Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.


Introduction
Despite advances in understanding the diversity and systematics of bacilli, studying their hydrolytic enzymes with bioengineering interest and their characterization has received more attention. Of particular interest, Virgibacillus is a genus of Gram-positive bacteria belonging to the wider family of Bacillaceae within the Firmicutes phylum [1]. e genus was named by Heyndrickx et al. [2]. At the time of writing, the genus Virgibacillus comprises 35 species with validly published names (http://www.bacterio.net/ virgibacillus.html). Most members of genus Virgibacillus are mostly isolated from saline environments like marine sediment, soil, fish sauce fermentation, and lake [3][4][5][6]. e genus showed the ability to produce a great variety of extracellular hydrolytic enzymes. For instance, Virgibacillus sp. strain SK37, Virgibacillus halodenitrificans strain RSK, Virgibacillus sp. strain CD6, and Virgibacillus dokdonensis strain VITP14 have been shown to produce extracellular proteases [7][8][9][10]. However, information regarding stability and compatibility with laundry detergents and molecular modeling and structural characteristics, as well as the docking study of proteases from Virgibacillus is still very limited.
Peptidases or proteinases are known as enzymes able to cleave the array of proteins ingested into smaller peptide fragments in aqueous environments, but some peptidases perform slightly the peptide synthesis bonds in microaqueous media [11]. According to the Enzyme Commission (EC), peptidases belong to group 3 of the hydrolases and subgroup 4, hydrolysis of peptide bonds, but can still be classified according to the catalytic action (endo-or exopeptidase), active site, charge, molecular size, and substrate specificity.
In the last 50 years, proteases and other enzymes in laundry detergents switched from being minor additives to key ingredients [12,13]. In common with all enzymes, external factors such as type of media, pH, and temperature are important for the catalytic efficiency, activity, stability, and proper functioning of proteases [14]. erefore, the search for novel alkaline-stable and thermostable peptidases from extremophilic microorganisms and the engineering of the already available enzymes have been the major areas of research in this field over the years [15,16]. Valuable characteristics of peptidases from polyextremophiles and their applications are gathered from a wide literature survey [17][18][19]. Salt-stable peptidases are the most widely used biocatalysts in laundry and dishwashing detergents where the high salt concentration is the desirable assurance of protease used in detergent, as NaCl is a key ingredient in the granulation process prior to addition of the protease in the detergent formulation [20,21]. Research is in progress to isolate halophilic microorganisms that produce enzymes with this desired property [22,23]. e assessment of bacterial diversity of an Algerian saline lake revealed the presence of a novel specie of a genus Virgibacillus, namely Virgibacillus natechei sp. nov., strain FarD T with unusual phenotypic and genotypic characteristics [5]. In fact, the strain FarD T was mesophilic, moderately halophilic, and alkaliphilic. is strain grew in the presence of NaCl concentrations ranging from 1 to 200 g/L, with an optimum at 100 g/L. e temperature range for growth was (15-40°C), with optimal growth occurring at 35°C. e pH range for growth was from 6 to 12, with an optimum at 7 [5]. No enzymatic research regarding this new species has been found in the literature, and for the first time with the current study, a research into the purification, characterization and biotechnological applicability of a new peptidase enzyme from strain FarD T was investigated.
Herein, the current research was undertaken to purify, characterize, and to express for the first time, a new peptidase secreted from the culture supernatant of the moderately halophilic bacterium Virgibacillus natechei strain FarD T and explore its promising potential enzymatic performance as a bioadditive for peptide synthesis biocatalysis and laundry detergent composition.

Materials.
e raw material of shrimp shell was obtained in fresh conditions from a fishery market located at Sfax, Tunisia. Column chromatography materials were purchased from Agilent Technologies, Lawrence, Kansas, MO, USA. A Amersham LMW protein marker was purchased from GE Healthcare Europe GmbH, Freiburg, Germany. DNA molecular markers for electrophoresis and substrates were purchased from Invitrogen, Carlsbad (CA, USA) and Sigma Chemicals Co. St. Louis (MO, USA), respectively. All the microbiological media components were a product of Bio-Rad Laboratories (Hercules, CA, USA e type strain of Virgibacillus natechei is FarD T (DSM 25609 T or CCUG 62224 T ) [5]. As reported previously by the authors [5], strain FarD T was maintained at 35°C on Sehgal and Gibbons medium (SG) containing the following (in g/L): casamino acids, 7.5; yeast extract, 10; sodium glutamate, 1; trisodium citrate, 3; MgSO 4 ·7 H 2 O, 20; KCl, 2; FeSO 4 ·7 H 2 O, 0.036; and MnCl 2 ·4 H 2 O, 0.00036 supplemented with 100 g/L of NaCl at pH 7. After incubation at 35°C, a halo of casein degradation was revealed around the colony growth onto skimmed milk agar plates, as well described previously by the authors [17,18]. e proteaseproducing isolate was propagated on SG agar plates at 35°C and conserved at − 80°C in SG medium where 20% of glycerol was added.

Peptidase Production.
e preculture of strain FarD T was carried out in a 1 L Erlenmeyer flask containing 100 mL of SG liquid medium at pH 7 supplemented with 100 g/L NaCl and incubated at 35°C overnight. is preculture was used to inoculate the culture using the optimized ELM media at pH 7.4 containing only 60 g/L white shrimp shell by-product in the Erlenmeyer flask. e initial A 600 nm of the culture was 0.1. e flasks were incubated at 35°C for 36 h with shaking at 200 rpm in 1000 mL Erlenmeyer flasks with a working volume of 100 mL. Bacterial growth of strain FarD T and cell dry weight were estimated as detailed recently [24]. e cell pellet was removed before each assay, and the obtained clear supernatant was served as a peptidase source in the subsequent investigations.

Peptidase Activity Assay.
e peptidase activity was assayed by the addition of 0.5 mL of an appropriately diluted enzyme in 0.1 M glycine-NaOH buffer at pH 9, supplemented with 2 mM CaCl 2 (buffer A), to a 0.5 mL of 10 g/L casein. e reaction mixture was incubated for 15 min at 60°C. e reaction was stopped by adding 0.5 mL of 200 g/L TCA. In fact, the liberated tyrosine from casein was measured using the Kembhavi method [25]. One unit (U) of peptidase was defined as the amount of enzyme releasing 1 µg of tyrosine released under the assay conditions detailed.
Peptidase activity present in the laundry detergent solution was determined through the method proposed by Boulkour Touioui et al. [26] which used the N,N-dimethylated casein (DMC) as a substrate and 2,4,6-trinitrobenzene sulfonic acid (TNBSA) as a colour indicator. One unit of protease activity was defined as the amount of enzyme required to catalyze the cleavage of 1 µmole of peptide bond from DMC per minute under the experimental conditions used. e absorbance was measured at 450 nm.

Peptidase Purification
Procedure. 500 mL of a 36 h culture of strain FarD T was centrifuged for 20 min at 10,000 ×g. e clear supernatant containing extracellular peptidase was used as the crude enzyme preparation and was submitted to the following purification steps. Proteins were precipitated to 20% with solid (NH 4 ) 2 SO 4 and then centrifuged at 10,000 ×g for 20 min. e obtained supernatant was saturated up to 80% with (NH 4 ) 2 SO 4 and centrifuged, the precipitate was resuspended in a minimal volume of buffer B composed with 50 mM MOPS and 2 mM CaCl 2 at pH 7, and dialyzed overnight. Next, the obtained supernatant was loaded and applied to an HPLC system using a ZORBAX PSM 300 HPSEC column previously equilibrated with buffer B. Pooled fractions, containing peptidase activity, were concentrated for further analysis.

Analytical Methods.
Protein concentration was estimated through the method of Bradford [27], using bovine serum albumin as a reference. e molecular weight of the purified peptidase SAPV was carried out by SDS-PAGE, as described by Laemmli [28]. Casein zymography staining was estimated by incorporating azo-casein (10 g/L) into the separating gel before polymerization, as detailed elsewhere [29]. Bands of purified SAPV were separated on SDS gels, transferred to a ProBlott membrane, and then the NH 2terminal sequence analysis was performed by automated Edman degradation using an Applied Bio system Model 473A gas-phase sequencer.

Effects of Inhibitors, Reducing Agents, and Metal
Ions on SAPV Stability. Preincubation of the purified SAPV with specific inhibitors, reducing agents, and various and divalent metal ions was investigated and described by Jaouadi et al. as well [29]. e remaining peptidase activity was measured after preincubating the purified SAPV with each compound for 1 h at 40°C. e remaining SAPV activity was measured by optimum assay. For the effect of metal ions, the nontreated SAPV, or dialyzed, was considered as control 100%. For the inhibitors and reducing agents on SAPV stability, the peptidase solution without inhibitor was considered as control.
Using casein as a substrate, the effect of temperature on SAPV was investigated at 30-80°C at pH 9. Its thermostability was examined by incubation for 600 min at 40-70°C at pH 9 with and without 2 mM CaCl 2 . Aliquots were withdrawn at desired time intervals to test the remaining activity.
e nonheated SAPV was considered as a control. Samples were taken out sequentially at various time intervals and residual activity was analyzed by performing the assay under standard assays.

Determination of the Effect of Polyols on SAPV Stability.
e effect of variety of polyols, with concentration of 100 g/L, on SAPV thermostability was carried out by its incubation at 80°C for 30 min with and without different polyols. e combination of glycerol (100 g/L) and calcium (2 mM) on the thermostability of SAPV peptidase was monitored at 80°C for 450 min. Peptidase assays were carried out under the standard experimental assay conditions.

2.2.9.
Effect of NaCl and KCl on SAPV Activity. SAPV was preincubated for 1 h with various concentrations of NaCl and KCl from 0.5 to 5 M at pH 9. e activity of SAPV without NaCl or KCl was taken as 100%. e residual peptidase activity was measured by performing the assay under standard conditions.

Molecular Study of SAPV Protease
(1) Cloning and Expression of the SapV Gene. Based on the high homology found in peptidase S8 gene from Virgibacillus massiliensis DSM 28587, two primers (F-MS40 and R-MS41) were designed to generate ∼1.4 kb PCR (Polymerase Chain Reaction) fragment encoding the peptidase SAPV. e PCR was carried out in a MultiGene ™ OptiMax ermal Cycler, 96 × 0.2 mL (Diamed Lab Supplies Inc., Canada), and the mixture (100 µL) was composed of 250 ng of DNA template, amplification buffer, 30 pg of each primer, and 2 U of pfu (Biotools, Madrid, Spain). e PCR cycling parameters were 94°C for 5 min, followed by 35 cycles of 94°C for 60 s, 58°C for 60 s, and 72°C for 45 s as well as 72°C for 10 min. e PCR product was purified and cloned into pCR-Blunt, pUT57, and pTrc99A Vectors, previously digested with EcoRI, leading to the pSM40, pSM41, and pSM42 plasmids, respectively, using the E. coli BL21(DE3) pLysS [29].
For the recombinant SAPV production, the E. coli BL21(DE3)pLysS/pSM41 and E. coli BL21(DE3)pLysS/ pSM42 were induced by the addition of 5 mM IPTG after reaching an absorbance (A 600 ) of about 0.7. e enzyme crude extracts were obtained from the extracellular fraction by the modified procedure described by Abe et al. [30]. e extracellular recombinant enzyme (rSAPV) was purified to homogeneity using the same steps applied for the purification of the native enzyme.
(2) Sequence Analyses and Bioinformatics Tools. e nucleotide sequence of sapV gene was determined at least with three positive clones on both strands, using the automated DNA sequencer ABI PRISM ® 3100-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Multiple and alignments analyses of nucleotide and protein sequences were performed using the available program tools at the ExPASy database (https://www.expasy.org/).

Enzymatic Performance of the Purified Peptidase rSAPV
(1) Substrate Specificity Determination. e substrate profile specificity of rSAPV from E. coli BL21(DE3)pLysS/pSM42 was determined using natural and modified protein substrates, as well as esters and synthetic peptides. Enzymatic activities were determined on each substrate based on standard conditions, previously described elsewhere [31,32].

(2) Kinetic Characterization of Purified and Commercial
Proteases. Kinetic parameter constants, K m and V max , were determined by Lineweaver-Burk plots using casein and Suc-F-A-A-F-pNA as substrates at different concentrations ranging from 0.10 to 50 mM for 15 min at optimum pH and temperature of each commercial or purified peptidases (rSAPV, ermolysin type X, Alcalase 2.4 L FG, and Bioprotease N100L) at a final concentration of proteins 1.5 mg/ mL.
(3) Effects of Organic Solvents on rSAPV, SPVP, and ermolysin Type X Peptidases. Various organic solvents, with different log P values at 50% (v/v), were tested as well detailed in a previously study [17]. e peptidase enzymes used were rSAPV, ermolysin type X, and SPVP. Enzyme assays were carried out under the standard assay conditions for each used peptidase (60°C and pH 9 for rSAPV, 70°C and pH 8 for ermolysin type X, and 60°C and pH 10 for SPVP).

(4) Comparison of Stability and Compatibility with Laundry
Detergents between rSAPV and Commercial Proteases. Using DMC as a substrate, the compatibility of rSAPV, Alcalase 2.4 L FG, and Bioprotease N100L enzymes with a wide range of commercialized detergents was assayed. So, to simulate washing conditions, detergent solutions were prepared in tap water (as laundry washing has to be done in tap water) at a concentration of 7 g/L and then heated for 1 h at 70°C to destroy the indigenous protease or enzyme activity anterior to the addition of the enzymes. en, residual peptidase activity was analyzed by performing the assay under standard protocol. e peptidase activity of a control (without any detergent), incubated under similar conditions, was taken as 100%.

(5) e Effectiveness of Protein Stain Removal from Cotton
Fabrics. To estimate the stain removal capabilities of rSAPV, Alcalase 2.4 L FG, and Bioprotease N100L proteases, clean white cotton cloth pieces (5 cm × 5 cm) were soaked and dried with chocolate, egg, and blood. e used blood was freshly obtained from a local municipal slaughterhouse and collected from cow into anticoagulant heparin tubes (Sfax municipal slaughterhouse, permission was obtained from this slaughterhouse to use these animal parts). e stained cloth pieces were shake-incubated separately for 1 h with litre beakers containing a total volume of 100 mL of tap water, Class detergent (7 g/L, in tap water), and detergent added with 500 U/mL of rSAPV peptidase or with 500 U/mL of commercial proteases (Bioprotease N100L and Alcalase 2.4 L FG), followed by rinsing with water. en, the washed cloth pieces were dried. Visual examination of various pieces was also carried out to show the effect of each used peptidase in the removal of proteinaceous stains. e untreated stained piece of cloth was taken as a control.

Producing Strain FarD T Peptidase Using Powder from
White Shrimp Shell By-Product. Strain FarD T was cultured for 36 h at 35°C in Erlenmeyer flasks in optimized ELM media. A high level of peptidase production (16,000 U/mL) was obtained with this bio waste as the sole carbon and nitrogen source (Figure 1(a)). us, the powder from white shrimp shell by-product is a source for the development of the bacterium and the synthesis of metabolites as well. While Virgibacillus pantothenticus strain MTCC 6729 was demonstrated to produce only 18.2 U/mL using casein at 40°C after 72 h of incubation at pH 9 [33], Virgibacillus dokdonensis strain VIT P14 was demonstrated to produce 996 U/ mL using zobell marine broth [34]. To an increasing extent, by-products from shrimp shell by-product are considered by researchers as a "raw material" and not a "waste material." Yet, the biochemists and microbiologists used it as a cheap substrate for the production of bioactive substances such as enzymes and peptides [35][36][37]. Annamalai recorded the production of halostable peptidase enzyme from halophilic bacteria using marine bio-wastes such as squid pen powder and crab and shrimp shells [38,39].

SAPV Purification.
e summary of the peptidase SAPV purification is tabulated in Table 1. e recovery yield and purification fold of SAPV from strain FarD T are higher than those of peptidases from Aeribacillus pallidus strain VP3 and from Virgibacillus dokdonensis strain VIT P14 [17,34]. In fact, the protease from strain VIT P14 was purified with a purification fold of 5.3 and by a low recovery yield of about 9, while the SPVP was purified by a recovery yield and purification fold, respectively, of about 22% and 18. e specific activity displayed by SAPV (74,652 U/mg) was significantly high compared with that reported from strain VP3 (41,250 U/mg) and strain VIT P14 (102 U/mg). Based on these results, SAPV enzyme  seems very promising and can be tested in several biotechnological applications.

Molecular Weight Determination.
e SDS-PAGE analysis of the pooled fractions from the HPLC column showed one band, with apparent molecular mass, about 31 kDa (Figure 1(b)). Similarly, Rajeswari et al. reported that the purified protease from strain VIT P14 had a molecular weight of 36 kDa [34]. e proteases from extremophilic bacteria showed a molecular weight ranged from 40 to 130 kDa [40]. Zymogram activity staining revealed the presence of one prominent zone of caseinase activity for the purified peptidase (Figure 1(c)). e SAPV preparation was a homogeneous pure enzyme with high purity confirmed by a symmetrical peak with Rt of 8.687 min (Figure 1(d)), matching with the protein of ∼31 kDa (Figure 1(b)). e results mentioned above strongly suggested that SAPV is a monomeric enzyme comparable to previous investigation for peptidases from strains VP3 [17] and VIT P14 [34].

NH 2 -Terminal Amino Acid Sequence Analysis of SAPV.
e blotted peptidase SAPV was submitted to NH 2 -terminal sequencing, which allowed the identification of 25 residues, which are EQTVPWGIDYIGKAAAHQLGIFGKG. e sequence showed uniformity, indicating that SAPV was isolated in the pure form. e NH 2 -terminal sequence of SAPV was compared with other closely related peptidase sequences and noted to share greatest homology with those from Bacillus spp. and related genus. So, the amino acid resemblance of the NH 2 -terminal sequence between these proteases is an indication of the existence of a molecular relationship and that all these enzymes seem to derive from the expression of an ancestral gene. As illustrated in Table 2, the identity reached 80% with peptidase S8 from Virgibacillus massiliensis DSM 28587, which contains five modified residues at positions T3L, G7N, K13S, A14T, and F22S, respectively. ese results and comparison with the nearest proteases indicated that SAPV enzyme from Virgibacillus natechei strain FarD T is a new peptidase.

Effects of pH on SAPV Activity and Stability.
According to the results presented in Figure 2(a), SAPV possesses an optimum activity at pH 9. e relative activity at pH 8 and 10 were, respectively, 90 and 74%. At pH levels of 5 and 6, the SAPV activity was 38 and 58%, respectively, while the two proteases produced by Virgibacillus dokdonensis strain VITP14 and Virgibacillus pantothenticus strain MTCC 6729 showed, respectively, an optimal pH at 10 and 7 [10,33,34]. e protease activity secreted by strain MTCC 6729 lost 20% of its activity at pH 12 and was reduced to 27% at pH 6 [33]. ese differences mentioned between the proteases of the bacteria produced by the same genus indicated that the range of pH activity is in relation with the source of the strain from which it has been isolated. e effect of pH on the stability of SAPV was determined at 60°C. e pH stability profile of SAPV, illustrated in Figure 2(c), indicated that the peptidase showed a maximum activity at pH 7, 8, 9, and 10, respectively, until 75, 60, 45, and 15 min. e half-lives (t 1/2 ) of SAPV are 195, 165, 120, 75, and 30 min, respectively, at pH of 7, 8, 9, 10, and 11. At pH values between 7 and 10, SPVP activity is 100% after 24 h of incubation [17]. In agreement to the current study, SAPRH protease, produced by Bacillus safensis strain RH12, which has an optimum pH at 9 and stable in the pH range of 7-12, has been applied in the laundry industry [24]. Rather, according to Gupta et al., a protease which is useful for detergent application should be active in a pH range between 8 to 12 [41]. So, SAPV can greatly be tested as a bioadditive in solid and liquid laundry detergents.

Effects of Temperature on SAPV Activity and Stability.
As shown in Figure 2(b), SAPV retained its activity over a wide range of temperature between 30 and 80°C with a maximum activity at 60°C. With 2 mM of Ca 2+ , SAPV activity was enhanced to 135 and 120%, respectively, at 50 and 70°C, with an optimum at 60°C. 35% of its activity was retained at 80°C. Without 2 mM of Ca 2+ , SAPV was observed to retain only 72 and 50% of activity, respectively, at 60 and 70°C, with an optimum at 50°C.
In agreement to the current study, SPVP has an optimum temperature at 60°C [17], the same of SAPV. Otherwise, the two peptidases produced by Virgibacillus dokdonensis strain VITP14 and Virgibacillus pantothenticus strain MTCC 6729 showed, respectively, an optimal temperature at 40 and 50°C [10,33,34]. e SAPRH protease successfully incorporated in commercial liquid laundry detergent (Class) of the STE JMAL (EJM)-Laundry Detergent Industry (Tunisia) showed the same optimum temperature of that of SAPV [24]. e calcium-dependent Experiments were conducted three times, and ± standard errors are reported. b One unit of peptidase activity was defined as the amount of enzyme required to release 1 µg tyrosine per minute under the experimental conditions used. c Amounts of protein were estimated by the method of Bradford [27].
stability of SAPV was determined by incubating the peptidase at different temperatures from 40 to 70°C for 600 min in the presence and absence of 2 mM Ca 2+ at pH 10 ( Figure 2(d)). e t 1/2 of SAPV in the absence of any additive was 510, 360, 210, and 90 min at 40, 50, 60, and 70°C, respectively. Adding calcium to 2 mM, the t 1/2 of SAPV was enhanced to 600, 420, 270, and 150 min, respectively. is behavior was similar to what has already been reported for SPVP protease, when the t 1/2 was ameliorated from 6 to 8 h when adding 2 mM of Ca 2+ [17]. All the evoked results suggested the high thermostable nature of SAPV peptidase in alkaline conditions.

Effects of Inhibitors and Metal Ions on Peptidase
Stability of SAPV. e findings in Table 3 revealed that SAPV peptidase is completely inhibited by DFP and PMSF, well-known serine peptidase inhibitors. e strong inhibition by those two inhibitors suggested that a serine residue was involved in the active site of the peptidase SAPV. Likewise, the protease from strain VIT P14 showed a complete inhibition against PMSF after an incubation at 37°C for 30 min [34]; however, the protease from strain MTCC 6729 retained 35% of its activity against 5 mM PMSF and totally inhibited against 10 mM PMSF [33]. e essential serine residue was often described for the alkaline salt-stable proteases from the halophilic Bacillus strains [22,23], while the peptidase SAPV retained all of its activity towards the trypsin competitive reagents, the chymotrypsin alkylating agent, the zinc protease inhibitor, and the acid and thiol reagents. In fact, the SBTI, TLCK, TPCK, benzamidine, DTNB, LD-DTT, 2-ME, NEM, iodoacetamide, leupeptin, pepstatin A, MIA, EPNP, and 1,10-phenanthroline monohydrate had almost no effect on the activity. is behavior is also detailed in SPVP protease [17]. e protease from strain MTCC 6729 is slightly inhibited by β-ME and LD-DTT, which are two thiol inhibitory proteases [33]. In addition, the peptidase from strain VIT P14 showed a moderate inhibition with LD-DTT. In fact, the relative activity is 89% in the presence of LD-DTT [34]. With the ethylene-diaminetetraacetic acid (EDTA) and ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), as chelating agent inhibitors, the SAPV peptidase retained 86 and 74% of its activity, respectively, in the presence of 10 mM EDTA and 2 mM EGTA, which suggests that no metal cofactors were required. Or, the two inhibitors are widely used to sequester metal ions. So, the moderate inhibition by EDTA and EGTA suggested that SAPV peptidase is not a metalloprotein.
ese observations indicate that SAPV enzyme showed a potential for use as a detergent bioadditive. Indeed, the chelating agents function as water softeners in commercial detergents and assist in stain suppression [42]. In Table 3, the influence of several metal ions on SAPV peptidase is tabulated. e addition of Ca 2+ increased SAPV activity by 290%. e Ca 2+ has been shown as ions that speed up the peptidase activity. Similarly, the addition of 2 mM Ca 2+ increased SPVP activity by 283% of the control [17]. Recently, with a docking study, two calcium binding sites have been shown at SAPRH protein, which play a significant role in stabilizing the enzymatic activity by binding at specific sites [24]. In the same context, SAPV peptidase is improved by Fe 2+ , Cu 2+ , and Zn 2+ at 2 mM in comparison with the control by 210, 125, and 120%, respectively. However, the Fe 2+ and Cu 2+ have an inhibitory effect on the peptidase activity from train VIT P14 [34]. e heavy metals: Co 2+ , Hg 2+ , and Cd 2+ completely inhibited SAPV activity. ese observations were similar to that described to the SPVP protease which is totally inhibited by Co 2+ , Cd 2+ , and Hg 2+ at 2 mM. is phenomenon is in contrary to what has been described in the peptidase produced by strain VIT P14, which, Co 2+ showed a stimulatory effect on activity [34].

Impact of Polyols on SAPV Activity and ermal Stability.
e results shown in Figure 2(e) mentioned that the highest level of SAPV peptidase was registered with glycerol followed by mannitol and PEG 1500 as additives, at a concentration of 100 g/L. Over and above, the thermal stability was more effective by the presence of calcium at 2 mM and xylitol at 100 g/L together. In fact, the half-life of SAPV at 80°C without any additive is 90 min; contrariwise, it is 420 min by the combined effect of calcium and glycerol (Figure 2(f )). e addition of polyols has already improved the thermal stability of SPVP. In fact, the half-life times at 80°C was determined to be, respectively, 9 h with calcium at 2 mM and xylitol compared with 2 h in the absence of any additive [17]. Researchers have assumed this phenomenon of enhanced stability of enzymes with polyols at high temperatures is related to their hydroxyl groups [24,43].    Results showed that more than 50% of residual activity was obtained at the concentration of 0.5-5 M of both osmolytes (Figures 2(g) and 2(h)). At 5 M NaCl and KCl, peptidase   activity decreased progressively and retained 62 and 52% of its residual activity, respectively. A similar kind of high-salt tolerance and osmotic pressure is a distinctive quality of halophiles proteases, which have a number of future purposes in industrial bioprocess [22,23,44].

Cloning and Heterologous Expression of the sapV Gene
3.6.1. Cloning and Sequencing of the sapV Gene. e PCR product corresponding to the entire SAPV coding region was cloned and sequenced leading to the pSM40. It revealed an open reading frame (ORF) of 1158 bp encoding 386 amino acids (Figure 3). is ORF was checked as the gene encoding SAPV peptidase enzyme, as given by the Edman degradation mechanism, the deduced amino acid sequence was noted to comprise the 25 NH 2 -terminal amino acid sequence of the purified enzyme. e signalP expected a signal peptide (pre-sequence) of 27 amino acids bounded with the signal peptidase recognition (SPR) site D-A-A-V, marking that a group of robustly hydrophobic amino acids was maintained. Belonging to the signal sequence, the prosequence consisting of 84 amino acids had to be brooked by autoproteolytic processing in the periplasm. e active mature SAPV peptidase consisted of 276 amino acids having a predicted molecular weight of 29,556.68 Da. e apparent molecular weight of the purified enzyme (∼31 kDa) determined by SDS-PAGE and HPLC gel filtration chromatography was in good agreement with the predicted value. e typical triad catalytic residues (D32, H64, and S219) in the active site and three serine peptidase signatures (amino acid residues 28-39, 64-75, and 216-226) [45] were also conserved in the sapV gene. e amino acid sequence deduced from the nucleotide sequence of the sapV gene was compared with that of other known subtilisin superfamily of serine peptidases S8/S53. e amino acid composition of SAPV indicated that it was devoid of cysteine and cystine residues. e most significant feature of the amino acid composition of SAPV was its high Asx amount (Asp and Asn residues), compared with other subtilisins. SAPV contained 37 amino acid residues of Asx (17 Asp and 20 Asn), corresponding to 13.4 mol%, whereas SAPB [29], subtilisin Novo [46], and subtilisin Carlsberg [47,48] possess exclusive 12, 10.2, and 9.45 mol%, respectively.

Expression of the sapV Gene in E. coli and Characterization of Recombinant Enzyme rSAPV.
To express SAPV, the corresponding gene was cloned downstream of PT7 and Ptac promoters in pSM41 and pSM42, respectively and then transformed into E. coli BL21(DE3)pLysS strain. No alkaline peptidase activity was found in the periplasmic fraction, neither in the intracellular fraction for all recombinant strains. Relatively elevated quantities of specific activity of 310 and 1,250 U/mg were, nevertheless, detected in the extracellular fractions of E. coli BL21(DE3)pLysS/ pSM41 and E. coli BL21(DE3)pLysS/pSM42, respectively. Found on this investigation, the SAPV peptidase was most efficiently expressed with the construction of Ptac-sapV (pSM42). e latest was, therefore, preserved for the purification of the recombinant peptidase (rSAPV). Extracellular rSAPV (pSM42) was purified using the same steps taken for the native enzyme from strain FarD T . All the biochemical properties from rSAPV were almost analogous to those of the original one. e recombinant enzyme, rSAPV, can be readily prepared on a large-scale for biotechnological sectors.
e , with 10 amino acids of difference. Even so, one amino acid in the signal peptide, and 9 amino acids (T3L, G7N, K13S, A14T, F22S, K36N, W65K, T214L, and L215A) in the mature SAPV were recorded to differ from the peptidase S8 from Virgibacillus massiliensis. In spite of displaying high levels of homology, this peptidase S8 proved relatively dissimilar properties. As a matter of fact, they indicated a sharply different physicochemical and kinetic properties as compared with SAPV. It can, therefore, be inferred that the substitutions in the mature SAPV, noted to differ from the other peptidases, synchronously enhanced the alkaline pH, thermostability, and catalytic parameters of the enzyme. e ability of proteolytic enzyme to hydrolyze and act upon a specific class of substrates with flawless precision and efficiency is a fundamental feature of biotechnology application. e substrate specificity is frequently assigned to the amino acid residues foregoing the peptide bond they broke. e relative hydrolysis rates of various protein substrates were investigated to elucidate the amino acid preference/substrate specificity of rSAPV enzyme from E. coli BL21(DE3)pLysS/pSM42 (Table 4). When assayed with esters as substrates, rSAPV showed high level of peptidase activity towards ATEE, followed by BTEE, which are two N-terminal-and C-terminal-protected L-tyrosine. Contrariwise, no esterase activity detected with BAEE, BCEE, and TAME as substrates which are N-terminal and C-terminal protected L-arginine and cysteine. us, rSAPV exhibits both amidase and esterase activities and preferentially hydrolyze peptide bonds that are contributed by hydrophobic and cationic amino acids at P 1 . e specificities for synthetic substrates at N-terminal residues to the cleavage site (P 1 , P 2 , etc) were also investigated, see Table 4. rSAPV largely preferred Suc-F-A-A-F-pNA peptide with 100% of activity. e relative peptidase activities are 87, 75, 62, and 52% on Suc-A-A-P-F-pNA, Suc-A-A-V-A-pNA, Suc-A-A-P-M-pNA, and Suc-L-L-V-Y-pNA, respectively. So, rSAPV peptidase chiefly preferred hydrophobic substrates, especially those with aromatic amino acids occupying the P 1 and P 4 positions of pNA peptides. When alanine amino acid is in the P 1 position, the relative activity of rSAPV is 38, 35, and 21 for Suc-A-A-P-L-pNA, Suc-A-A-F-pNA, and Suc-A-A-V-pNA, respectively. us, rSAPV was closely dependent in terms of specificity for position P 1 , but also with regard to the effects of amino acid residues neighboring the cleavage site.

Determination of Kinetic Parameters.
e kinetic parameters of rSAPV peptidase, SPVP, ermolysin type X, and Alcalase 2.4 L FG enzymes were estimated by assaying the enzymatic activity using an increasing concentration of casein and Suc-F-A-A-F-pNA at pH 9 and 60°C, see Table 5. Nevertheless, the K m and V max values of rSAPV were estimated to be 0.313 mM and 74,652 U/mg, respectively. Lower K m value indicates more affinity towards the substrate. With casein as substrate, the k cat /K m of rSAPV was at least 2.6, 10.3, 7.9, and 9.8 folds higher, respectively, than those observed for SPVP, ermolysin type X, Alcalase 2.4 L FG, and Bioprotease N100L, while with Suc-F-A-A-F-pNA as the substrate, the k cat /K m of rSAPV was at least 1.9, 9.3, 6.9, and 9.2 folds higher, respectively, than those observed for SPVP, ermolysin type X, Alcalase 2.4 L FG, and Bioprotease N100L (Table 5).

Effects of Organic Solvents on Peptidase Activity and
Stability. e effects of organic solvents on the activity and stability of the purified SPVP and ermolysin type X proteases are shown in Figure 4(a). When compared with SPVP and ermolysin type X peptidases, rSAPV enzyme is naturally stable and exhibits high peptidase activities in the presence of organic solvents. So, it would be very useful for organic synthetic reactions [17]. In upgrading environmentally mild chemical synthesis, the proteases are foreseen as supreme enzyme as the protease in nonaqueous media offers various novel characteristics as compared with traditional aqueous enzymatic transformation [49,50]. e proteolytic enzymes have attracted a high deal of attention in organic synthesis because they have many advantages associated with the application of enzymes for the synthesis of peptides and esters [51][52][53]. Very few research studies are characterized in the literature concerning the screening of microorganisms which produce organic solvent-stable proteases [54]. Poor stability and low catalytic activity of enzymes are the limitations of their usage in organic solvents, which tend to strip water from protein and thereby disrupt noncovalent forces and decrease enzyme activity and stability. e ability to use enzymes in nonaqueous solvents expands the potential applications of biocatalysts in chemical transformations, which is useful for many industries. Enzymatic reactions in organic solvents provide numerous industrially attractive advantages, such as increased solubility of nonpolar substrates, reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, alternation of substrate specificity and enantioselectivity, and elimination of microbial contamination.

Stability and Compatibility of Peptidase with Laundry
Detergents. As illustrated in Figure 4(b), rSAPV peptidase is compatible with all the tested commercial detergents at concentration of 7 g/L. It exhibited better stability with Class followed by Dipex, Nadhif, Det, Dixan, Skip, iSiS, Ariel, EcoVax, and OMO. rSAPV retained 100% of its initial activity with Class (vs 86% for Bioprotease N100L and 80% for Alcalase 2.4 L FG) even after 1 h incubation at 40°C. e SPVP protease retained about 60-100% of its initial activity in various commercial detergents [17]. e protease from Virgibacillus pantothenticus showed excellent compatibility in presence of the locally available detergents. It was noted to be stable in the presence of UltraVim, Ghari, and Wheel after 3 h of incubation at 40°C [33]. In addition, the protease from strain VIT P14 is stable in the presence of some detergents (Henko, Ariel, Rin, Tide, Surfexcel, and Technobright) [34].
us, the present study aimed at exploiting the application of a peptidase from moderately halophilic strain which revealed compatibility and stability under various liquid and solid detergents tested and therefore has a great potential in many laundry formulations.

Removal of Blood Stains from Cotton Fabrics.
Protein stains like chocolate, egg, and blood have been hard to eliminate with commercial detergent. Some stains could only be dealt with at high temperatures and even then the stain was only partly removed. Detergent enzymes especially with proteases offered a key and are being persistently improved to digest proteinaceous stains. e ability of proteases to hydrolyse the various proteinaceous stains has attracted the interest of industrialists in detergent market. As shown in Figures 4(c)-4(e), the visual comparison of the washed cloth revealed that the combinations of every enzyme individually with commercial detergents (Class) yielded fairly good results of its ability to remove blood, egg, and chocolate stains. In fact, a limited washing performance was observed with tap water only or with detergent (Class) only. e supplementation of SAPV enzyme or commercial proteases (Bioprotease N100L or Alcalase 2.4 L FG) in Class detergent seems to enhance the cleaning process as evidenced by rapid stain removal. us, SAPV peptidase was characterized by its strong hydrolytic effect against blood and chocolate which are recalcitrant stains. Similarly, protease from strain VIT P14 demonstrated its potential use as a bioadditive detergent in the removing of the blood stains from cotton cloth [34]. Although reported the usefulness of thermostable alkaline peptidase from Bacillus and related genus for the removal of protein stains from cotton cloth in the presence and absence of detergents [17,24,31], we believe that the SAPV peptidase form is more effective.

Conclusions
In this research, a SAPV peptidase was produced by utilizing shrimp shell by-product as the energy and carbon sources, and the peptidase was successfully purified to homogeneity by two steps. Biochemical properties of the purified SAPV enzyme demonstrated that it has a high optimum working temperature and pH, which could make the peptidase suitable for industries applications. is was supported by cloning amino acid sequence inspection and homology modeling of the gene encoding SAPV peptidase, which is endowed with a number of characteristics that are highly valued for the detergent industry and peptide synthesis. ese observations inspired us to explore other enzymes from strain FarD T . e structure-function relationship of SAPV is now underway to improve the properties of this peptidase. Further works will be investigated in our laboratories to perform the crystallization of SAPV enzyme.
Data Availability e datasets generated and/or analyzed during the current study are available on the GenBank repository, https://www. ncbi.nlm.nih.gov/genbank/. e GenBank accession number for the nucleotide sequence of the sapV gene referred to in the text is MN094794. Other datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
is article does not contain any studies with human participants or animals performed by any of the authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.