A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101

Accepted Manuscript Title: A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101 Author: Maroua Omrane Benmrad Mouna Ben Elhoul Nadia Zaraˆı Jaouadi Sondes Mechri Hatem Rekik Sidali Kourdali Mohamed El Hattab Abdelmalek Badis Samir Bejar Jaouadi Bassem PII: DOI: Reference:

S0141-8130(16)30561-X http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.06.025 BIOMAC 6198

To appear in:

International Journal of Biological Macromolecules

Received date: Accepted date:

8-2-2016 9-6-2016

Please cite this article as: Maroua Omrane Benmrad, Mouna Ben Elhoul, Nadia Zaraˆı Jaouadi, Sondes Mechri, Hatem Rekik, Sidali Kourdali, Mohamed El Hattab, Abdelmalek Badis, Samir Bejar, Jaouadi Bassem, A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.06.025 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.

Page 1 of 46

A novel organic solvent- and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101

Maroua Omrane Benmrada, Mouna Ben Elhoula, Nadia Zaraî Jaouadia, Sondes Mechria, Hatem Rekika, Sidali Kourdalib, Mohamed El Hattabc, Abdelmalek Badisb,c, Samir Bejara, Bassem Jaouadia,*

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

National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA) 11,

Bd Amirouche PO Box 67, BouIsmaïl, 42415, Tipaza, Algeria c

Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida

1, Road of Soumaâ, PO Box 270, 09000 Blida, Algeria

* Corresponding author. Tel.: +216 99 53 52 53; fax: +216 74 870 451. E-mail addresses: [email protected]; [email protected] (B. Jaouadi).

1

Page 2 of 46

Research Highlights 

A novel protease (SPTC) from Trametes cingulata strain CTM10101 was studied.



SPTC with a molecular mass of 31405.16-Da was purified and characterized.



The optimum pH and temperature values for activity were pH 9 and 60 °C, respectively.



SPTC displayed higher enzymatic performance than Flavourzyme® 500L and Thermolysin.



SPTC is a potential candidate for peptide synthesis and detergent formulations.

Abstract

A protease-producing fungus was isolated from an alkaline wastewater of chemical industries and identified as Trametes cingulata strain CTM10101 on the basis of the ITS rDNAgenesequencing. The fungus was found to hyper-produce extracellular protease when it was grown at 30°C in potato-dextrose-broth (PDB) optimized media (13500 U/ml). The pure serine protease isolated by Trametes cingulata (designated SPTC) was purified by ammonium sulfate precipitation-dialysis, heat-treatment and UNO S-1 FPLC cation-exchange chromatography. The physico-chemical characterization was also conducted. The MALDITOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 31405.16-Da.

The

enzyme

had

an

NH2-terminal

sequence

of

ALTTQTEAPWALGTVSHKGQAST, thus sharing high homology with those of fungalproteases. The optimum pH and temperature values of its proteolytic activity were pH 9 and 60°C, respectively, and its half-life times at 60 and 70°C were 9and 5-h, respectively. It was completely inhibited by PMSF and DFP, which strongly suggested its belonging to the serine

2

Page 3 of 46 protease family. Compared to Flavourzyme®500L from

Aspergillus oryzae and

Thermolysin,typeX from Geobacillus stearothermophilus, SPTC displayed higher levels of hydrolysis, substrate specificity, and catalytic efficiency as well as elevated organic solvent tolerance and considerable detergent stability. Finally, SPTC could potentially be used in peptide synthesis and detergent formulations.

Keywords: Trametes cingulata; Organic solvent; Detergent.

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Page 4 of 46 1. Introduction

Thanks to their specificity, enzymes have long been used as a substitute to chemicals to improve the efficiency, eco-friendliness, cost-effectiveness, and profitability of a wide range of industrial systems and bioprocesses. The world enzyme demand is predicted to be 6.8% per year and reathed to $8 billion in 2015 [1]. Among the world sale of industrial enzymes of about US $ 300–600 million per annum, 75% of them are hydrolytic enzymes, of which twothirds are proteolytic enzymes. Proteases (EC 3.4.21-24 and 99) represent one of the earliest known families of enzymes that have long been used to catalyze the hydrolysis of peptide bonds in aqueous solutions and the synthesis of peptides in the presence of organic solvents. High-alkaline proteases, accounting alone for about 40% of the total worldwide enzyme sales [2], belong to the proteases family and constitute one of the most important groups of industrial enzymes. They have applications in different industries, namely detergent, food, leather, pharmaceutical, silk, and for recovery of silver from used X-ray films [2, 3]. Thermoalkaline proteases represent one of the most commonly used groups of alkaline proteases, particularly because they can function at a pH range of 7-12 and a temperature range of 3580°C. The particular importance of alkaline proteases in the detergent industry, for instance, emanates from their central contribution as cleaning additives to detergent formulations [4]. Since enzymes, including proteases, are, however, labile catalysts that easily lose their catalytic activity in the presence of organic solvents [5], increasing attention has been given to the search for solvent-stable proteases. The growing demand for proteases with specific properties has incited biotechnologists to explore new sources of proteases. The industrial demand for proteolytic enzymes, with the appropriate specificity and stability to pH, temperature, metallic ions, and compatibility with detergent compounds like surfactants, bleaching agents, and organic solvents continues to boost the search for new enzyme sources. Proteases with high activity and stability in a high

4

Page 5 of 46 alkaline range and high temperatures are interesting for bioengineering and biotechnological applications. In general, microbial proteases are extracellular in nature and directly secreted into the fermentation broth by the producer, thus simplifying downstream processing, purification, and recovery of the enzyme as compared to proteases obtained from plants and animals [6]. Alkaline proteases used industrially are produced by a wide range of microorganisms including bacteria, moulds, and yeasts. Nowadays, the use of enzyme (protease)-based detergents is preferred over the conventional synthetic ones in view of their cleaning properties, better performance at lower washing temperature and pollution alleviation [7, 8]. In this case, many fungal proteases are applied. Microbial proteases represent one of the three largest groups of industrial enzymes and account for approximately 65% of the total enzyme sale in the world. They are also the leaders of the industrial enzyme market worldwide [2, 9]. Proteases from microbial origin have long been used in industry. They are mostly of fungal origins due to their biochemical diversity and the possible increase and ease of their production by environmental and/or genetic manipulations [10]. They are easily extracted and separated from mycelium. Besides, fungi produce a larger spectrum of hydrolases as bacteria and present a large specificity towards substrates. Furthermore, fungal proteases are an extracellular enzyme that allows the separation of the mycelium from the fermentation medium by simple filtration. Moreover, these mold strains are Generally Regarded As Safe (GRAS) and may grow on cheaper substrates [11-13]. The amount of produced proteases varies greatly with the used strains and media. Distinct proteases from different strains, which catalyze the same reactions, allow flexibility in the choice of fermentation conditions since they may have different stabilities and different pH and temperature optima [2]. Alkaline proteases are particularly important because they are both stable and active at high pH solutions as well as in the presence of surfactants and oxidizing agents. They are also

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Page 6 of 46 important since to reduce the harsh chemicals employed in laundry detergent, thus eliminating the toxicity in wash. They are produced by a wide range of microorganisms including bacteria, moulds, yeasts and mammalian tissues. Most of the commercial alkaline proteases were isolated from Bacillus species [9, 14, 15]. A considerable number of fungal species are known to produce large amounts of extracellular alkaline proteases such as Microsporum canis [16], Alternaria alternata [17], Conidiobolus coronatus [18], Arthrobotrys olgospora [19], Aspergillus fumigatus [20], Aspergillus clavatus [21], Aspergillus nidulans [22], Aspergillus parasiticus [23], and Trichoderma harzianum [24]. The majority of studies so far performed on Trametes have focused on their lignin degradation [25, 26]. Besides, the relatively low stability and catalytic activity under relevant operational conditions, namely high temperature and pH values and the presence of detergents, have often precluded the adoption of fungal enzymes into wide-scale applications. In this context, the isolation and screening of protease-producing strains from naturally occurring habitats or alkaline wastewater is expected to provide new valuable fungal strains for the production of organic solvent-stable proteases in highly alkaline conditions. Accordingly, the present study reports, for the first time, on the purification and biochemical characterization of a novel alkaline protease, nominated SPTC, from the fungus Trametes cingulata strain CTM10101 isolated from the chemically-contaminated wastewater samples of the nitrogen-phosphate-potash (NPK) Chemical Company of Sfax (Tunisia). It also provides basic information on the potential use of the SPTC enzyme as a potential candidate for future applications in detergent formulations and non-aqueous peptide biocatalysis.

2. Materials and methods

2.1. Substrates, chemicals, and commercial proteases

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Page 7 of 46 Unless specified, all substrates, chemicals and reagents purchased from Sigma Chemical Co. were of the analytical grade or highest available purity (St. Louis, MO, USA). Flavourzyme® 500L, a commercial fungal protease/peptidase complex was supplied by Novozymes A/S (Bagsvaerd, Denmark). It is produced by the submerged fermentation of a selected strain of Aspergillus oryzae and contains both endoprotease and exopeptidase activities. Thermolysin type X, a commercial thermostable extracellular metalloendopeptidase from Geobacillus stearothermophilus, purchased from Sigma-Aldrich Inc. Fluka, Chemical Co. (St. Louis, MO, USA).

2.2. Isolation and growth conditions of protease-producing fungal strains Soil samples were collected from different Tunisian sites to isolate protease-producing microorganisms. They were then dispersed in sterile distilled water. Alternatively, the protease activity was screened in strains available in the fungi collection of the Centre of Biotechnology of Sfax (CBS). The treated samples and strains were plated onto skimmed milk agar plates containing (g/l): peptone, 5; yeast extract, 3; skimmed milk 250 ml; and bacteriological agar, 20 at pH 7.4. The plates were then incubated for 12 h at 30 °C to obtain colonial growth. The colonies with a clear zone formed by the hydrolysis of milk casein were evaluated as protease producers and several proteolytic fungal strains were isolated. Fungi were maintained on potato-dextrose-agar (PDA) plates at -20 °C. The CTM10101 strain, available from the CBS fungi collection and previously isolated from contaminated soil samples at the NPK Chemical Company of Sfax, Tunisia, showed a large clear zone of hydrolysis, and was, therefore, selected for further experimental analysis. The protease-producing isolate CTM10101 was propagated on PDA plates at 30 °C, and inocula were prepared from 7-day-old colonies by flooding with 10 ml of sterile distilled water and scraping off the agar plates. A 1-ml spore suspension (108 spore/ml) of strain CTM10101 was added to a 1000-ml Erlenmeyer flask with baffles containing 200 ml of a 7

Page 8 of 46 complete PDB liquid medium at pH 5.6 containing (g/l): lentil flour, 10; yeast extract, 2; glucose,10; KCl,0.5; KH2PO4, 1; and K2HPO4. The flasks were incubated at 30 °C for 3 days with shaking at 200 rpm. The medium was sterilized by autoclaving at 120 °C for 20 min.

2.3. PCR amplification and ITS of 18S rDNA gene sequencing, and molecular phylogenetic analysis The genomic DNA of the isolate fungal strain CTM10101 was extracted from freeze-dried mycelia according to hexadecyltrimethylammonium bromide (CTAB) method as described by Li [27]. The gene encoding ITS region of the ribosomal DNA (rDNA) was amplified by the fungi identification polymerase chain reaction (PCR) kit using primers: ITS1 (5′TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [13]. The PCR product was inserted into the pMOSBlue vector using a pMOSBlue blunt-ended cloning kit (GE Healthcare/Amersham Biosciences Corp., Piscataway, NJ, USA). The nucleotide sequences of both strands of the cloned ITS rDNA gene sequence were sequenced in both directions with universal primers: U-19 (5′-GTTTTCCCAGTCACGACG-3′) and T7 (5′-TAATACGACTCACTATAGGG-3′) using BigDye Terminator Cycle Sequencing Ready Reaction kits and the automated DNA sequencer ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Multiple nucleotide sequence alignment was performed using the BioEdit version 7.0.2 software program and CLUSTALW program at the European Bioinformatics Institute server (http://www.ebi.ac.uk/clustalw). Phylogenetic and molecular evolutionary analyses were conducted via the molecular evolutionary genetics analysis (MEGA) software version 4.1. Distances and clustering were calculated using the neighbor-joining method. Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by performing 100 re-samplings.

2.4. Assay of proteolytic activity

8

Page 9 of 46 The protease activity was assayed by a modified caseinolytic Peterson’s method protocol [28]using Hammerstein casein (Merck, Darmstadt, Germany) as a substrate. Unless otherwise stated, a suitably diluted enzyme solution (0.5 ml) was mixed with 2.5 ml 50 mM glycineNaOH buffer supplemented with 2 mM CaCl2 at pH 9 (Buffer A) containing 10 g/l casein, and incubated for 15 min at 60 °C. The reaction was stopped by adding 2.5 ml 20% TCA. The mixture was left at room temperature for 30 min, and the precipitated proteins (non-digested) were removed by centrifugation at 10,000 × g for 20 min. Afterwards, 0.5 ml of the clear supernatant were mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu’s phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control. One unit of caseinolytic activity was defined as the amount of enzyme that hydrolysed the substrate and produced 1 µg of amino acid equivalent to tyrosine per min under the assay conditions. The proteolytic activity present in the laundry detergent solution was evaluated by the method of Boulkour Touioui et al. [29] using N,N-dimethylated casein (DMC) as a substrate. 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. SPTC purification procedure Five hundred ml of a 72-h culture of Trametes cingulata strain CTM10101was centrifuged for 30 min at 10,000 × g to remove microbial cells. The supernatant containing extracellular protease was used as the crude enzyme preparation and submitted to the following purification steps. It was initially saturated up to 40% with solid (NH4)2SO4 and then centrifuged at 10,000 × g for 20 min. The obtained supernatant was saturated up to 60% with solid (NH4)2SO4, re-centrifuged, re-suspended in a minimal volume of buffer A, containing 10 mM NaCl (Buffer B), and dialyzed overnight against the repeated changes of 9

Page 10 of 46 the same buffer. The sample thus obtained was deposited on a UNO S-1 column (7 × 35 mm) (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using Fast protein liquid chromatography (FPLC) system previously equilibrated with 20 mM Histidine buffer supplemented with 2mM CaCl2 at pH 5.5. The proteins were eluted with the same buffer containing an increasing concentration of NaCl of 0 to 500 mM at a rate of 30 ml/h. Fractions of each peak were collected manually and analyzed by measuring absorbance at 280 nm and the proteolytic activity on casein. Pooled fractions containing protease activity were concentrated in centrifugal micro-concentrators (Amicon Inc., Beverly, MA, USA) with 10-kDa cut-off membranes and stored at -20 °C for further analysis.

2.6. Proteins measurement, electrophoresis, and analytical methods Protein concentration was determined at 595 nm using a Dc protein assay kit purchased from Bio-Rad Laboratories (Hercules, CA, USA) based on the method of Bradford [30]. The total protein concentration of the enzyme solution was estimated from a calibration curve that was constructed using bovine serum albumin (BSA). The analytical SDS-PAGE was performed using 10% (w/v) acrylamide in gels as described by Laemmli [31]. The protein bands were visualized with Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) staining. Casein zymography staining was performed as described by Jaouadi et al. [32]. The molecular mass of the purified protease was analyzed in the linear mode by Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) using a Voyager DE-RP instrument (Applied Biosystems/PerSeptive Biosystems, Inc., Framingham, MA, USA). Data was collected with a Tektronix TDS 520 numeric oscillograph and analyzed using the GRAMS/386 software (Galactic Industries Corporation, Salem, NH, USA).

2.7. Amino acid sequencing

10

Page 11 of 46 Bands of purified SPTC enzyme 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. Amino acid residues were detected as individual signals.

2.8. Biochemical characterization of 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:

phenylmethanesulfonyl fluoride (PMSF); diiodopropyl fluorophosphates (DFP); soybean trypsin inhibitor (SBTI); N-p-tosyl L-lysine chloromethyl ketone (TLCK); N-p-tosyl Lphenylalanine chloromethyl ketone (TPCK); benzamidine hydrochloride hydrate; 2mercaptoethanol (2-ME);

LD-dithiothreitol (LD-DTT);

monoiodoacetic acid (MIA); DTNB;

1,2-epoxy-3-(p-nitrophenoloxy) propane (EPNP); N-ethylmalemide (NEM); iodoacetamide; leupeptin; pepstatin A; 1,10-phenanthroline monohydrate; EDTA, ethylene diamine (βaminoethyl ether)-N,N,N',N'-tetraacitic acid; and EGTA, ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacitic acid, as well as various divalent and monovalent metallic ions on protease stability were investigated by pre-incubating 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.

2.8.2. Determination of the optimum pH and stability The effect of pH was determined using casein as a substrate. Protease activities were assayed over a pH range of 2 to 13 at 70 °C. The pH stability of SPTC was determined by

11

Page 12 of 46 pre-incubating the enzyme in buffer solutions with different pH values for 120 h at 40 °C. Aliquots were then withdrawn, and residual enzymatic activities were determined under standard assay conditions. The following buffer systems, supplemented with 2 mMCaCl2, were used at 50 mM: glycine-HCl for pH 2-5, 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.Duringthe preparation of an aqueous casein solution from a lyophilized powder, the mixture should be gently inverted several times to introduce casein into the buffer solution. In fact, casein is soluble in alkaline pH as it forms sodium caseinate by stirring and heating the solution till about 50 °C for 15 min. However, casein is rather very difficult to dissolve in acidic pH. Hence, to guarantee a maximum of solubilization in acidic environment, the solution need to be stirred and heated in the presence of 10 mM ammonium hydroxide till about 70 °C for 30 min. After wood, casein was stored at 4 °C inconvenient aliquots with no additional treatment.

2.8.3. Determination of optimum temperature and thermal stability The effect of temperature on enzyme activity was examined at 30-100 °C at pH 9 in the presence and absence of 2 mM CaCl2. For the thermal stability, the half-life times of the purified protease SPTC were determined after incubation at 40, 50, 60, 70, and 80 °C for 24 h in the presence and absence of 2 mM CaCl2. Aliquots were withdrawn at the desired time intervals 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. Substrate specificity The substrate specificity of SPTC was determined using natural (casein, gelatin, keratin, ovalbumin, BSA, hemoglobin and myoglobin) and modified (azo-casein, keratin azure, azo albumin, and collagen types I and II) protein substrates as well as ester [N-benzol-L-arginine

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Page 13 of 46 ethyl ester (BTEE), N-acetyl-L-tyrosine ethyl ester monohydrate (ATEE), N-benzol-L-arginine ethyl ester (BAEE), (TAME) and S-benzyl-L-cysteine ethyl ester hydrochloride (BCEE)] and synthetic peptide [N-succinyl-L-Ala-L-Ala-L-Pro-L-Met-p-nitroanilide, N-succinyl-L-Ala-LAla-L-Pro-L-Phe-p-nitroanilide,

N-succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide,

methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide,

N-

N-succinyl-L-Ala-L-Ala-L-Val-L-

Ala-p-nitroanilide, N-succinyl-L-Ala-L-Ala-L-Phe-p-nitroanilide, N-succinyl-L-Ala-L-Ala-LVal-p-nitroanilide, N-succinyl-L-Ala-L-Pro-L-Ala-p-nitroanilide, N-succinyl-L-Ala-L-Ala-LAla-p-nitroanilide, and N-succinyl-L-Tyr-L-Leu-L-Val-p-nitroanilide] substrates. Enzymatic activities were determined on each substrate according to standard conditions previously described by Zaraî Jaouadi et al. [33].

2.9. Performance evaluation of the purified proteases

2.9.1. Kinetic measurements of SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases Kinetic parameters were calculated from the initial rate activities of the purified SPTC enzyme using casein and Suc-(Ala)2-Val-Ala-pNAas substrates at different concentrations ranging from 0.10 to 50 mM at 50 °C and pH 9 for 10 min. The purified enzymes used were, SPTC (This study); Flavourzyme® 500 L, and Thermolsyin, 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 velocity (Vmax) values were calculated using the Hyper32 software. The value of the turnover number (kcat) was calculated using the following equation: kcat 

Vmax [E]

where [E] refers to the active enzyme concentration and Vmax to maximal velocity.

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Page 14 of 46 2.9.2. Determination of hydrolysis degree of SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases Casein hydrolysis was conducted at 60 °C and pH 9. The pH was kept constant throughout hydrolysis by adding NaOH 5 N. An amount of 4 g of casein was dissolved in 100 ml of 50 mM glycine-NaOH buffer containing 2 mM CaCl2 at pH 9 and then treated with 500 U of the purified enzymes, namely SPTC, Flavourzyme® 500 L, and Thermolsyin. The degree of hydrolysis (DH) was calculated for each case from the amount of base (NaOH) added to keep the pH constant during hydrolysis [34] as previously described by Zaraî Jaouadi et al. [33].

2.9.3. Effects of organic-solvents on protease activity and stability of SPTC and Thermolysin type X proteases Various organic solvents, with different Log P values (50%, v/v), were tested by shaking at 150 strokes per min and 40 °C for 3 and 180 days to evaluate their effects on protease activity and stability, respectively. The proteases used were SPTC and Thermolysin type X. The relative and residual caseinolytic activities were assayed under the same conditions at 60 °C and pH 9. The activity of the enzyme without any organic solvent was taken as 100%.

2.9.4. Stability and compatibility of SPTC and Flavourzyme® 500 L with laundry detergents The stability and compatibility of the purified SPTC and Flavourzyme® 500 L proteases with some currently commercialized liquid and solid detergents were investigated. The list of liquid detergents included Dixan, Nadhif (Henkel-Alki, Tunisia), Ariel (Procter & Gamble, Switzerland), OMO, and Skip (Unilever, France). The solid detergents used were Dipex, Ecovax (Klin Productions, Sfax, Tunisia), Alyss (EJM, Sfax, Tunisie), and Detech (SOTUP, Sfax, Tunisia).

14

Page 15 of 46 In order to investigate their stability and compatibility, the mentioned commercial detergents were diluted in tap water to obtain a final concentration of 7 mg/ml (to simulate washing conditions) [35]. The endogenous proteases 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 (SPTC and Flavourzyme® 500 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 9 and 60 °C using DMC as a substrate. The enzyme activity of a control (without any detergent), incubated under similar conditions, was taken as 100%.

2.9.5. Removal of blood stain from cotton fabrics New cotton cloth pieces (6 × 6 cm) were stained with blood stains and used to simulate the washing condition and determine the efficiency of SPTC as a biodetergent additive compared to the commercially protease Flavourzyme® 500 L. The endogenous proteases found in Skip liquid laundry detergent were inactivated by heating the diluted detergents for 1 h at 70 °C prior to the addition of the purified tested enzymes. The stained cloth pieces were shake-incubated (250 rpm) in different wash treatments at 40 °C for 1 h in 1-litre beakers containing a total volume of 100 ml of: tap water, Skip detergent (7 mg/ml, in tap water), and detergent added with SPTC (500 U/ml) or commercial Flavourzyme® 500 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 bloodstained piece of cloth was taken as a control.

2.10. Statistical analysis All determinations were performed in 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

15

Page 16 of 46 (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, USA).

2.11. Nucleotide sequence accession number The data reported in this work for the ITS nucleotide sequence of the 18S rDNA gene has been deposited in the DDBJ/EMBL/GenBank databases and assigned under accession number KU681024.

3. Results and discussion

3.1. Screening of alkaline protease-producing fungi In the current study, fifty fugal strains that were either newly isolated from Tunisian wastewater samples, or from diverse alimentary product rich in protein such as lentils, wheat, and barley, or obtained from the CBS fungi collection, 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 diameter of the clear zone and that of the colony served as an index for the selection of strains with high protease production ability. Seven isolates exhibited a ratio that was higher than 3, with the highest ratio being 5. Strain CTM10101 (newly isolated from a contaminated wastewater sample at the NPK Chemical Company of Sfax, South Tunisia), exhibited the highest extracellular protease activity (about 13500 U/ml) after 72 h of incubation in an optimized medium and was, therefore, retained for all subsequent studies.

3.2. Identification and molecular phylogeny of the microorganism The findings revealed that the CTM10101 isolate was a white rot basidiomycete. In order to identify CTM10101 strain, the internal transcribed spacer region of 18S rDNA (551 bp) 16

Page 17 of 46 was amplified, cloned in pMOSBlue vector and sequenced. The ITS nucleotide sequence was analyzed with the GenBank database using BLAST program and showed 99 and 98% homology with ITS of Trametes cingulata MUCL:40167 (accession no. JN645075) and Trametes marianna BJFC12714 (accession no. KC848334) strains, respectively. Multiple sequence alignment was performed with CLC Gene Workbench software version (2.0.2). The phylogenetic tree was constructed using neighbor-joining method (data not shown). The multiple alignment and phylogenetic tree showed that CTM10101 strain is Trametes cingulata (accession no. KU681024). The strain was inoculated in Erlenmeyer flasks with baffles and incubated at 30 °C for 72 h. The cultures were then filtrated and centrifuged and the supernatant was used as the enzyme solution. 3.3. Purification of alkaline protease The SPTC enzyme was purified from the culture supernatant according to the procedure described in Section 2.The supernatant was obtained by the centrifugation of a 72-h old culture of the Trametes cingulata strain CTM10101 (data not shown) using broth (500 ml) as a crude enzyme solution. The protein elution profile obtained at the final purification step indicated that the protease was eluted at 100-180 mM NaCl (fractions number 31 to 37) (Fig. 1A). Position Figure 1 here The results of the purification procedure are summarized in Table 1. The purified enzyme preparation contained about 20% of the total activity of the crude and had a specific activity of 94000 U/mg (Table 1) using casein as substrate. Position Table 1 here

3.4. Molecular weight determination of SPTC enzyme

17

Page 18 of 46 The homogeneity of the purified enzyme was checked by SDS-PAGE and protein staining analyses. A single protein band was obtained for the pooled fractions of the purified enzyme. The purified SPTC had a molecular weight of about 31 kDa (Fig. 1B), which is less than those previously reported for other fungal proteases (Aspergillus parasiticus, 36 kDa [36]; Aspergillus clavatus ES1, 32 kDa [21]; and Purpureocillium lilacinum LPS # 876, 37 kDa [37]). The exact molecular mass of the purified SPTC was confirmed by MALDI-TOF mass spectrometry as being 31405.16 Da. Zymogram activities staining revealed one zone of caseinolytic activity for the purified sample co-migrating with proteins with an estimated molecular mass of 31 kDa (Fig. 1C). Taken together, these observations suggest that SPTC is a monomeric protein comparable to those previously reported for other proteases from fungal strains [21-24, 37-39].

3.5. NH2-terminal amino-acid sequence determination of SPTC enzyme The sequence determined for the first 23 NH2-terminal amino acid of SPTC from the Trametes cingulata strain CTM10101, ALTTQTEAPWALGTVSHKGQAST, showed 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 Swiss-Prot database, using the BLASTP and tBlastn search programs (Table 2). The sequence showed homology with those found for other fungal proteases, reaching 87% identity with the alkaline serine protease Tvsp1 from Trichoderma virens strain Gv29-8, and 80% identity with the serine protease Prb1 from Trichoderma hamatum strain LU593. The NH2-terminal amino acid of SPTC differed from Tvp1 and Prb1 proteases by two and three amino acids, respectively; the Thr6 residue in SPTC was a Ser3 in the Prb1 protease from Trichoderma hamatum strain LU593. It also differed by the Asn7 and Ala11 residues, which was substituted by a Gln7 and Gln11 in the Tvp1 and Prb1 proteases. Besides, it showed 73, 72, and 67% homology with proteases from Tolypocladium inflatum strain 29910, 18

Page 19 of 46 Aspergillus clavatus strain NRRL 1, and Aspergillus versicolor strain 4647, respectively. The NH2-terminal amino acid of SPTC differed from those of the three last proteases by five amino acid residues at positions 6, 7, 11, 14, and 15 (Table 2). Position Table 2 here

3.6. Biochemical characterization of purified protease

3.6.1. Effects of inhibitors and metallic ions on protease stability of SPTC enzyme Proteases can be classified on the basis of their sensitivity to various inhibitors [40]. Accordingly, further assays were performed to evaluate the effects that natural and synthetic inhibitors, as well as chelating agents and group-specific reagents, might have on protease activity determined as the molar ratio of inhibitor/enzyme = 100. The relative inhibitory effects of various compounds are listed in Table 3. The findings indicated that enzyme activity was strongly inhibited by PMSF and DFP, which are well-known inhibitors of serine proteases. This suggested that a serine was involved in the catalytic activity. Other inhibitors, such as TPCK and TLCK, NEM a chymotrypsin alkylating agent, benzamidine and aprotinin, a trypsin competitive reagents and SBTI, and a soybean trypsin inhibitor, did not show inhibitory effects. The acid reagents (Leupeptin and Pepstatin A) had almost no effect on enzyme activity. In the presence of 10 mM EDTA and 1 mM EGTA, as chelating agents and metalloprotease inhibitors, the protease retained 93 and 91% of its activity, respectively, which suggests that no metallic cofactors were required. Extensive dialysis against buffer A containing 1 mM EGTA did not reduce the activity of the enzyme (data not shown). Insensitivity to chelators would be a valuable property for potential application in detergent formulations because these agents are added as water softeners and stain removers [41]. In fact, serine-proteases are known to contain two calcium binding sites and the removal of Ca2+ from the strong binding site is associated with a significant decrease in thermal stability. The

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Page 20 of 46 role of Ca2+ is probably related to the stabilization of the activated form of the SPTC and the preservation of its structure against autolysis. Similar fungus protease activity effects were previously reported for EDTA and Ca2+ [21]. Several metallic ions were also assayed for their effects on SPTC activity (Table 3). The activity of the enzyme was essentially unaffected by monovalent cations (Li+, K+, and Na+). Its activity was enhanced by 170and 219%, following the addition of MnCl2 and CaCl2 at 2 mM, as compared to the control, respectively. This result indicates that the enzyme requiresCa2+ and Mn2+for optimal activity. Furthermore, the enzyme was slightly activated by Mg2+ and Zn2+and underwent no significant inhibition in the presence of Co2+. Protease activity was, however, completely inhibited by Ni2+, Cd2+, and Hg2+ and moderately inhibited by Fe2+and Ba2+. Likewise, the purified proteases ES1 [21], SAPB [32], and KERAB [42] were previously reported to be totally activated byCa2+, Mg2+, and Mn2+ but strongly inhibited by Cd2+, Ni2+, and Hg2+ (5 mM). The increase in protease activity with Ca2+, Mg2+, and Mn2+ indicated that the metallic ions exerted a protective effect for the enzyme against thermal denaturation, thus playing a vital role in maintaining its active confirmation at higher temperature [43]. The toxic metallic ions exerted toxicity by binding to a variety of organic ligands, causing the denaturation of proteins, including enzymes. The inhibitory effect of heavy metallic ions is well documented in the literature. Mercury ions are, for instance, known to react with protein thiol groups (converting them to mercaptides) as well as histidine and tryptophan residues [44]. Position Table 3 here

3.6.2. Effects of pH on enzyme activity and stability The results presented in Fig. 2A show that SPTC protease is active in a wide pH range between 6 and 11, with optimal activity being obtained at pH 9. The relative activities at pH 5 and 12 were 50%. As far as pH stability analysis is concerned, the purified enzyme was 20

Page 21 of 46 incubated in buffers at various pH values. The pH stability profile showed that SPTC was highly stable in the pH range of 7-12. The half-life times of SPTC at pH 7, 8, 9, 10, 11, and 12 were 22, 20, 18, 16, 10, and 6 h, respectively (Fig. 2B). The high activity and stability exhibited by SPTC at high pH solutions is, in fact, a very important attribute that provided further support for its potential strong candidacy for future application in detergent formulations since laundry detergents generally operate at a pH of 711 [41]. The pH stability displayed by the title protease was found to exceed the ones reported for currently commercialized detergent proteases, including subtilisin BPN' and Savinase™, which generally have a pH optimum of 10.5 and pH stability at 8-10 [15].Yet, the protease Hnsp from Hirsutella rhossiliensis strain OWVT-1 displayed an optimal pH at 7 [45]. Position Figure 2 here

3.6.3. Effects of temperature on protease activity and stability The optimum temperature for protease activity at pH 9 was 50 °C in the absence of CaCl2 and 60 °C in the presence of 2 mM Ca2+, using casein as a substrate (Fig. 2C). The half-life times of SPTC in the absence of any additive were 18, 10, 7, 4, and 1 h at 40, 50, 60, 70, and 80 °C, respectively. The addition of different concentrations of CaCl2 (1 to 10 mM) enhanced the thermostability of the enzyme. The maximal thermostability was achieved with 2 mM Ca2+. As shown in Fig. 2D, the half-life times of the purified protease at 40, 50, 60, 70, and 80°C increased to 24, 14, 9, 5, and 2h in the presence 2 mM CaCl2. In fact, Ca2+ was previously reported to improve microbial protease activity and stability [32, 46].

3.6.4. Substrate specificity profile The substrate specificity of proteases is often attributed to the amino acid residues preceding the peptide bond they hydrolyze. The relative hydrolysis rates of various substrates were investigated to elucidate the amino acid preference/substrate specificity of SPTC (Table

21

Page 22 of 46 4). The highest activity was observed with casein, azo-casein, gelatin, keratin, and keratin azure. A relatively high activity against hemoglobin, myoglobin, and albumin was also observed. The purified SPTC was noted to exhibit esterase and amidase activities on BTEE and ATEE, but not on BAEE, BCEE, and TAME. It also displayed a preference for aromatic and hydrophobic amino acid residues, such as Met, Phe, Leu, Val, and Ala, and the carboxyl side of the splitting point in the P1 position. SPTC was, therefore, active against methionine peptide bonds, a quality that was previously demonstrated for SAPB [32], KERAB [42], SAPDZ [35], and SAPB-N99Y [47-49]. When Suc-(Ala)n-pNA was used as the synthetic oligopeptide substrate, a minimum length of two residues was required for hydrolysis. Enzymatic activity was noted to largely depend on secondary enzyme substrate contacts with amino acid residues (P2, P3, etc.) more distant from the scissile bond, as illustrated by the differences observed between the kinetic parameters of Suc-(Ala)2-Val-Ala-pNA and Suc(Ala)2-Pro-Phe-pNA. SPTC was noted to attain the highest hydrolysis rates of 100 and 97% with Suc-(Ala)2-Val-Ala-pNA and Suc-(Ala)2-Pro-Phe-pNA, respectively. Its preference for lager hydrophobic amino acids could presumably be due to the active site cleft or the crevice lined with hydrophobic amino acid residues. Position Table 4 here

3.6.5. Determination of kinetic parameters SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases exhibited the classical kinetics of Michaelis-Menten for the two substrates used. The order of the catalytic efficiency (kcat/Km) values of each enzyme was almost the same, i.e., Suc-(Ala)2-Val-Ala-pNA >casein(Table 5). When casein was used as a natural substrate, SPTC was noted to exhibit kcat/Km values that were 2.29 and 5.84 times higher than those Flavourzyme® 500 L, and Thermolysin type X, respectively. When Suc-(Ala)2-Val-Ala-pNA was used as a synthetic

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Page 23 of 46 substrate, SPTC was also noted to exhibit kcat/Km values that were 4.71 and 11.77 times higher than those Flavourzyme® 500 L, and Thermolysin type X, respectively (Table 5). Position Table 5 here

3.7. Performance evaluation of the purified protease

3.7.1. Determination of hydrolysis degree The hydrolysis curves of casein protein after 4 h of incubation are shown in Fig. 3A. The purified enzymes were used at the same levels of activity for the production of protein hydrolysates from casein and for the subsequent comparisons of hydrolytic efficiencies. The casein was noted to attain high hydrolysis rates during the first hour. The enzymatic reaction was then noted to decrease and reach a subsequent steady-state phase where no apparent hydrolysis took place. As shown in Fig. 3A, the purified SPTC was the most efficient, with 30% protease used during hydrolysis, followed by Thermolysin type X (24%), Flavourzyme® 500 L with is the least efficient (11%) one. These findings indicate that SPTC can be useful for the preparation of protein hydrolysates. Position Figure 3 here

3.7.2. Effects of organic solvents on protease activity and stability of SPTC and Thermolysin type X proteases Various organic solvents were examined for their effects on the activity and stability of the purified SPTC and Thermolysin type X proteases. The enzyme solutions containing 50% (v/v) organic solvent were shake-incubated at 40 °C (Table 6). When compared to the control, SPTC and Thermolysin type X were noted to retain at least 155, 147 and 120% of their activities of incubation in the presence of hexane as a hydrophobic solvent and 89, 106, and 65% of their activities in the presence of isopropanol as a hydrophilic solvent, respectively.

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Page 24 of 46 Particularly, SPTC is not stable at acetonitrile (hydrophilic organic solvent) as STAP [50] and Prot-2 [51]. This result may be due to their effect on the enzyme itself or on the substrate used for testing activity. But, SPTC were found to be notdeactivated by ethyl acetate as STAP which was previously reported to be a quite harmful polar aprotic solvent to other solventstable proteases from Pseudomonas aeruginosa strain CTM50182 [35] and Bacillus licheniformis strain RSP-09-37 [52]. In media containing organic solvents, enzyme deactivation could presumably be due to the disruption of the protein molecule hydrophobic core due to the change of medium hydrophobicity [53, 54]. In particular, polar solvents (such as ethyl acetate), which can penetrate into the protein, are more capable of inducing structural changes for the interaction between the active site and substrate than non-polar solvents, as previously reported by Serdakowski and Dordick [55]. This suggests that the stability of enzymes in the presence of organic solvents depends on both the enzyme and the nature of the organic solvent being used. The activity and stability shown by SPTC in the presence of isopropanol, dimethylformamide (DMF), chloroform, n-heptane, and, n-hexane were higher than those of Thermolysin type X, which further supports the potential candidacy of the SPTC enzyme for future application as a biocatalyst for the synthesis of peptide reactions in low water activity systems. Position Table 6 here

3.7.2. Stability and compatibility of SPTC and Flavourzyme® 500 Lwith laundry detergents The data presented in Fig. 3B show that compared to Flavourzyme® 500 L, SPTCwas extremely stable and compatible with the commercial liquid detergents used, retaining 100% of its initial activity with Nadhif and 99% with Ariel (vs 100% for Flavourzyme® 500 L) even after 120 h incubation at 40 °C. The data illustrated in Fig. 3B also show that, compared to Flavourzyme® 500 L, SPTC was highly stable in the presence of solid laundry detergents at a concentration of 7 mg/ml. The alkaline proteases exhibited higher stability in Skip and 24

Page 25 of 46 Dixan (100%) than in Detech. However, Flavourzyme® 500 Lwere noted to be less stable in the presence of Skip, retaining 75% of its initial activity. Incubated under the same conditions in the presence of Ariel, STAP [50], Ve2-20-91 [56], VM10 [57], and SSR1 [58] proteases retained only 95, 72, 42, and 37% of their initial activities, respectively. These findings provide further support for the usefulness of SPTC in future industrial applications as a cleaning bio-additive in detergent formulations.

3.7.3. Removal of blood stains from cotton fabrics Several pieces of stained cotton cloth were incubated at different conditions and used to evaluate the performance of SPTC in terms of its ability to remove blood stain. A shown in Fig. 3C, a limited washing performance was observed with detergent (Skip) only. The supplementation of SPTC or commercial protease Flavourzyme® 500 L in detergent seems to improve the cleansing process as evidenced by rapid blood stain removal when compared to detergent alone. In fact, SPTC facilitated the release of proteinacious materials in a much easier way than the currently used Flavourzyme® 500 L protease. Furthermore, the combination of SPTC with the Skip detergent resulted in the complete stain removal (Fig. 3C). These findings further support the usefulness of SPTC in future industrial applications as a cleaning bioadditive in detergent formulations. Detergent proteases have the ability to hydrolyze large insoluble proteins. This is an important property of detergent enzymes as they are required to act on protein substrates attached to solid surfaces, making them attractive additives for hard-surface cleaners. They could also help in the removal of proteinaceous stains that are often encountered in the laundry. Several works have reported the usefulness of alkaline proteases in the facilitation of blood stains removal from cotton cloth [59, 60]. The common detergents contain hazardous chemicals for effectual washing performance which can be substituted by the enzymes in modern detergent formulations attributed to efficient washing along with being eco-friendly 25

Page 26 of 46 and fabric friendly. Although Abidi et al. (2008) reported the usefulness of alkaline protease from Botrytis cinerea for the removal of blood stains from cotton cloth in the presence and absence of detergents [61], we believe that the SPTC enzyme from Trametes cingulata strain CTM10101 is more effective.

4. Conclusions

In the present study, the extracellular alkaline protease produced by Trametes cingulata strain CTM10101 was proven to have several novel properties of prominent industrial value, in particular, alkaline pH and temperature stability. Compared to commercial Flavourzyme® 500 L and Thermolysin type X proteases, SPTC showed high catalytic efficiency, better hydrolysis degree, and elevated tolerance to organic solvents as well as an excellent stability and compatibility with a wide range of commercialized detergents. Overall, the findings indicate that SPTC is bestowed with a number of promising properties that might open new opportunities for the synthesis of non-aqueous peptides and laundry detergent formulations. Further studies, some of which are currently underway in our laboratories, are needed to investigate the structure-function relationships of the enzyme using site-directed mutagenesis and 3-D structure modeling.

Acknowledgments

This work was in part supported by a grant from the Tunisian Ministry of Higher Education and Scientific Research contract program CBS-LMBEE/code: LR15CBS06_20152018, and the Tunisian-Algerian project JAOUADI/BADIS_TNDZ-MicrooZymes_20122016/code: TA/04/2012. 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.

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Page 27 of 46 We extend our thanks to Mrs. S. Kchaou (CTM Unit-CBS) for the gift of the filamentous fungi strain CTM10101 and Prof. H. Mejdoub and Mr. C. Bouzid (USCR/SP-FSS, Sfax Faculty of Science) for the sequencing of the NH2-terminale. Special thanks are also due to Prof. L. Mahfoudhi from the English Department at the Sfax Faculty of Science, University of Sfax (Sfax, Tunisia) for constructively proofreading and language polishing services.

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Page 34 of 46 [49] B. Jaouadi, N. Aghajari, R. Haser, S. Bejar, Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis, Biochimie 92 (2010) 360-369. [50] M. Ben Elhoul, N. Zaraî Jaouadi, H. Rekik, W. Bejar, S. Boulkour Touioui, M. Hmidi, A. Badis, S. Bejar, B. Jaouadi, A novel detergent-stable solvent-tolerant serine thiol alkaline protease from Streptomyces koyangensis TN650, Int. J. Biol. Macromol. 79 (2015) 871-882. [51] F. Abidi, J.-M. Chobert, T. Haertlé, M.N. Marzouki, Purification and biochemical characterization of stable alkaline protease Prot-2 from Botrytis cinerea, Process Biochem. 46 (2011) 2301-2310. [52] R. Sareen, U.T. Bornscheuer, P. Mishra, Synthesis of kyotorphin precursor by an organic solvent-stable protease from Bacillus licheniformis RSP-09-37, J. Mol. Catal. B-Enzym. 32 (2004) 1-5. [53] N. Annamalai, M.V. Rajeswari, S.K. Sahu, T. Balasubramanian, Purification and characterization of solvent stable, alkaline protease from Bacillus firmus CAS 7 by microbial conversion of marine wastes and molecular mechanism underlying solvent stability, Process Biochem. 49 (2014) 1012-1019. [54] G. Careri, Cooperative charge fluctuations by migrating protons in globular proteins, Prog. Biophys. Mol. Biol. 70 (1998) 223-249. [55] A.L. Serdakowski, J.S. Dordick, Enzyme activation for organic solvents made easy, Trends Biotechnol. 26 (2008) 48-54. [56] V.H. Raval, S. Pillai, C.M. Rawal, S.P. Singh, Biochemical and structural characterization of a detergent-stable serine alkaline protease from seawater haloalkaliphilic bacteria, Process Biochem. 49 (2014) 955-962.

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Page 35 of 46 [57] M. Venugopal, A.V. Saramma, Characterization of alkaline protease from Vibriofluvialis strain VM10 isolated from a mangrove sediment sample and its application as a laundry detergent additive, Process Biochem. 41 (2006) 1239-1243. [58] J. Singh, N. Batra, R.C. Sobti, Serine alkaline protease from a newly isolated Bacillus sp. SSR1, Process Biochem. 36 (2001) 781-785. [59] B. Jaouadi, S. Ellouz-Chaabouni, M. Ali, E. Messaoud, B. Naili, A. Dhouib, S. Bejar, Excellent laundry detergent compatibility and high dehairing ability of the Bacillus pumilus CBS alkaline proteinase (SAPB), Biotechnol. Bioproc. Eng. 14 (2009) 503512. [60] K. Bouacem, A. Bouanane-Darenfed, H. Laribi-Habchi, M.B. Elhoul, A. Hmida-Sayari, H. Hacene, B. Ollivier, M.-L. Fardeau, B. Jaouadi, S. Bejar, Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis, Int. J. Biol. Macromol. 81 (2015) 299-307. [61] F. Abidi, F. Limam, M.M. Nejib, Production of alkaline proteases by Botrytis cinerea using economic raw materials: assay as biodetergent, Process Biochem. 43 (2008) 12021208.

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Page 36 of 46 Figure legends

Fig. 1. Purification of the protease SPTC from Trametes cingulata strain CTM10101. (A) Chromatography of the protease SPTC on FPLC system using UNO S-1. The column (7 × 35 mm) (Bio-Rad Laboratories, , Inc., Hercules, CA, USA) was equilibrated with buffer C. Adsorbed material was eluted with a linear NaCl gradient (0 to 500 mM in buffer C at a flow rate of 30 ml/h, and assayed for protein content at 280 nm (○) and protease activity (●) as described in Section 2. (B) SDS-PAGE 10% of the purified protease. Lane 1, total cell extract. Lane 2, sample after ammonium sulfate fractionation (40-60%). Lane 3, purified SPTC (50 µg) obtained after UNO S-1 FPLC chromatography (fractions 31-37). Lane 4, protein markers. (C) Zymogram caseinolytic activity staining of the purified protease.

36

Page 37 of 46

37

Page 38 of 46 Fig. 2. Effects of pH on the activity (A) and stability (B) of the purified protease SPTC from Trametes cingulata strain CTM10101. The activity of the enzyme at pH 9 was taken as 100%. Effects of the thermoactivity (C) and thermostability (D) of SPTC. The enzyme was pre-incubated in the presence and absence of CaCl2 at temperatures ranging from 40 to 80 °C. The activity of the non-heated enzyme was taken as 100%.

38

Page 39 of 46 Fig. 3. Performance evaluation of the purified protease SPTC from Trametes cingulata strain CTM10101. (A) Hydrolysis curves of casein proteins treated with various purified enzymes. The purified proteases used were: SPTC (●), Flavourzyme® 500 L (∆), and Thermolysin type X (◊). (B) Stability of SPTC and Flavourzyme® 500 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. Vertical bars indicate standard error of the mean (n = 3). (C) Washing performance analysis test of SPTC in the presence of the commercial detergent Skip. (a) Cloth stained with blood washed with tap water; (b) blood-stained cloth washed with Skip detergent (7 mg/ml), (c) blood-stained cloth washed with Skip added with Flavourzyme® 500 L (commercial enzyme, 500 U/ml), (d) blood-stained cloth washed with Skip added with SPTC (500 U/ml). I: untreated cloths (control) and II: treated cloths.

39

Page 40 of 46

40

Page 41 of 46

Table 1 Flow sheet purification of SPTC from Trametes cingulata strain CTM10101.

Purification stepa

Total activity b

Crude extract

(units) × 10 675 ± 37

(NH4)2SO4 fractionation (40-60%)

4

Total protein b,c

Specific activity b

Activity recovery

Purification

rate (%) 100

factor (fold) 1

(mg) 450 ± 21

(U/mg of protein) 15000 ± 273

575 ± 28

187 ± 19

30749 ± 416

85

2.05

Heat treatment (30 min at 70 °C)

500 ± 24

93 ± 10

53763 ± 699

74

3.58

UNO S-1 FPLC

141 ± 16

15 ± 3

94000 ± 851

20

6.27

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 [30].

41

Page 42 of 46 Table 2 Alignment of the NH2-terminal amino acid sequence of the purified SPTC from Trametes cingulata strain CTM10101 with the sequences of other fungal proteases. Enzyme

Origin

NH2-terminal amino acida

SPTC (This work)

Trametes cingulata strain CTM10101

ALTTQTEAPWALGTVSHKGQAST

Serine protease Tvsp1 (AAO63588)

Trichoderma virens strain Gv29-8

ALTTQTGAPWGLGTV

87

Serine proteinase Prb1 (AAA34211)

Trichoderma hamatum strain LU593

ALTTQSGAPWGLGTV

80

Serine alkaline protease Protn (Q8TGV7)

Tolypocladium inflatum strain 29910

ALATQHGAPWGLGTV

73

Serine protease Alp2 (XM_001274134)

Aspergillus clavatus strain NRRL 1

ALTTQSGAPWGLGSISHK

72

Serine alkaline protease ES1 (ACX47962)

Aspergillus clavatus strain ES1

ALTTQSGAPWGLGSI

67

Alkaline serine protease (GU827714)

Aspergillus versicolor strain 46472

ALTTQSDAPWGLGAI

67

Alkaline protease Alp1 (AAT85626)

Aspergillus viridinutans strain MK246

SLTTQKGAPWGLGSI

64

Alkaline serine protease (AF413104)

Ophiostoma piceae strain 123-142

ALTTQSGSTWGLGTI

60

a

Identity (%)b

Amino acid sequences for comparison were obtained using the program BLASTP (NCBI,

NIH, USA) database. The protein ID is bracketed. b

Residues not identical with SPTC from Trametes cingulata strain CTM10101 protease are

indicated in black box.

42

-

Page 43 of 46 Table 3 Effects of various inhibitors, reducing agents, and metallic ions on SPTC 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 9 at 60 °C. Inhibitor/reducing agent/metallic ions

Concentration

None

–

100 ± 2.5

PMSF

5 mM

0 ± 0.0

DFP

5 mM

0 ± 0.0

SBTI

3 mg/ml

101 ± 2.5

TLCK

1 mM

105 ± 2.6

TPCK

1 mM

107 ± 2.6

10 mM

109 ± 2.7

-ME

5 mM

95 ± 2.4

LD-DTT

1 mM

90 ± 2.2

MIA

50 µM

97 ± 2.4

DTNB

10 mM

102 ± 2.5

EPNP

5 mM

92 ± 2.2

NEM

2 mM

101 ± 2.5

Iodoacetamide

5 mM

99 ± 2.5

Leupeptin

50 µg/ml

94 ± 2.3

Pepstatin A

10 µg/ml

98 ± 2.5

1,10-Phenanthroline monohydrate

10 mM

90 ± 2.2

EDTA

10 mM

93 ± 2.3

EGTA

1 mM

91 ± 2.2

2+

Ca (CaCl2)

2 mM

219 ± 5.5

Mn2+ (MnCl2)

2 mM

170 ± 3.7

2 mM

108 ± 2.6

Ba (BaCl2)

2 mM

79 ± 1.9

Cu2+ (CuCl2)

Benzamidine

2+

Mg (MgCl2) 2+

2 mM

63 ± 1.6

2+

2 mM

110 ± 2.6

2+

Co (CoCl2)

2 mM

99 ± 2.5

Fe2+ (FeCl2)

2 mM

81 ± 1.9

2 mM

0 ± 0.0

2 mM

100 ± 2.5

Zn (ZnCl2)

Ni2+ (NiCl2), Cd2+ (CdCl2), Hg2+ (HgCl2) +

+

+

Li (LiSO4), K (KCl), Na (NaCl) a

Residual activity (%)a

Values represent means of four independent replicates, and ± standard errors are reported.

43

Page 44 of 46 Table 4 Substrate specificity profile of SPTC from Trametes cingulata strain CTM10101. Concentration

Absorbance (nm)a

Relative activity (%)b

Casein

30 g/l

660

100 ± 2.5

Gelatin

30 g/l

660

91 ± 2.2

BSA

30 g/l

660

83 ± 2.1

Myoglobin

30 g/l

660

75 ± 2.0

30 g/l

660

66 ± 1.7

Hemoglobin

30 g/l

660

60 ± 1.6

Keratin

30 g/l

660

55 ± 1.4

Azo-casein

25 g/l

440

100 ± 2.5

Azoalbumin

25 g/l

440

94 ± 2.3

Keratin azure

25 g/l

440

65 ± 1.7

Collagen type I

10 mg/ml

490

0 ± 0.0

Collagen type II

10 mg/ml

490

0 ± 0.0

BTEE

10 mM

253

100 ± 2.5

ATEE

10 mM

253

96 ± 2.4

BAEE

10 mM

253

0 ± 0.0

BCEE

10 mM

253

0 ± 0.0

TAME

10 mM

253

0 ± 0.0

Suc-(Ala)2-Val-Ala-pNA

5 mM

410

100 ± 2.5

Suc-(Ala)2-Pro-Phe-pNA

5 mM

410

97 ± 2.4

Suc-Phe-(Ala)2-Phe-pNA

5 mM

410

96 ± 2.4

Suc-(Ala)2-Pro-Met-pNA

5 mM

410

80 ± 1.9

Suc-(Ala)2-Pro-Leu-pNA

5 mM

410

61 ± 1.6

Suc-Ala-Pro-Ala-pNA

5 mM

410

51 ± 1.4

Suc-(Ala)2-Phe-pNA

5 mM

410

38 ± 1.2

Suc-(Ala)2-Val-pNA

5 mM

410

13 ± 1.0

Suc-(Ala)3-pNA

5 mM

410

10 ± 1.0

Suc-Tyr-Leu-Val-pNA

5 mM

410

0 ± 0.0

Substrate Natural protein

Ovalbumin

Modified protein

Ester

Synthetic Peptide

a

Values represent means of three independent replicates, and ± standard errors are reported.

b

The activity of each substrate was determined by measuring absorbance at specified wave

lengths according to the relative method reported elsewhere [33].

44

Page 45 of 46 Table 5 Kinetic parameters of purified proteases: SPTC, Flavourzyme® 500 L, and Thermolysin type X for hydrolysis of natural protein (casein) and synthetic peptide (AAVA).

Vmax (× 103 U/mg)

kcat (× 103 min-1)

kcat/Km (× 103 min-1 mM-1)

Substrate

Enzyme

Km(mM)

Casein

SPTC

0.475 ± 0.02

94.075 ± 856

62.717

132.036

Flavourzyme® 500 L

0.546 ± 0.04

47.125 ± 566

31.417

57.540

Thermolysin type X

0.613 ± 0.05

20.764 ± 323

13.843

22.582

Suc-(Ala)2-

SPTC

0.735 ± 0.06

285.561 ± 997

190.374

259.012

Val-Ala-pNA

Flavourzyme® 500 L

1.093 ± 0.09

90.126 ± 701

60.084

54.971

Thermolysin type X

1.277 ± 0.10

42.138 ± 511

28.098

21.998

Values represent means of three independent replicates, and ± standard errors are reported.

45

Table 6 Effects of organic solvents on the activity and stability of the purified SPTC and Thermolysin type X proteases. The non-treated enzyme was considered as 100%. The activity is expressed as a percentage of activity level in the absence of organic solvents.

Organic solvent

Log P

(50%, v/v)

Relative activity (%)

Residual activity (%)

SPTC

SPTC

Thermolysin type X

Thermolysin type X

None

–

100 ± 2.5

100 ± 2.5

100 ± 2.5

100 ± 2.5

N-Hexadecane

8.8

111 ± 2.6

104 ± 2.5

103 ± 2.5

96 ± 2.4

N-Decane

5.6

107 ± 2.6

97 ± 2.4

99 ± 2.5

78 ± 2.0

N-Octane

4.5

123 ± 3.5

130 ± 3.6

109 ± 2.6

110 ± 2.6

N-Heptane

4.0

105 ± 2.5

144 ± 3.7

85 ± 2.1

130 ± 3.6

N-Hexane

3.5

155 ± 4.1

147 ± 4.0

120 ± 3.4

135 ± 3.6

Cyclohexane

3.2

230 ± 6.1

140 ± 3.7

189 ± 4.8

115 ± 2.6

Toluene

2.5

106 ± 2.6

87 ± 2.1

82 ± 2.1

66 ± 1.8

Chloroform

2.0

70 ± 2.0

105 ± 2.5

56 ± 1.6

80 ± 2.1

1-Hexanol

1.8

109 ± 2.6

70 ± 2.0

82 ± 2.3

50 ± 1.4

1-Buthanol

0.88

170 ± 4.5

60 ± 1.8

135 ± 3.6

45 ± 1.3

Ethyl acetate

0.73

130 ± 3.6

0 ± 0.0

105 ± 2.5

0 ± 0.0

Isopropanol

0.28

89 ± 2.3

106 ± 2.6

65 ± 1.8

88 ± 2.1

Isopropyl alcohol

0.05

121 ± 3.4

105 ± 2.5

101 ± 2.5

92 ± 2.4

Acetonitrile

- 0.15

0 ± 0.0

0 ± 0.0

0 ± 0.0

0 ± 0.0

Ethanol

- 0.24

93 ± 2.4

86 ± 2.1

76 ± 2.0

73 ± 2.0

Methanol

- 0.76

63 ± 2.6

90 ± 2.4

45 ± 1.3

79 ± 2.1

DMF

- 1.03

146 ± 4.0

127 ± 3.5

119 ± 3.4

117 ± 2.6

DMSO

- 1.35

115 ± 2.6

150 ± 4.0

95 ± 2.4

131 ± 3.6

Values represent means of three independent replicates, and ±standard errors are reported.

46