Accepted Manuscript Title: Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R Authors: Sondes Mechri, Mouna Kriaa, Mouna Ben Elhoul Berrouina, Maroua Omrane Benmrad, Nadia Zaraˆı Jaouadi, Hatem Rekik, Khelifa Bouacem, Amel Bouanane-Darenfed, Alif Chebbi, Sami Sayadi, Mohamed Chamkha, Samir Bejar, Bassem Jaouadi PII: DOI: Reference:
S0141-8130(17)30280-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.051 BIOMAC 7217
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
20-1-2017 23-2-2017 10-3-2017
Please cite this article as: Sondes Mechri, Mouna Kriaa, Mouna Ben Elhoul Berrouina, Maroua Omrane Benmrad, Nadia Zaraˆı Jaouadi, Hatem Rekik, Khelifa Bouacem, Amel Bouanane-Darenfed, Alif Chebbi, Sami Sayadi, Mohamed Chamkha, Samir Bejar, Bassem Jaouadi, Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.03.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R
Sondes Mechri a, Mouna Kriaa b, Mouna Ben Elhoul Berrouina a, Maroua Omrane Benmrad a, Nadia Zaraî Jaouadi a, Hatem Rekik a, Khelifa Bouacem c, Amel Bouanane-Darenfed c, Alif Chebbi d, Sami Sayadi d, Mohamed Chamkha d, Samir Bejar a, Bassem Jaouadi a,*
a
Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of
Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia b
Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax
(CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia c
Laboratory of Cellular and Molecular Biology (LCMB), Microbiology Team, Faculty of
Biological Sciences, University of Sciences and Technology of Houari Boumediene (USTHB), PO Box 32, El Alia, Bab Ezzouar, 16111 Algiers, Algeria d
Laboratory of Environmental Bioprocesses (LEBP), LMI COSYS-Med, Centre of
Biotechnology of Sfax (CBS),University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia
* Corresponding author. E-mail Jaouadi).
addresses:
[email protected],
[email protected]
(B.
Highlights ► Using statistical methodology, protease production by strain C250R was optimized. ► RSM was used to determine optimum levels of screened factors and their interaction. ► New extracellular protease from strain C250R was purified (SAPLF) and characterized. ► Optimum pH and temperature values for activity were pH 10 and 70 °C, respectively. ► SAPLF displayed a higher catalytic efficiency than SPVP and Alcalase Ultra 2.5 L. ► SAPLF offers an interesting potential for its use in the laundry detergent industry.
ABSTRACT
In this study, we aimed to optimize the cultural and nutritional conditions for protease production by Lysinibacillus fusiformis strain C250R in submerged fermentation process using statistical methodology. The most significant factors (gruel, wheat bran, yeast extract, and FeSO4) were identified by Plackett-Burman design. Response surface methodology (RSM) was used to determine the optimum levels of the screened factors and their interaction. Under the optimized conditions, protease yield 3100 U/mL was 4.5 folds higher than those obtained by the use of the initial conditions (680 U/ml). Additionally, a new extracellular 51 kDa-protease, designated SAPLF, was purified and biochemically characterized from strain C250R. It shows optimum activity at 70°C and pH 10. Its half-life times at 70 and 80 °C were 10 and 6-h, respectively. Irreversible inhibition of enzyme activity of SAPLF with serine protease inhibitors demonstrated that it belongs to the serine protease family. Interestingly, its catalytic efficiency was higher than that of SPVP from Aeribacillus pallidus strain VP3 and Alcalase Ultra 2.5 L from Bacillus licheniformis. This study demonstrated that SAPLF has a high detergent compatibility and an excellent stain
removal compared to Alcalase Ultra 2.5 L; which offers an interesting potential for its application in the laundry detergent industry.
Keywords: protease; Lysinibacillus fusiformis; detergent formulations.
1. Introduction
Microbial life is present not only in familiar world for example, in air, soil, and lakes, but also in geothermal oil field. Petroleum reservoirs are sites with respect to their physicochemical characteristics (halophilic and thermophilic). They are ecological niches from which taxonomically, physiologically, and phylogenetically unusual microbes can be isolated [1, 2]. These are main source for a biotechnological and industrial process. They host a variety of products namely extracellular enzymes, biopesticides, biosurfactants, biopolymers and other renewable resources and are ecofriendly [3]. Over billions of years, prokaryotic microbes developed a wealth of physiologies and molecular adaptations that enabled their survival in virtually every environmental niche, some so extreme and inhospitable that no other life forms could coexist [4]. In the course of evolution, microorganisms faced extreme environmental challenges and adapted their metabolic pathways to survive in diverse environments [5]. The literature indicates that these microorganisms are considered as an important source of enzymes with unconventional biochemical and molecular characteristics, and unique metabolic capabilities are the major points of attractions in biotechnological applications [6, 7]. Of particular interest, proteases (EC 3.4.21-24 and 99; peptidyl-peptide hydrolases) are enzymes that hydrolyse proteins via the addition of water across peptide bonds and catalyze peptide synthesis in organic solvents and in solvents with a low water content [8, 9]. They are
a highly complex group of enzymes that differ in their substrate specificity, catalytic mechanism and active site [10, 11]. Proteases constitute one of the commercially important groups of enzymes, accounting for nearly 65% of the whole enzyme market and are frequently used in detergent, leather, pharmaceuticals, food and biotechnology industries [12, 13]. Proteases that are due to be used in these industries should have some special characteristics that may not be found in their traditional counterparts, for example, a higher thermostability, enhanced activity, and extreme pH profile [14]. Detergent industries are the primary consumers of enzymes, in terms of both volume and value [15]. So, there is always a need for newer enzymes with novel properties that can further widen the scope of enzymebased detergents [16]. Proteases are vital to improving wash performance in detergent formulation [17]. Most often, the detergent protease belongs to serine protease that acts by cleaving peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site of the enzyme. They catalyze the reaction using catalytic triad consisting of serine, histidine, and aspartate. Each amino acid in the catalytic triad has the specific role during catalysis. These three amino acids may not be closely situated in the sequence but they form the triad after protein folding [18]. Extensive investigations on extremophiles have therefore been carried out as the large diversity of such organisms makes it possible to find, within these groups of extreme organisms, the appropriate isolate that produces the best possible protease for a specific application. This has forced the manufacturers to look for microorganisms originating from peculiar environments that could possibly fulfill the conditions imposed by the biotechnological process. Several other researchers have also described proteases secreted by bacteria isolated from petroleum reservoirs. Likewise, Aeribacillus pallidus strain VP3 isolated from a geothermal oil field in Tunisia has the ability to produce a thermostable
protease with a potential use in detergent formulations and non-aqueous peptide biocatalysis [19]. Enzyme production by microorganisms is generally affected by growth conditions (pH, temperature, agitation) and culture medium composition. The optimal design of culture conditions is a crucial aspect for enzyme production in commercial practice [20]. It has an important impact on the economy and process practicability. Response surface methodology is a useful model for studying the effect of several factors influencing the responses by varying them simultaneously and carrying out a limited number of experiments [21]. Many scientific works have reported satisfactory optimization of protease production from microbial sources [22] using a statistical approach [23, 24]. The current study is intended to scrutinize a novel extracellular protease named SAPLF secreted from Lysinibacillus fusiformis strain C250R isolated from a geothermal oil field, located in Sfax, Tunisia, a niche of extremophiles. The main goal of our study is to optimize the cultural and nutritional conditions to produce the maximum level of protease, to purify and to unravel the functional characteristics of protease enzyme to find its suitability as detergent additive.
2. Materials and methods
2.1. Substrates, chemicals, and used proteases All substrates and chemicals were reagent grade unless specified otherwise. General reagents were obtained from commercial suppliers. The serine protease SPVP from Aeribacillus pallidus strain VP3, had previously been purified and characterized [19]. The Alcalase Ultra 2.5 L, a commercial bacterial protease/peptidase complex was supplied by Novozymes Biopharma DK A/S (Bagsvaerd, Denmark). It is produced by submerged
fermentation of a selected strain of Bacillus licheniformis. The enzyme was separated and purified from the production organism.
2.2. Microorganism Strains were previously isolated from the production water (an oil/water mixture) of the Ramoura oil field (onshore) [25], with a temperature of 95 °C, and salinity of 106 g/1. Strains were isolated from enrichment cultures inoculated with the production water. In fact, a 5 mL sample from the production water was used to inoculate 45 mL of basal medium containing 1% (v/v) of crude oil in the presence of various NaCl concentrations. Then one of them, which revealed attractive potentiality to degrade crude oil (1%, v/v) identified as Lysinibacillus fusiformis strain C250R, was selected in this study.
2.3. Optimization of enzyme production
2.3.1 Influence of carbon source and nitrogen source For a high production of protease, the best carbon source and nitrogen source were chosen using the traditional method ‘one variable at a time’. The experiments were carried out in 250 mL Erlermeyer flasks containing 25 mL of production liquid medium. After sterilization, the flasks were inoculated with inoculums and maintained under different conditions using various carbon sources at a concentration of 10 g/l (casein, gelatin, gruel, glucose, starch, and maltose) and different organic and inorganic nitrogen sources at a concentration of 2 g/l (yeast extract, soya flour, meat extract, beef extract, (NH4)2SO4, and (NH4)3FeSO3). The initial optical density (OD) at 600 nm of the culture was 106 CFU/ml.
2.3.2 Screening of factors The Plackett–Burman design was used to screen variables that significantly affect Lysinibacillus fusiformis C250R protease production. Hence, seventeen variables (incubation
temperature, initial pH, speed of agitation, gruel, sucrose, lentil flour, wheat bran, yeast extract, urea, NaNO3, crude oil, NaCl, CaCl2, FeSO4, MnSO4, trace elements and KH2PO4) were studied at two widely spaced levels. The low (–1) and high (+1) levels of each factor and the design matrix are listed in Table 1. The effect of each variable was carried out by the determination of the contrast coefficient (b) obtained from the difference between the measurements average made at the high (+) and the low (−) levels of the variable.
2.3.3 Response surface methodology using Box–Behnken design Based on the results of the Plackett–Burman design, four variables (gruel (X1), wheat bran (X2), yeast extract (X3) and FeSO4 (X4)) were found to have a greater influence on protease production by the Lysinibacillus fusiformis strain C250R. Then, the Box–Behnken experimental design was performed to determine the level of the selected variables that influence protease production. These variables were prescribed into three levels, coded -1, 0 and 1. The realized modeling was based on the second–order polynomial equation: n
n
i 1
i 1
n 1
n
y b0 bi xi bii xi2 bij xij i j j i 1
(eq 1)
Where y is the protease activity, b0 is the offset term, bi is the linear effect, bii is the squared effect, bij is the first order interaction effect and xi is the independent variable. The predicted response was calculated using the model involving only significant parameters. The insignificant model terms were eliminated using the step-by-step procedure.
2.4. Assay of proteolytic activity The protease activity was assayed by a modified caseinolytic Peterson’s method protocol using Hammerstein casein (Merck, Darmstadt, Germany) as a substrate [26]. Enzyme solution (0.5 mL) suitably diluted was mixed with 0.5 mL 100 mM glycine-NaOH buffer
supplemented with 2 mM CaCl2 at pH 10 (Buffer A) containing 10 g/l of casein, and incubated for 15 min at 70 °C. The reaction was stopped by the addition of 0.5 mL TCA 20% (w/v). The mixture was allowed to stand at room temperature for 15 min and then centrifuged at 10,000g for 15 min to remove the precipitate. The acid soluble material was estimated spectrophotometrically at 280 nm. A standard curve was generated using solutions of 0-100 µg/l tyrosine. One unit (U) of protease activity was defined as the amount of enzyme, which liberated 1 mg tyrosine per minute under the experimental conditions used. The proteolytic activity in presence of the laundry detergent solution was evaluated by the method suggested by Touioui Boulkour et al. [27] using N,N-dimethylated casein (DMC) as a substrate. Unless otherwise stated, a suitably diluted enzyme solution (0.5 mL) was mixed with 1 mL laundry detergent, 2 mL 100 mM borate-NaOH buffer (pH 9) containing 10 g/l DMC, and 0.25 mL of 10 g/l 2,4,6-trinitrobenzene sulfonic acid (TNBSA) as a colour indicator. The mixture was shake-incubated at 70 °C for 25 min, and the reaction was stopped by the addition of a 2.5 mL of cold water for 15 min. The precipitate was then removed by centrifugation at 10,000g for 15 min. Absorbance was measured at 450 nm. One unit of protease activity was defined as the amount of enzyme required to catalyze the liberation of 1 µmole of product from DMC per min under the experimental conditions used.
2.5. SAPLF purification The culture from Lysinibacillus fusiformis strain C250R (500 mL), harvested at an early stationary growth phase, was centrifuged at 9,000 rpm for 30 min. The supernatant containing extracellular protease was used as the crude enzyme preparation and submitted the following purification steps. The supernatant was heat-treated for 5 min at 80 °C and insoluble material was removed by centrifugation at 9,000g for 15 min. Proteins were precipitated between 20 and 60% ammonium sulphate saturation. The precipitate obtained after centrifugation at 9,000g for 30 min was suspended, in a minimal volume of 20 mM MOPS buffer at pH 7
supplemented with 2 mM CaCl2 (Buffer B), and dialyzed overnight against the repeated changes of the same buffer. The clear supernatant was loaded and applied to a UNO Q-6 Fast protein liquid chromatography (FPLC) (Bio-Rad Laboratories, Inc., Hercules, CA, USA) equilibrated with buffer B. The column was rinsed with 500 mL of the same buffer. Adsorbed material was eluted with a NaCl linear gradient from 0 to 500 mM supplemented to the buffer B at a rate of 60 mL/h. The pooled fraction of the protease activity was eluted between 200 and 350 mM NaCl. The fractions containing protease activity were pooled and then applied to high-performance liquid chromatography (HPLC) system using a Zorbax PSM 300 (26.2 × 250 mm), Agilent Laboratories, pre-equilibrated with 25 mM MOPS buffer at pH 7.4 supplemented with 2 mM CaCl2 (Buffer B). Proteins were separated by isocratic elution at a flow rate of 40 mL/h with buffer B and detected using a UV/VIS Spectrophotometric detector at 280 nm. The fractions containing protease activity were eluted with a retention time of 6.7 min. The pooled fractions containing protease activity were concentrated in centrifugal micro-concentrators (Amicon Inc., Beverly, MA, USA) with 30-kDa cut-off membranes and stored at -20 °C in a 20% glycerol (v/v) solution for further analysis.
2.6. Protein measurement, electrophoresis, and analytical methods SDS–PAGE was performed using a 5% stacking gel and 12% resolving gel under reducing conditions as described by Laemmli [28]. Protein concentration was determined using Bio-Rad protein assay kit, based on the method of Bradford using bovine serum albumin as the standard [29]. The molecular mass estimated for the native and purified protease was determined by PAGE under denaturating and non-denaturating conditions. Protein bands were visualized by Coomassie brilliant blue R-250 Bio-Rad Laboratories (Hercules, CA, USA), staining. Enzyme activity was detected in situ in SDS polyacrylamide gels containing 10 g/l casein. After electrophoresis, proteins were re-natured by soaking the gel for 2 h with 100 mL of 2.5% Triton X-100 in buffer A at room temperature using a
horizontal shaker to replace SDS and separation buffer in the gel. The incubation of the washed gel at 70 °C for 15 min in buffer A produced a casein cleared zone at the location of the proteolytic band.
2.7. Amino acid sequencing Bands of purified SAPLF were separated on SDS gels and transferred to a ProBlott membrane (Applied Biosystems, Foster City, CA, USA), and the NH2-terminal sequence analysis was performed by automated Edman’s degradation using a protein sequencer (Applied Biosystems Protein sequencer ABI Procise 492/610A) equipped with 140 C HPLC system per standard operating procedures. Residues of amino acids were detected as individual signals.
2.8. Biochemical characterization of the purified protease
2.8.1. Effects of inhibitors, reducing agents, and metallic ions on protease stability The effects of some specific inhibitors and reducing agents such as diiodopropyl fluorophosphates (DFP), phenylmethanesulfonyl fluoride (PMSF), soybean trypsin inhibitor (SBTI), N-p-tosylL-lysine chloromethyl ketone (TLCK), N-p-tosylL-phenylalanine chloromethyl ketone (TPCK), benzamidine hydrochloride hydrate, 5,5-dithio-bis-(2-nitro benzoic acid) (DTNB), N-ethylmalemide (NEM), iodoacetamide, leupeptin, pepstatin A, and EDTA, as well as various divalent and monovalent metallic ions on protease stability were investigated by preincubating the purified enzyme for 1 h at 40 °C, in the presence of metallic ions or each inhibitor. Enzyme assays were carried out under standard assay conditions. Protease activities, measured with casein as a substrate in the absence of any metallic ions or reagent, were taken as control (100%).
2.8.2. Determination of the optimum pH and stability
The effect of pH was determined using casein as a substrate. Investigation for the optimum pH of purified protease activity was carried out in nine different buffers at pH range of 3–13 at 70 °C. The pH stability of SAPLF was determined by pre-incubating the enzyme in buffer solutions with different pH values for 24 h at 40 °C. Enzymatic activities were determined according to standard conditions previously detailed [30]. Aliquots were then withdrawn, and residual enzymatic activities were determined under standard assay conditions. The following buffer systems, supplemented with 2 mM CaCl2, were used at 100 mM: glycine-HCl for pH 3–5, MES for pH 5–6, HEPES for pH 6–8, Tris-HCl for pH 8–9, glycine-NaOH for pH 9–11, bicarbonate-NaOH for pH 11–11.5, Na2HPO4-NaOH for pH 11.5–12, and KCl-NaOH for pH 12–13.
2.8.3. Determination of optimum temperature and thermal stability The effect of temperature on enzyme activity was examined at 30–90 °C at pH 10 in the presence and absence of 2 mM CaCl2. Thermal stability was determined by incubating SAPLF at 50–80 °C and pH 10 for 24 h in the presence and absence of 2 mM CaCl2. Aliquots were withdrawn after each 2 h to test the remaining activity under standard conditions. The non-heated enzyme, which was left at room temperature, was considered as control (100%).
2.8.4. Effect of polyols on protease stability To study the influence of polyols on thermo stability of protease, the SAPLF was preincubated for 1 h at 80 °C in the presence and absence of some polyols, at 10% (w/v) of concentration, such as mannitol, ploy(propylene glycol) (PEG) 1000, 1500, and 6000, as well as glycerol, sorbitol, and xylitol. Enzyme assays were carried out under standard assay conditions. Enzymatic activities were determined according to standard conditions previously described by Jaouadi et al. [30].
2.8.5. Kinetic measurements of SAPLF, SPVP, and Alcalase Ultra 2.5 L Kinetic parameters were calculated from the initial rate activities of the purified enzymes using casein and Suc-Ala-Ala-Pro-Phe-pNA, as substrates at different concentrations ranging from 0.10 to10 mM at 60 °C and pH 10 for 10 min. The purified enzymes were SAPLF (this study), SPVP from Aeribacillus pallidus strain VP3, and Alcalase Ultra 2.5 L (commercial enzyme) at a final concentration of proteins 1.5 mg/ml. Each assay was carried out in triplicate, and kinetic parameters were estimated by Lineweaver–Burk plots. Kinetic constants, Michaelis–Menten constant (Km), and maximal reaction rate (Vmax) values were obtained using the Hyper32 software. The value of the turnover number (kcat) was calculated using the following equation:
kcat
Vmax [E]
(eq 2)
where [E] refers to the active enzyme concentration, Vmax refers to the maximum reaction rate and
kcat
is
defined
of substrate molecules
per
as
the
second
maximum that
a
number
single
of
catalytic
chemical site
conversions
executes
for
a
given enzyme concentration.
2.9. Performance evaluation of the purified proteases
2.9.1. Stability and compatibility of SAPLF and Alcalase Ultra 2.5 L with laundry detergents In order to confirm the potential of SAPLF as a detergent additive, its compatibility and stability towards some commercial laundry detergents available in the local market such as Dixan and Nadhif (Henkel, Tunisia) was checked. The solid detergents used were Ariel (Procter & Gamble, Switzerland), OMO (Unilever, France), Ecovax (Klin Productions, Sfax, Tunisia), and Alyss (EJM, Sfax, Tunisia). In order to check their stability and compatibility with detergents, the mentioned commercial detergents were diluted in tap water to obtain a
final concentration of 7 mg/ml (to simulate washing conditions). The endogenous proteolytic enzymes present in these laundry detergents were inactivated by heating the diluted detergents for 1 h at 65 °C, prior to the addition of the purified enzymes (SAPLF and Alcalase Ultra 2.5 L). A 500 U/ml of each purified protease was shake-incubated with each laundry detergent for 1 h at 40 °C, and residual activity was determined at pH 10 and 70 °C using DMC as a substrate. The enzyme activity of a control (without any detergent), incubated under similar conditions, was taken as 100%.
2.9.2. Removal of blood stain from cotton fabrics With the aim to simulate the washing condition and to determine the efficacy of SAPLF for use as a bio-detergent additive, new cotton cloth pieces (6 × 6 cm) was stained with blood. The stained cloth pieces were shake-incubated (200 rpm) in different wash treatments at 40 °C for 1 h in 1-litre beakers containing a total volume of 50 ml of: tap water, Ariel detergent (7 mg/ml, in tap water), and detergent added with SAPLF (500 U/ml) or commercial Alcalase Ultra 2.5 L (500 U/ml). After treatment, the cloth pieces were taken out, rinsed with water, dried and submitted to visual observation to examine the stain removal effects of the enzymes. The untreated blood-stained piece of cloth was taken as a control.
2.10. Statistical analyses The data were analyzed using SPSS (Version 11.0.1 2001, LEAD Technologies, Inc., and USA). The response surface was plotted using Microsoft Excel software (Version 2007, Microsoft Inc., USA). All determinations were performed at least three independent replicates, and the control experiment without protease was carried out under the same conditions. The experimental results were expressed as the mean of the replicate determinations and standard deviation (mean ± SD). The statistical significance was evaluated using t-tests for two-sample comparison and one-way analysis of variance
(ANOVA) followed by t-test. The results were considered statistically significant for P values of less than or equal to 0.05. The statistical analysis was performed using the R package Version 3.1.1 (Vanderbilt University, Nashville, TN, USA).
3. Results and discussion
3.1. Screening of alkaline protease-producing bacteria from Tunisian oil fields Four aerobic bacterial strains (Lysinibacillus fusiformis strain C250R, Aeribacillus pallidus strain VP3, Achromobacter xylosoxidans strain C350R, and Halomonas luta strain C2SS100) among eight strains that were previously isolated from Tunisian onshore oil fields were identified as protease producers based on their patterns of clear zone formation on casein-containing media at pH 7.4. The ratio of the clear zone diameter and that of the colony served as an indicator for the selection of strains with high protease production ability. In a previous work, a protease named SPVP secreted from Aeribacillus pallidus strain VP3 was studied [19]. The aim of this research is to optimize, purify, and characterize the protease SAPLF secreted from a bacterium called Lysinibacillus fusiformis strain C250R that displayed an extracellular protease activity (about 680 U/ml) in an initial medium.
3.2. Effect of carbon and nitrogen sources Protease production was tested in the initial medium containing (g/l): yeast extract (as nitrogen source), 1; CaCl2, 2; KH2PO4, 1; FeSO4, 10; trace elements 1%; and 10 g/l of different carbon and energy sources: casein, gelatin, gruel, starch, glucose and maltose. As shown in Fig. 1A, the best carbon source for protease production was gruel, (680 U/ml); followed by gelatin (659 U/ml). In general, both organic and inorganic nitrogen sources were used efficiently by Bacillus species, for protease production. In the present study, different organic (yeast extract, meat extract, beef extract, and soya flour) and inorganic [ammonium
sulphate, (NH4)2SO4 and ferric ammonium sulphate, NH4Fe(SO4)2] nitrogen sources, at a concentration of 2 g/l, were also tested in a medium containing gruel at 10 g/l as a sole carbon and energy source. As shown in Fig. 1B, the best nitrogen source for protease production was yeast extract (750 U/ml) followed by meat extract (400 U/ml) and beef extract (304 U/ml). In the light of these results, the gruel and yeast extract turns out to be more adequate for the production of protease by Lysinibacillus fusiformis strain C250R. In fact, the gruel and yeast extract are two sources of amino acids and growth factors for the bacterium. They also contain vitamins and cofactors essential for the development of the bacterium and for the synthesis of metabolites as well. Position Figure 1 here
3.3. Screening of significant variables using Plackett–Burman design The Plackett–Burman design is widely used in biotechnological production because it allows the screening of main factors from a large number of variables that can be retained in the further optimization process [31]. In this study, we first used the Plackett–Burman design to screen the most influential variables on Lysinibacillus fusiformis C250R protease production. Seventeen different variables including some operational parameters and medium components were screened using a 19 runs matrix. Levels of the factors tested for the production of proteases by strain C250R using Plackett–Burman methodology are presented in Table 1. Position Table 1 here
The data showed a wide variation of protease activity production ranging from 680 to 3045 U/ml. This variation proved the importance of this step in selecting the most influent factors and the level of the others. The main effect of each variable was estimated by
evaluating the contrast coefficient (b). Result analysis showed that gruel, wheat bran, yeast extract and FeSO4 were the most significant variables that present the largest contrast coefficient. In fact, wheat bran, yeast extract and FeSO4 concentration had a positive effect on protease production, while the gruel concentration exhibited a negative effect. The design matrix with the corresponding results of Plackett–Burman experiments is presented in Table 2. For further optimization, the level of the most significant factors was optimized by RSM using Box-Behnken design while insignificant ones were used in all trials at their optimum level. The other variables with positive effect (speed of agitation, crude oil, MnSO4, trace elements and KH2PO4) were fixed at high level while variables with a negative effect (temperature, initial pH, sucrose, lentil flour, urea, NaNO3, NaCl, and CaCl2) were maintained at low level. Position Table 2 here
3.4. Level optimization of the main factors using response surface methodology (RSM) Based on the results given above, four factors namely gruel, wheat bran, yeast extract and FeSO4 were chosen as critical variables affecting protease production. The design matrix with the corresponding results of Box–Behnken experiments, as well as the predicted results is presented in Table 3. Position Table 3 here
The model was established after the regression analysis and was predicted by the following equation: Y = 8991.906 – 2239.733 × X1 + 147.683 × X2 + 348.229 × X3 – 2419.875 × X4 + 173.160 × X1 × X1+ 12.739 × X1 × X2 + 77.699 × X1 × X4 – 4.264 × X2 × X2 + 12,5 × X2 × X3 – 40.900 × X2 × X4 – 63.468 × X3 × X3 + 39.874 × X3 × X4 + 273.437 × X4 × X4 (eq 3)
When Y is the protease activity (U/ml), X1, X2, X3 and X4 are respectively gruel (g/l), wheat bran, (g/l), yeast extract (g/l) and FeSO4 (g/l).The regression analysis result showed that the F-value was 3.417 with a very low probability value (P < 0.001) indicating the significance of the model. The closeness of experimental and predicted protease activity can be expressed by the determination coefficient of (R2 = 0.88) which stipulates that only 12% of the total variation could not be explained by the model. Adjusted R Square (predicted R2) of 0.774 explains the good agreement between the experimental and the predicted results. The model equation related to protease production shows an important negative linear effect for gruel (X1) and FeSO4 (X4) and significant interactions between gruel and FeSO4 (X1X4), wheat bran and FeSO4 (X2X4) and yeast extract and FeSO4 (X3X4). These results were confirmed by the Student’s t-test (α = 0.05). Response surface plot was generally the graphical representation of the regression equation, from which the response (protease production) is plotted against any two variables, while other variables were fixed at their middle levels. Response surface plots can directly reflect the impact of various factors on the response. Contour map traits can reflect the strength of the interaction between the two factors: an oval contour indicates that the interaction between the two factors is strong, whereas a circular contour indicates that the interaction between the two factors is weak [32]. Fig. 2 shows the mutual interaction between the gruel and FeSO4 (X1 X4) and yeast. An increase in protease production was recorded in the minimum levels of the two factors. Thus, the protease activity variation was observed with the simultaneous decrease or increase of these two factors. Position Figure 2 here
3.5. Model validation The model validity was examined by additional experiments using the optimal culture conditions: temperature 30 °C, initial pH 7, speed of agitation 180 rpm, gruel (5 g/l), wheat
bran (15 g/l), yeast extract (5 g/l), crude oil (4%, v/v), NaCl (20 g/l), FeSO4 (2 g/l), MnSO4 (2 g/l), trace elements (3%, v/v), and KH2PO4 (2 g/l). Protease production yield (3100 U/ml) was absolutely more important than that obtained during the preliminary study (680 U/ml). Thus, protease activity was multiplied by a factor of 4.55 fold. The expected result (3114.75 U/ml) was very close to the experimental result (3100 U/ml). By optimizing the medium composition and the culture conditions, not only the production of proteases was enhanced but also the cost of enzyme production was reduced, since two cheap and readily available complex substrates (gruel and wheat bran) were used. Also, the present study is the first contribution towards the use of crude oil as a complex organic source for the production of alkaline protease by Lysinibacillus fusiformis strain C250R isolated from a geothermal oil field.
3.6. Purification of SAPLF After validation of RSM, the protease production was undertaken in the optimal medium. So, a supernatant obtained by centrifugation of the Lysinibacillus fusiformis strain C250R culture broth (500 mL) was used as the crude enzyme solution. The protein elution profile obtained at the final purification step indicated that the protease was eluted with retention time of 6.7 min on a Zorbax PSM 300 column using HPLC system (Fig. 1A). In addition, this preparation was a homogeneous enzyme with high purity as it exhibited a unique symmetrical elution peak, corresponding to a protein of nearly 51 kDa on HPLC gel filtration chromatography (Fig. 1A). The results of the purification procedure are summarized in Table 4. The purified enzyme preparation contained about 18.3% of the total activity of the crude and had a specific activity of 94333 U/mg (Table 4). Position Table 4 here
3.7. Molecular weight determination of SAPLF
The purity of the enzyme was confirmed by obtaining a single band of molecular weight 51 kDa in both SDS–PAGE (Fig. 3B) and casein zymography (Fig. 3C). This result is unique as only a few reports are available on the haloalkaliphilic thermostable protease having such a high molecular weight. The estimated molecular weight of SAPLF enzyme (51 kDa), is different from other reported proteases of Bacillus species such as Aeribacillus pallidus strain VP3 (29 kDa) [19], Bacillus firmus strain CAS 7 (21 kDa) [33], Bacillus sp. strain B001 (28 kDa) [34] and Bacillus cereus strain TKU006 (33 kDa) [35]. Sareen et al. [36] reported the production of thermostable protease of molecular mass of 55 kDa from Bacillus licheniformis strain RSP-09-37. Further, the activity staining showing a zone of clearance at the same position as observed in the zymography analysis confirmed, the purity and molecular weight of the enzyme (~ 51 kDa). Overall, these observations suggest that SAPLF is a monomeric protein comparable to those previously reported for other proteases from Bacillus strains [19, 33-36]. Position Figure 3 here
3.8. NH2-terminal amino-acid sequence determination of SAPLF The NH2-terminal sequencing of the blotted purified SAPLF allowed the identification of 27 residues, VPSGPYGPIDIKADKVIEDGFKMDEYF, showing uniformity, thus indicating that it was isolated in a pure form. This sequence was submitted to comparisons with the existing protein sequences in the GenBank non-redundant protein database and the SwissProt database, using the BLASTP and tBlastn search programs, respectively. Table 5 shows the alignment of NH2-terminal sequences of SAPLF and other subtilisins from Bacillus strains. The SAPLF sequence showed homology with those found for other alkaline serine proteases, reaching 80% identity with the serine protease (SP) from Lysinibacillus fusiformis strain RB-21 and Lysinibacillus sphaericus strain C3-41 and 76% identity with SPVP from
Aeribacillus pallidus strain VP3 (Table 5). Besides, it showed 63-70% identities with proteases or subtilisins from Bacillus strains. These results indicated that SAPLF protease is a new proteolytic enzyme. Position Table 5 here
3.9. Biochemical characterization of purified protease
3.9.1. Effects of inhibitors, metallic ions and salt on protease stability of SAPLF enzyme Enzyme inhibitors are substances that alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. In this context, effect of 2 mM of various inhibitor ions was examined on protease activity as the molar ratio of inhibitor/enzyme = 100. The relative inhibitory effects of various compounds are listed in Table 6. Inhibition of protease activity in presence of DFP and PMSF suggested that the coproduced protease is a member of serine-protease family of enzymes. The literature indicated that almost one-third of all proteases can be classified as serine proteases, named for the nucleophilic serine residue at the active site [37]. In fact, all serine proteases are inhibited by DFP and PMSF that covalently modify a catalytic serine residue in enzyme’s active site [38]. The observation corroborates with the early report of Bouacem et al. [39]. Other inhibitors, such as TPCK and TLCK (chymotrypsin alkylating agents), SBTI, and benzamidine (a trypsin competitive reagent), did not display any inhibitory effects. Furthermore, the thiol (DTNB, NEM, iodoacetamide, and leupeptin) and acid (pepstatin A) reagents have almost no effect on enzyme activity. SAPLF has lost 74% of its original activity in the presence of 10 mM EDTA, as a chelating agent and metalloprotease inhibitor which chelates metallic ions at the active site of the purified protease possibly serving as cofactors for the protease. This property is highly valued in detergent additives since chelators are a common ingredient in most laundry detergents. They have a number of different functions such as reducing water
hardness, assisting in keeping particulate soil in suspension and the removal of certain stains, thus complementing the action of the anionic surfactants [40]. Therefore, the presence of both chelators and enzymes in a liquid detergent presents a challenge. As the most commonly used serine-proteases within the detergent industry depend on two calcium binding sites, to maintain conformational stability and function at elevated temperatures [40, 41]. Metals may be part of the active sites of enzymes and participate directly in catalysis [42]. So, the effect of metallic ions (Ca2+, Fe 2+, Cu 2+, Mn2+, Mg2+, Zn2+, Na+, Co2+,Cd+, and Li+) on the protease activity was studied at a concentration of 2 mM. The addition of ZnSO4, FeSO4 and CaCl2 at 2 mM enhanced enzymatic activity by 180, 227 and 340% as compared to the control, respectively. These phenomena indicate that the enzyme requires metallic ions as cofactors. In fact, those bivalent ions (Ca2+, Fe2+, and Zn2+) apparently promoted enzyme activity by stabilizing its structure and protecting it against thermal denaturation, thus playing a vital role in maintaining its active conformation at higher temperatures. In addition, specific calcium binding sites that influence the protein activity and stability apart from the catalytic site were described for the following proteases: KERQ7 from Bacillus tequilensis strain Q7 [43], SAPDZ from Bacillus circulans strain DZ100 [44], SAPB from Bacillus pumilus strain CBS [30] and BM1 from Bacillus mojavensis strain A21 [45]. Another reason for the increase activity in the presence of calcium may be due to stabilization of enzyme in its active conformation rather than it being involved in the catalytic reaction. It probably acts as a salt or ion bridge via a cluster of carboxylic groups as has been suggested for subtilisins and thereby maintains the rigid conformation of the enzyme molecule [46]. However, protease activity was completely inhibited by cadmium and cobalt. Inhibition studies by heavy metallic ions have been well described in the literature. Indeed, heavy metals can bind to native proteins and inhibit their biological activity [47]. Taking the example of SAPDZ from Bacillus circulans strain DZ100 was totally inhibited by Hg2+, Ni2+, and Cd2+ [44].
Moreover, enzyme activity was unaffected in the presence of 2 mM NaCl. From these results, we suggest that SAPLF is a good candidate for detergent additives as NaCl is used as one of the core material components during the granulation of protease for detergent additives. Position Table 6 here
3.9.2. Effects of pH on enzyme activity and stability Fig. 4A shows that SAPLF displayed activity over a broad range of pH (3–13), with an optimum at pH 10. The relative activities at pH 5 and 12 were 40 and 60%, respectively. These results are in accordance with earlier works [19, 39]. In fact, the optimum pH range of microbial alkaline proteases is generally between pH 9 and 11, with a few exceptions of higher pH optima of 9-11 [48], 11.5 [49, 50], pH 11–12 [51, 52], and pH 12–13 [44, 53]. The pH stability profile of SAPLF illustrated in Fig. 4B indicated that the purified enzyme was highly stable in the pH range of 5–10. SAPLF was noted to be more efficient at alkaline pH compared to major commercial detergent enzymes [39, 54]. The half-life times of SAPLF were 22, 20, 18, 16, 12, and 8 h at pH 7, 8, 9, 10, 11, and 12, respectively. Meanwhile, the half-life times of the purified protease are more than 24 h at pH 5 and 6. The remarkable activity and stability over a wide pH range reveals the highly alkaline nature of SAPLF, which makes it suitable for applications in alkaline environments and with detergents. For this reason, the activity at high alkaline pH is a prerequisite for protease application in detergent formulations. Position Figure 4 here
3.9.3. Effects of temperature on protease activity and stability To estimate the optimum temperature of the enzyme, the activity was determined at different temperatures (30–90 °C) at pH 10. The enzyme had a broad temperature range
between 50 and 80 °C and the optimum temperature was observed to be around 70 °C in the presence of 2 mM Ca2+ and 60 °C in the absence of CaCl2 (Fig. 4C). The activity of protease enzymes in broad temperature ranges is a desired characteristic for their application in detergent formulations [36, 48, 55-57]. The addition of different concentrations of calcium from 1 to 10 mM enhanced the thermostability of the enzyme. The maximal thermostability was achieved with calcium at 2 mM. As shown in Fig. 4D, the half-life times of SAPLF without calcium at 2 mM were 22, 16, 8, and 4 h at 50, 60, 70, and 80 °C, respectively. The half-life times of the purified protease at 50, 60, 70, and 80 °C increased to 24, 18, 10, and 6 h with 2 mM CaCl2. In fact, thermostable extremozymes can be used in industrial reactions that are not feasible at ambient temperature. Higher temperature is preferable in many chemical reactions due to higher solubility of substrates, lower viscosity, better mixing, faster reaction rate, and decreased risk of microbial contamination. Moreover, thermostable enzymes are easy to purify by heat treatment [5].
3.9.4. Effect of polyols on the thermal stability As shown in Fig. 5A, the addition of polyols, like mannitol, PEG 1000, PEG 1500, PEG 6000, glycerol, and xylitol at concentration of 100 g/l increases the activity of the protease to nearly the same as the control. The results displayed in Fig. 5A indicate that the highest levels of enzymatic activity were reached with mannitol as an additive. Furthermore, thermostabilization was more effective with calcium at 2 mM and mannitol at 100 g/l since the half-life times at 80 °C was determined to be respectively 12 h compared to 6 h in the absence of any additive. Several other researchers have also shown improvement the thermostability of proteases in the presence of polyols [39, 58]. The protective impact of polyols is linked to their hydroxyl groups. The addition of certain polyols plays a vital role in maintaining hydrophobic interactions within protein molecules. It has further been suggested
that the protective role of polyols is due to their capability to form hydrogen bonds that support and stabilize the native conformation of the enzyme to make it more resistant to heat treatment [19]. Position Figure 5 here
3.9.5. Determination of kinetic parameters SAPLF, SPVP, and Alcalase Ultra 2.5 L proteases exhibited the classical kinetics of Michaelis-Menten for the two used substrates: casein (as natural protein) and Suc-Ala-AlaPro-Phe-pNA (as synthetic peptide). The order of the catalytic efficiency (kcat/Km) values of each enzyme was almost the same, i.e., Suc-Ala-Ala-Pro-Phe-pNA > casein (Table 7). When casein was used as a natural substrate, SAPLF was noted to exhibit kcat/Km values that were 2.59 and 5.96 times elevated than those of SPVP and Alcalase Ultra 2.5 L, respectively. When Suc-Ala-Ala-Pro-Phe-pNA was used as a synthetic substrate, SAPLF was also noted to exhibit kcat/Km values that were 2.34 and 7.10 times higher than those of SPVP and Alcalase Ultra 2.5 L, respectively (Table 7). Position Table 7 here
3.10. Performance evaluation of the purified protease
3.10.1. Stability and compatibility of SAPLF and Alcalase Ultra 2.5 L with laundry detergents The use of proteases as detergent additives represents a major application of industrial enzymes. To be suitable, they must be active under thermophilic (60 °C) and alkalophilic (pH 9–11) conditions, as well as in the presence of the various laundry detergents [59]. According to Fig. 5C, SAPLF protease was extremely stable and compatible with the commercial solid detergents used, retaining 84% of its initial activity with Ecovax and 85% with Alyss even
after 1 h incubation at 40 °C. However, Alcalase Ultra 2.5 L was noted to be less stable in the presence of Eovax and Alyss, retaining respectively only 75% and 77% of its initial activity. In addition, SAPLF was highly stable in the presence of liquid laundry detergents at a concentration of 7 mg/ml (Fig. 5C). The alkaline protease exhibited higher stability in Ariel, Dixan, than in Nadhif and Skip. However, Alcalase Ultra 2.5 L was noted to be less stable in the presence of Nadhif, retaining only 80% of its initial activity (vs 93% for SAPLF). Detergent stable proteases with variable stability in the presence of different detergents have been studied by several other researchers [34, 60]. Mechri et al. [19] reported an alkaline protease from Aeribacillus pallidus strain VP3 with 60–100% residual activity when incubated with 7 mg/ml commercial detergents for 1 h at 45 °C. These results support the potential of SAPLF as an ideal candidate for use in laundry detergent.
3.10.2. Removal of blood stains from cotton fabrics With the progress of urbanization, there is growing interest in detergent industries, especially to prepare bio-formulations effective for removal of various types of stains on the fabric [61]. Removal of proteinaceous stains such as that of blood, grass, and sauces always remained a challenge to laundry workers in addition to households. The role of protease as an additive in detergent formulations has long been maintained. In laundry detergents, they account for approximately 25% of the total worldwide sales of enzymes [59]. The success of subtilisins in the field of industrial detergents is based on their stability and high substrate specificity. The application of protease SAPLF in detergents was represented in the Fig. 5D. The stained cloth was washed with prepared detergent solutions without and with SAPLF. We noticed a limited washing performance with detergent (Ariel) only. The supplementation of SAPLF or commercial protease Alcalase Ultra 2.5 L in detergent seems to improve the cleansing process as evidenced by rapid blood stain removal when compared to detergent alone. In fact, SAPLF facilitated the release of proteinacious materials in a much easier way
than the currently used Alcalase Ultra 2.5 L protease. Furthermore, the combination of SAPLF with the Ariel detergent resulted in the complete stain removal (Fig. 5D). These results confirm the newly isolated alkaline protease SAPLF from Lysinibacillus fusiformis strain C250R as an effective cleaner of stains. Alkaline proteases have been used in the detergents to hydrolyze and clean proteinaceous stains on garments [62]. The effectiveness of alkaline proteases in stain removal has been demonstrated with various washing conditions and in different detergent compositions [58, 63] but according to the reported illustration, we believe that SAPLF is more effective. Jaouadi et al. [63] reported the stain removal using protease in cotton fabric to remove blood stains completely. The alkaline SAPB protease produced by Bacillus pumilus strain CBS was capable of cleaning stains but its mixture with detergent was proved to be more effective. Additionally, the protease isolated from mutant Bacillus licheniformis used in stained cloth increased ability to remove blood stain from cotton cloth [64].
4. Conclusions
The study is the first contribution towards the use of crude oil as an organic substrate for the production of a serine alkaline protease by Lysinibacillus fusiformis strain C250R, previously isolated from a geothermal oil field (Sfax, Tunisia). In this research, Plackett– Burman and Box–Behnken designs were employed to optimize the medium and culture conditions for the production of detergent stable protease SAPLF by strain C250R. The extracellular protease (SAPLF) was purified to homogeneity and biochemically characterized. The results revealed an optimum activity at 70 °C and pH 10. The SAPLF protease also displayed high levels of activity and stability over a wide range of temperature and pH, which are highly valued in the detergent industry. Compared to the protease SPVP from Aeribacillus pallidus strain VP3 and the commercial Alcalase Ultra 2.5 L enzyme,
SAPLF showed high catalytic efficiency, excellent stability and compatibility with a wide range of commercialized laundry detergents, making it particularly suitable for detergent formulations. Accordingly, further studies, some of which are currently underway in our laboratories, are needed to clone the encoding gene and to explore the structure-function relationships of the enzyme using site-directed mutagenesis and 3-D structure modeling.
Acknowledgments
This research was supported by a grant provided by the Ministry of Higher Education and Scientific Research of Tunisia under the contract program CBS-LMBEE/code: LR15CBS06 2015-2018. The authors would like to express their gratitude to Mr. K. Walha, Mrs. N. Kchaou, and Mrs. N. Masmoudi (Analysis Unit-CBS) for their technical assistance. We extend our thanks to Pr H. Mejdoub and Mr. C. Bouzid (USCR/SP-FSS, Sfax Faculty of Science) for the NH2-terminal amino acid sequencing of the SAPLF protein. Special thanks are also due to Pr. K. Maaloul from the English Department at the Sfax Faculty of Science, University of Sfax (Sfax, Tunisia) for constructive proofreading and language polishing services. The authors are also very much grateful to the editors and the anonymous reviewers for their valuable comments and efforts during the revision of the present manuscript.
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Figure legends
Fig. 1. Effects of carbon sources at 10 g/l (A) and nitrogen sources at 2 g/l (B) on protease production by Lysinibacillus fusiformis strain C250R. Fig. 2. Response surface plot of proteolytic enzyme production showing the interactive effects of the FeSO4 and gruel concentrations (A), FeSO4 and wheat bran concentrations (B) and FeSO4 and yeast extract concentrations (C). Fig. 3. Purification of SAPLF protease from Lysinibacillus fusiformis strain C250R. (A) Chromatography profile of the protease SAPLF on FPLC system using UNO Q-6. The column (12 × 53 mm) (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was equilibrated with buffer B. Adsorbed material was eluted with a linear NaCl gradient (0 to 500 mM in buffer B at a flow rate of 60 mL/h, and assayed for protein content at 280 nm. (D) Size exclusion HPLC chromatography of the purified SAPLF from using a HPLC Zorbax PSM 300 (26.2 × 250 mm) equilibrated with buffer B, shows a single and symmetrical peak of approximately 51 kDa (retention time = 6.7 min). (D) SDS-PAGE 12% of the purified protease SAPLF. Lane 1, purified SAPLF (50 µg) obtained after HPLC Zorbax PSM 300. Lane 2, sample after UNO Q-6 FPLC chromatography (fractions 200-350 mM NaCl). Lane 3, dialyzed-sample after ammonium sulfate fractionation (20-60%). Lane 4, sample after heat treatment (5 min at 80 °C). Lane 5, total cell extract. Lane 6, protein markers. (D) Zymography showing the caseinolytic activity staining of the purified protease SAPLF (50 µg). Fig. 4. Physico-chemical proprieties of the purified SAPLF from Lysinibacillus fusiformis strain C250R. Effects of pH on the activity (A) and stability (B) of the purified protease SAPLF. The activity of the enzyme at pH 10 was taken as 100%. Effects of the thermoactivity (C) and thermostability (D) of SAPLF. The enzyme was pre-incubated in the
presence and absence of CaCl2 at temperatures ranging from 50 to 80 °C. The activity of the non-heated enzyme was taken as 100%. Each point represents the mean of three independent experiments. Fig. 5. Performance evaluation of the purified protease SAPLF from Lysinibacillus fusiformis strain C250R. (A) Stability of SAPLF in the presence of various polyols at 100 g/l. Enzyme activity of the control sample, without additive, was incubated under similar conditions, and taken as 100%. Vertical bars indicate standard error of the mean (n = 3). (B) Effect of the thermostability of SAPLF at 80 °C. The enzyme was pre-incubated in the absence (Δ) or presence of additive: 2 mM Ca2+ (▲); 100 g/l mannitol (♦); and 2 mM Ca2+ and 100 g/l mannitol (●). The residual protease activity was determined from 0 to 12 h at 1 h intervals. The activity of the non-heated enzyme was considered as 100%. Each point represents the mean (n = 3) ± standard deviation. (C) Stability of SAPLF and Alcalase Ultra 2.5 L purified proteases in the presence of liquid and solid laundry detergents. Enzyme activity of the control sample, which contained no additive and incubated under similar conditions, was taken as 100%. Each point represents the mean of three independent experiments. (D) Washing performance analysis test of SAPLF in the presence of the commercial detergent Ariel. (a) Cloth stained with blood washed with tap water; (b) bloodstained cloth washed with Ariel detergent (7 mg/ml), (c) blood-stained cloth washed with Ariel added with Alcalase Ultra 2.5 L (commercial enzyme, 500 U/ml), (d) blood-stained cloth washed with Ariel added with SAPLF (500 U/ml). I: untreated cloths (control) and II: treated cloths.
A
Protease activity (U/ml)
800 700 600 500 400 300 200 100 0 Control
Casein
Gelatin
Gruel
Glucose
Starch
Maltose
Carbon sources (10 g/l)
B 800
Protaese activity (U/ml)
700 600 500 400 300 200 100 0 Control
(NH4)3FeSO3 (NH4)2SO4
Soya flour Yeast extract Meat extract Beef extract
Nitrogen sources ( 2 g/l)
Figure 1
A 2500-3000
1500-2000 1000-1500
2500
500-1000 0-500
2000 1500 1000
6 5
500
4
0 2,5
3
3
3,5
4
4,5
5
5,5
6
6,5
2 7
FeSO4 (g/l)
Protease activity (U/ml)
2000-2500
3000
7,5
Gruel (g/l)
B 1250-1500 1000-1250
1750
750-1000
1500
500-750 250-500
1250
0-250
1000 750 500
6 5
250 4
0 5
6
3
7
8
9
10
11
12
13
2 14
FeSO4 (g/l)
Protease activity (U/ml)
1500-1750
15
Wheat bran (g/l)
C 1250-1500 750-1000 500-750
1250
250-500 0-250
1000 750 500
6 5
250 4 0 3
3 3,5
4
4,5
5
5,5
Yeast extract (g/l)
Figure 2
6
2 6,5
7
FeSO4 (g/l)
Protease activity (U/ml)
1000-1250
1500
OD at 280 nm (AU)
Protease activity
Time (min)
B
OD at 280 nm (AU)
SAPLF
Retention time (min)
Conductivty (ms/cm)
A
C
D 1 2 3 4 5 6
~ 51 kDa SAPLF
97.0 66.0 45.0 30.0 20.1 14.4
Figure 3
1
MW (kDa) Caseinolytic activity of SAPLF
A
B 150
Residual protease activity (%)
Relative protease activity (%)
125
100
75
50
25
pH 5 pH 9
125
pH 6 pH 10
pH 7 pH 11
pH 8 pH 12
100 75
50 25 0
0
3
4
5
6
7
8
9
10
11
12
13
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
pH
C
D 150 50 °C 60 °C 70 °C 80 °C
SAPLF (0 mM Ca2+)
Residual protease activity (%)
Relative protease activity (%)
250 SAPLF (2 mM Ca2+)
200
150
100
50
0
125
(0 mM Ca2+) (0 mM Ca2+) (0 mM Ca2+) (0 mM Ca2+)
50 °C 60 °C 70 °C 80 °C
(2 mM Ca2+) (2 mM Ca2+) (2 mM Ca2+) (2 mM Ca2+)
100
75
50
25
0
30
35
40
45
50
55
60
65
70
Temperature (°C)
Figure 4
75
80
85
90
0
2
4
6
8
10
12
Time (h)
14
16
18
20
22
24
B
125
150
Residual protease activity (%)
Residual protease activity (%)
A
100
75
50
25
80 °C without additif SAPLF (80 °C + 2 mM Ca2+) SAPLF (80 °C + 10% mannitol) SAPLF (80 °C + 2 mM Ca2+ + 10% mannitol)
125
100
75
50
25
0
0
0
1
2
3
4
5
6
7
8
9
10
Time (h)
Polyols (10% w/v)
C
D
Residual protease activity (%)
125 SAPLF Alcalase Ultra 2.5 L
100
I
75
50
II 25
0 Control
Alyss
EcoVax
Ariel
Nadhif
OMO
Laundry detergents (7 mg/ml)
Figure 5
Dixan
Skip
I
a
b
c
d
11
12
Table 1 Levels of independent variables on the protease production of Lysinibacillus fusiformis strain C250R.
Code
of
Variables
Levels
variables
Lo w (-1)
Hig h (+1)
X1
Temperature (°C)
30
37
X2
pH
7
9
X3
Agitation (rpm)
X4
Gruel (g/l)
5
10
X5
Sucrose (g/l)
0
5
X6
Lentil flour (g/l)
0
5
X7
Wheat bran (g/l)
0
5
X8
Yeast extract (g/l)
1
3
X9
Urea (g/l)
0
2
X10
NaNo3 (g/l)
0
2
X11
Crude oil (%, v/v)
1
4
X12
NaCl (g/l)
20
50
X13
CaCl2 (g/l)
0
2
X14
FeSO4 (g/l)
1
3
X15
MnSO4 (g/l)
0
2
1
3
1
2
X16 X17
Trace
elements
(%, v/v) KH2PO4 (g/l)
15 0
180
Table 2 The experimental design using the Plackett-Burman method for screening of factors affecting protease production. R un
G lobal
X 1
X 2
X 3
X 4
X 5
X 6
X 7
X 8
X 9
X 10
X 11
X 12
X 13
X 14
X 15
X 16
X
Pr otease
17
activity (U/ml)a 1
+
+
+
+
+
+
+
+
+
+
+
2
+
-
+
-
-
+
+
+
+
-
+
3
+
-
-
+
-
-
+
+
+
+
-
4
+
+
-
-
+
-
-
+
+
+
+
5
+
+
+
-
-
+
-
-
+
+
+
6
+
-
+
+
-
-
+
-
-
+
+
7
+
-
-
+
+
-
-
+
-
-
+
8
+
-
-
-
+
+
-
-
+
-
-
9
+
+
-
-
-
-
+
+
-
-
+
+
-
+
-
-
-
-
+
+
-
-
1 0
+ -
+
-
+ +
+
+ -
+
+
+
+
+
+
-
-
-
-
+
-
-
+
-
+
-
-
+
-
+
+
-
+
-
+
+
-
+
+
+
+
-
-
+
+
+
-
-
+
+
+
+
0
-
+
11 37
-
-
16 82
-
-
11 82
-
-
0
+
-
11 82
-
+
72 8
+
-
0
+
-
91 0
+
+
30 45
a
7 1 8 1 9
+
-
+
-
-
-
-
+
+
+
+
-
+
-
+
-
-
-
-
+
+
+
+
-
+
-
+
-
-
-
+
+
+
+
+
-
+
-
+
-
-
+
-
+
+
+
+
-
+
-
+
-
+
-
-
+
+
+
+
-
+
-
+
+
+
-
-
+
+
+
+
-
+
-
+
+
-
-
-
-
+
-
-
-
-
+
-
+
-
-
+
+
+
-
-
-
+
+
-
-
-
+
+
-
-
-
+
-
-
-
-
15
Values
+
+
-
+
0 36 4
-
-
+
-
-
+
0
0 54 6
-
-
63 7
+
-
+
+
0 31 9
10
-
+
+ 00
35.
+
-
+
7.68
+
-
+
21.
+
-
13
+
-
-
-
93.37 74.
-
-
-
26
-
31
+
-
-
-
+
188.9 69
6.42 B
-
+
789
1
+
-
474
6
-
1.58
1
+
289.5 -
5
+
105
1
-
117.2 -12
4
-
0.63
1
-
8.32
3
-
88.42 -
1
+
437.8 -
2
-
337.3 2.4
1
+
211
1
+
7.263 -
1
represent means of three replicates. B:
Contrast
coefficient.
Table 3 The Box-Behnken design of RSM for optimization of the protease production by Lysinibacillus fusiformis strain C250R. R un
Grue
l (g/l)
Wheat bran (g/l)
Yeast extract (g/l)
FeSO 4 (g/l)
Protease (U/ml)
a
1
-1
-1
0
0
1091
2
-1
1
0
0
1272
3
1
-1
0
0
182
4
1
1
0
0
1000
5
0
0
-1
-1
910
6
0
0
-1
1
0
7
0
0
1
-1
1182
8
0
0
1
1
910
9
-1
0
0
-1
3100
1
-1
0
0
1
2636
1
1
0
0
-1
1910
1
1
0
0
1
3000
1
0
-1
-1
0
0
1
0
-1
1
0
0
1
0
1
-1
0
0
1
0
1
1
0
500
1
-1
0
-1
0
910
1
-1
0
1
0
0
1
1
0
-1
0
819
2
1
0
1
0
0
2
0
-1
0
-1
364
0
1
2
3
4
5
6
7
8
9
0
activity
1 2
0
-1
0
1
818
2
0
1
0
-1
1182
2
0
1
0
1
0
2
0
0
0
0
0
2
0
0
0
0
0
2
0
0
0
0
0
2
3
4
5
6
7 a
Values represent means of three replicates.
Table 4 Flow sheet purification of the SPALF protease from Lysinibacillus fusiformis strain C250R. Purification stepa
Total
Total b
activity (units) × 10 Crude extract
4
1550 ± 39
Heat treatment (5 min at 80
Specific activity
protein b,c
(mg) 328.5 11
(U/mg of protein) ±
b
Activity recovery rate (%)
2891.93
100
Purificat ion
factor
(fold) 1
1202 ± 30
200 ± 8
3819
80.4
1.32
1145 ± 28
22 ± 6
52045
73.8
11.03
UNO Q-6 FPLC
465 ± 12
7.5 ± 3
62000
30
13.14
HPLC (ZORBAX PSM 300)
283 ± 7
3±1
94333
18.3
20
°C) (NH4)2SO4 fractionation (2060%)
a
Experiments were conducted three times and ± standard errors are reported.
b
One unit (U) of protease 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 [29].
Table 5 Alignment of the NH2-terminal amino acid sequence of the purified SAPLF protease from Lysinibacillus fusiformis strain C250R with the sequences of other Bacillus proteases. Enzyme
NH2-terminal amino acida
Origin
SAPLF
(this
work)
Lysinibacillus
fusiformis
Lysinibacillus
fusiformis
VVFLRYIGIPDSVDLYKEDILKM
strain
VVFLRYIGIPDSVDLYKEDILKM
Lysinibacillus sphaericus strain
(WP_036225589) SPVP
-
80
DEYF
RB-21 SP
(%)
DEYF
C250R
SP (AJK86342)
VPSGPYGPIDIKADKVIEDGFKM
strain
Identity b
80
DEYF
C3-41 Aeribacillus pallidus strain VP3
APSGPYGPQGIKADKVHAQGFKG
76
AN SAPB
Bacillus pumilus strain CBS
AQTVPYGIPQIKAPAVHAQGY
70
Subtilisin E
Bacillus subtilis strain 168
AQSVPYGISQIKAPALHSQGY
67
Subtilisin
Bacillus
strain
AQTVPYGIPLIKADKVQAQGF
67
Bacillus amyloliquefaciens strain
AQSVPYGVSQIKAPALHSQGY
64
AQTVPYGMAQIKDPAVHGQGYKG
63
Carlsberg
licheniformis
ATCC 14580
Subtilisin Novo
ATCC 23350 SAPDZ
Bacillus circulans strain DZ100 AN a
Amino acid sequences for comparison were obtained using the program BLASTP
(NCBI, NIH, USA) database. b
Residues not identical with SAPLF protease from Lysinibacillus fusiformis strain
C250R are indicated in black box. The GenBank accession number is in parentheses.
Table 6 Effects of various inhibitors, reducing agents, and metallic ions on SAPLF stability. Protease activity measured in the absence of any inhibitor or reducing agent was taken as control (100%). The non-treated and dialyzed enzyme was considered as 100% for metallic ion assay. Residual activity was measured at pH 10 and 70°C. Inhibitor/reducing agent/metallic ions
ration
Residual activity (%)a
None
–
100 ± 2.5
DFP
2 mM
0 ± 0.0
PMSF
5 mM
0 ± 0.0
SBTI
3 mg/ml
104 ± 2.5
TLCK
1 mM
100 ± 2.5
TPCK
1 mM
108 ± 2.6
Benzamidine
5 mM
113 ± 2.7
DTNB
10 mM
103 ± 2.5
NEM
2 mM
99 ± 2.5
Iodoacetamide
5 mM
98 ± 2.5
Leupeptin
Pepstatin A
50 µg/ml 10 µg/ml
101 ± 2.5
97 ± 2.4
EDTA
10 mM
74 ± 1.8
Ca2+ (CaCl2)
2 mM
340 ± 8.5
2+
2 mM
227 ± 5.6
2+
Cu (CuSO4)
2 mM
118 ± 2.8
Mn2+ (MnCl2)
2 mM
100 ± 2.5
Mg2+ (MgCl2)
2 mM
124 ± 3.1
Zn (ZnSO4)
2 mM
180 ± 4.5
Na+ (NaCl)
2 mM
100 ± 2.5
Co2+ (CoCl2)
2 mM
0 ± 0.0
2 mM
0 ± 0.0
2 mM
0 ± 0.0
Fe (FeSO4)
2+
2+
Cd (CdCl2) +
Li (LiSO4)
a
Concent
Values represent means of four independent replicates, and ± standard errors are
reported.
Table 7 Kinetic parameters of purified proteases: SAPLF, SPVP, and Alcalase Ultra 2.5 L for hydrolysis of natural protein (casein) and synthetic peptide (Suc-Ala-Ala-Pro-Phe-pNA).
Substrate
Enzyme
Km
Vmax
(mM)a Casein
SAPLF
(×
103
U/mg)a
0.397±
kcat (× 103 min-1)
kcat/Km (× 103 min-1 mM-1)
94.300 ± 554
62.867
158.355
0.01 SPVP
0.450
±
41.200 ± 277
27.467
61.038
0.625
±
24.895 ± 151
16.597
26.555
0.518
±
166.011 ± 202
110.674
213.656
0.666
±
91.042 ± 510
60.694
91.132
0.852
±
37.199 ± 202
24.799
30.059
0.01 Alcalase Ultra 2.5 L Suc-Ala-
0.02 SAPLF
Ala-Pro-Phe-
0.02
pNA
SPVP 0.02 Alcalase Ultra 2.5 L a
0.07
Values represent means of four independent replicates, and ± standard errors are
reported.
