word count 14 to 12. And please let us know that if there is anything else you suggest in the proposed title, if something is not correct will be highly appreciated.

The maximum units in U/ml are added to the manuscript as per suggestion.

Mention the maximum enzyme activity in term of unit and rewrite the sentences.

3. Abstract: Line 14. Significance of partially-purified enzyme?

The partially purified enzymes are proved to be economically feasible and efficient for poultry field application. Because highly purified proteins are only required for the pharmaceutical application and if highly purified product will be used eventually compromise the cost effectiveness of the product.

4. The introduction section is lack of focus. Please be selective on the background content and rewrite the introduction part after a wider literature investment.

The introduction is improved as per suggestion and necessary additions are made to the manuscript.

5. Line 36 Mention how the application of protease in poultry feed is promising? Kindly highlight productivity

and improved health via control on poultry zoonotic pathogens.

A separate set of experiments were designed and executed to observe the zoonotic pathogen control. The data is part of another manuscript. We collected the fecal matter of the tested birds in triplicate from all of the groups and were cultured to identify unique microflora present in the bird fecal matter. Further the microbes were identified. We observed feeding enzyme containing formulation increased the growth of lactobacillus species which was further tested on the potentially zoonotic pathogens and tested the antimicrobial efficacy against these pathogens affirming that feeding these formulations support growth of microbes that have greater antimicrobial activity against the zoonotic pathogens.

6. Author must mention the types of immobilization methods in brief and advantage of the method by which immobilization has been reported in this study.

The types of immobilizations are mentioned in the introduction section.

Line 43 Consider the relevant works published within 5 year for possible citations such as

Sanjay KS PATEL, Raviteja PAGOLU, DIBYA BHOL, LEE Jung-Kul

Eco-Friendly Composite of Fe₃O₄-Reduced Graphene Oxide Particles for Efficient Enzyme Immobilization. ACS applied materials & interfaces, 9(3), 2213-2222.

More relevant study may be cited related to immobilization support.

The suggested recent papers on the enzyme immobilization and efficacy of enzyme has been mentioned in the manuscript to improve the introduction section.

7. Recheck entire manuscript for additional space, for instance line 45.

The additional spaces are reviewed carefully and changes are made as per suggestions.

8. If the protease producing strain has deposited in any microbial culture collection repository mention the ID number (section bacterial cultivation)

It has not been deposited and we are still working on the genome sequencing. The protein sequence has been submitted to NCBI.

9. This production medium (3L) was inoculated with 2% (v/v) B. licheniformis and incubated at

99 60°C for 7 days. Author must mention other production parameter such as RPM and pH for protease production.

Thank you for feedback. The suggested correction has been made to the manuscript.

it was written mistakenly wrong and now corrected in manuscript.

11. Protease activity method and calculation should be explained in brief for readers

The Goose formula has been added as reference for understanding.

Line 110 Please add space between numbers and unit, for instance 280 nm (space between 280 and nm, please make further corrections in all the text.)

The spacing among units and words is corrected.

12. Line 121 Digestion protocol not provided

The reference of digestion has been attached as link to the reference.

13. Section 2.4 what was the peptide concentration for MALDI-TOF MS/MS analysis?

The tryptic digest with the concentration of 1:1 (0.1 ug/ul) was added. This has been added in the manuscript as well.

14. Line 130 how the database search was performed? Any specific genera or generic search kindly mention?

The peptide confirmation was obtained by MS/MS analysis of a tryptic peptide peak with m/z 1108.496. MS/MS showed that this peptide is identical to the peptide covering amino acid residue 342-351 (KHPNLSASQVRN) of the apr gene product (Mascot score 80) (Fig 1B). The Unipept analysis revealed that peptide was hitherto solely found in B. licheniformis Subtilisin (37).

15. Line 132 Mention the peptide charge used in MASCOT search

A positive charge was selected as the mentioned in the reference number 26.

16. Kindly provide peptide sequence showing identity with protease of Bacillus licheniformis along with score obtained in MASCOT search.

The score of MASCOT is updated to be 80 and the sequence is added to the manuscript.

17. Reason for selection of clay as support material?

Already a part of poultry feed as mentioned in the Introduction omits the hassle of approving the content from the poultry feed federations

18. Line 148 significance of HCl pretreatment?

The use of HCl remove the unnecessary charged particles from the clay to make it more susceptible for binding of protease to the support material.

19. Line 168 correct as supernatant

The corrections have been made as per suggestions thanks for highlighting it.

The suggested corrections have been made to the reviewed document.

21. Section 3.1 before mentioning the fold activity, author should mention the actual enzyme activity in terms of IU/ml

The actual enzyme units have been updated in the manuscript as per suggestion.

22. Section 3.1 line 207 “highest enzyme activity observed on seventh day” is the activity reduced or stable ,what was the reason for the reduction of activity explain in brief (1-2)lines.

The experiment was a batch culture so after a specific duration when all of the nutrients are drained the overall specific activity tends to drop indicative of the fact the all of the nutrients have be used.

23. Line 221“The peptide confirmation was obtained by MS/MS analysis of a tryptic peptide

A reference is linked as per the information and additional information is also added as per my understanding. Any suggestions for improvements will be welcomed.

24. Section 3.3 line 235 Reason for better residual activity in bentonite immobilized subtilisin compared to other support used in the study?

The residual activity improvement has been highlighted in the discussion section.

25. Author studied the effect of change in buffer Molarity, and found at 1M strength the activity was maximum, at what temperature this activity was measured. Include this information.

The temperature has been updated in the manuscript as 50C.

These trends will be better discussed in a separate study of immobilization kinetics and parameter characteristic evaluation of free and adsorbed subtilisin.

27. Section 3.4.4, is not explained adequately. The adsorption of enzyme on a selected solid support should be explain properly.

The relevant information has been updated as per the requirement.

28. Line 331“only a limited amount of activity was lost (how much).” check the statement and rewrite.

5% loss

The testing is unique as the combination of support with enzyme is not reported.

31. Spell check the entire manuscript.

Checked and corrected

32. Check the units properly and follow standard units.

Checked and corrected

33. Check your reference format. It should be uniform.

Reviewer #3: Manuscript number: PONE-D-21-14945

Title: Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite

as a poultry feed supplement

Major

Q1. The authors should describe advantage of bentonite relevant with enzyme immobilization. This discussion would be very important to address originality of the manuscript.

The discussion section includes all the necessary information highlighting the immobilization of subtilisin to bentonite and its impact on the poultry in terms of weight gain.

Q2. Physico-chemical characterization of bentonite should be discussed.

A separate study is designed to discuss these characteristics, Raman, FTIR, XRD was performed with and without the protein immobilizations.

The carbon based diet formulation of proteases has a drawback of fungal contamination increasing the risks to birds.

Q4. Provide the supporting literature information about bentonite for enzyme immobilization (additional comparison Table) to justify the significance of this study.

The Bentonite is mostly used in feed as a wet liter controlling agent, making this easier to choose and found it to be a good immobilizing agent for subtilisin.

Q5. Was determined the full loading of immobilized enzyme prepared under the optimized immobilization conditions? This information must be clear in the manuscript.

Q6. The loading capacity of enzyme immobilizaed prepared and the amount of attached enzyme: What were the optimum conditions? Please use numbers, no qualitative statements (high, good, higher, etc).

The suggested comments have been addressed in the reviewed document and suggestion changes have been made.

Indeed, a point to ponder, these results will be better elaborated in another study designed to evaluate the comparative analysis of free and adsorbed subtilisin separately.

Q8. Comparison of the free and immobilized enzyme in terms of kinetic parameters: Authors need to compare these results with other results in the literature.

These parameters were studied and designed for another manuscript and shall be published with elaborated discussion of kinetic parameters and comparisons.

The suggested information has been added to the introduction sections as well.

Q10. The influence of metal ions and some inhibitors on the enzyme activity were examined?

The effects were studied in another studied to be published in another paper.

Q11. The effect of the detergents and organic solvents was studied?

The effect of detergent and organic solvents were tested in another study

The suggested changes have been made to the document.

The amount of bentonite was balanced out in the feed controls.

Attachment

Submitted filename: Response to reviewers --U.docx

Click here for additional data file.^{(28.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0269717.r003

Decision Letter 1

Sanjay Kumar Singh Patel

3 Feb 2022

PONE-D-21-14945R1Immobilization and docking studies of Carlsberg subtilisin for application in poultry industryPLOS ONE

Dear Dr. ali,

Please submit your revised manuscript by Mar 20 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Sanjay Kumar Singh Patel, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

The authors should satisfy the Reviewer 3.

Please address his comments as response carefully as per PONE-D-21-14945 (previous submission).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: Author has revised the manuscript according to comments. Manuscript is recommended for publication as it is.

Reviewer #2: The quality of the manuscript has improved as a result of these revisions. I believe the manuscript (PONE-D-21-14945) entitled "Immobilization and docking studies of Carlsberg subtilisin for application in poultry industry" is now acceptable for publication in the PLOS ONE journal.

The following are the minor comments:

Comment 1: Line 32 abstract section: "The highest free enzyme activity () was observed." Correct the sentence.

Comments 2: Follow the guidelines in the writing units, and check the spacing throughout the manuscript.

Comment 3: For a clearer presentation and a more polished paper, capitalization, abbreviation, spelling, and spacing should be revised. The author should check the manuscript carefully.

Reviewer #3: The revised manuscript's corrections are not fully improved. Therefore, I recommend the article can be rejected.

PLoS One. 2023 Aug 16;18(8):e0269717. doi: 10.1371/journal.pone.0269717.r004

Author response to Decision Letter 1

18 May 2022

PONE-D-21-14945

Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite as a poultry feed supplement

PLOS ONE

Reviewer 2:

Thank you for careful evaluation of the Manuscript. I have tried my best to make all the suggested changes to the manuscript.

Reviewer #3: Manuscript number: PONE-D-21-14945

Title: Immobilization and docking studies of Thermophilic Carlsberg Subtilisin on Bentonite

as a poultry feed supplement

Major

Q1. The authors should describe advantage of bentonite relevant with enzyme immobilization. This discussion would be very important to address originality of the manuscript.

The discussion section includes all the necessary information highlighting the immobilization of subtilisin to bentonite and its impact on the poultry in terms of weight gain. As bentonite is an inexpensive matrix that does not have toxicity and chemical reactivity towards poultry making it most suitable solid support material for immobilization of Carlsberg subtilisin enzyme for potential use in poultry industry. Moreover, Bentonite is already an approved ingredient used in Asian poultry industry with an intention to control wet droppings making it most ideal solid support.

Q2. Physico-chemical characterization of bentonite should be discussed.

A separate comparative study is designed which addresses the Kinetics of immobilization in comparison with free enzyme. Additionally, the immobilization was confirmed by performing the physico-chemical evaluation through Raman, FTIR, SEM and XRD. The study includes the MALDI-TOF MS/MS Analysis of the Carlsberg subtilisin with O18 tagging of immobilized and free enzyme further digested with trypsin to evaluate the precise amino acid involved in binding of the protein to the bentonite. A comparative study design to evaluate the amino acid attachment pattern. The study also includes the comparative analysis of metal ions and inhibitors effect on immobilized and free enzyme.

The carbon-based adsorption diet formulation of proteases has a drawback of fungal contamination increasing the risks to birds. As the temperate and humid regions have greater chances of fungal growth. During experimentation upon storage for more than two weeks, we observed mold growing on the product requiring the need to add some preservatives to the formulations. Whereas this study is designed in a way that no foreign or unapproved ingredient added to feed might increase the risks to the birds.

Q4. Provide the supporting literature information about bentonite for enzyme immobilization (additional comparison Table) to justify the significance of this study.

The Bentonite is mostly used in feed as a wet liter controlling agent, making this easier to choose and found it to be a good immobilizing agent for subtilisin.

Q5. Was determined the full loading of immobilized enzyme prepared under the optimized immobilization conditions? This information must be clear in the manuscript.

The optimized conditions were observed to make the loading for immobilization and is addressed in the manuscript as well. I have added the free enzyme and immobilized enzyme comparison to highlight the impact of immobilized subtilisin to the bentonite but the results are already a part of another study as mentioned earlier and writing again will get plagiarized.

Q6. The loading capacity of enzyme immobilizaed prepared and the amount of attached enzyme: What were the optimum conditions? Please use numbers, no qualitative statements (high, good, higher, etc).

The suggested comments have been addressed in the reviewed document and suggestion changes have been made. The result and discussion section contains enzyme units in U/mg.

The results of immobilization comparison and discussion is already a part of submitted paper as mentioned in Q4’s response. A full fledge study was designed stating the immobilization of enzyme to bentonite compared with free enzyme and discussed with respect to reported literature.

Q8. Comparison of the free and immobilized enzyme in terms of kinetic parameters: Authors need to compare these results with other results in the literature.

These parameters were studied and designed for another manuscript and shall be published with elaborated discussion of kinetic parameters and comparisons for the same study addressed in Q4.

The suggested information has been added to the introduction sections as well. The name of commercial enzyme supplement is given in methodology section referring to line number 229.

Q10. The influence of metal ions and some inhibitors on the enzyme activity were examined?

The effects were studied in another studied to be published in another paper as mentioned in Q4.

Q11. The effect of the detergents and organic solvents was studied?

The effect of detergent and organic solvents was tested in another study mentioned in Q4.

The suggested changes have been made to the document. Majorly All of the concerning questions are related to another study that I have already designed for the second paper mentioned in Q4.

The amount of bentonite was balanced out in the feed controls. A negative and positive controls were run in parallel to address the effect. Also, Bentonite is already being used in poultry feed to reduce the wet liter control. We added same amount of unabsorbed bentonite to evaluate the difference between both. We added same amount of bentonite to the commercial enzyme supplement test to reduce the false positive and negative results. The gist is highlighted in the manuscript line number 230-232.

________________________________________

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PLoS One. doi: 10.1371/journal.pone.0269717.r005

Decision Letter 2

Afsheen Aman

27 May 2022

Immobilization and docking studies of Carlsberg subtilisin for application in poultry industry

PONE-D-21-14945R2

Dear Dr. ali,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Afsheen Aman, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: I suggest it can be accepted for publication.

The revised manuscript's corrections are improved. The justification and discussion are clearer. Therefore, I recommend the article can be accepted.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

PLoS One. doi: 10.1371/journal.pone.0269717.r006

Acceptance letter

Afsheen Aman

11 Apr 2023

PONE-D-21-14945R2

Immobilization and docking studies of Carlsberg subtilisin for application in poultry industry

Dear Dr. Ali:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Afsheen Aman

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Carlsberg subtilisin produced by B. licheniformis PB1.

(PDF)

Click here for additional data file.^{(253.8KB, pdf)}

S1 Raw images

(PDF)

Click here for additional data file.^{(277.3KB, pdf)}

S1 File

(DOCX)

Click here for additional data file.^{(86.5KB, docx)}

Attachment

Submitted filename: Response to reviewers --U.docx

Click here for additional data file.^{(28.7KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(18.5KB, docx)}

Data Availability Statement

All relevant data are within the paper and its supporting information files.

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PERMALINK

Immobilization and docking studies of Carlsberg subtilisin for application in poultry industry

Anum Munir Rana

Bart Devreese

Stijn De Waele

Asma Rabbani Sodhozai

Maryam Rozi

Sajid Rashid

Abdul Hameed

Naeem Ali

Roles

Abstract

Introduction

Materials and methods

Chemicals

Protease production, purification, and extraction

Bacterial cultivation

Enzyme production

Enzyme extraction and purification

Protease activity assay

Electrophoretic analysis

Identification of proteins by MALDI-TOF/TOF mass spectrometry

Enzyme immobilization

Free enzyme optimization

Enzyme adsorption on selected solid supports

Enzyme desorption

Structural modeling

Fig 4. The best-docked pose of bentonite nanotube in the BSP binding cleft.

Supplementation of Carlsberg subtilisin-bentonite product to poultry as a growth enhancer

Statistical analysis

Results and discussion

Protease production, purification, and extraction

SDS PAGE, zymography and MALDI TOF-MS/MS

Fig 1.

Solid support selection for enzyme adsorption

Fig 2. Free and absorbed enzyme optimization for immobilization to a suitable solid support and desorption studies.

Optimization of conditions before adsorption

Effect of change in buffer molarity

Free enzyme temperature optimization

Influence of pH for enzyme activity

Free enzyme adsorption on a selected solid support

Enzyme desorption

Molecular docking analysis

Fig 3.

Fig 5.

Poultry application

Table 1. Comparison of enzyme immobilization to organic and inorganic solid supports.

Table 2. Weight gain in Broilers (46 Days) after addition to feed Carlsberg-subtilisin-bentonite complex, B&G enzyme product, and without enzyme.

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Sanjay Kumar Singh Patel

Roles

Author response to Decision Letter 0

Decision Letter 1

Sanjay Kumar Singh Patel

Roles

Author response to Decision Letter 1

Decision Letter 2

Afsheen Aman

Roles

Acceptance letter

Afsheen Aman

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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