Accepted Manuscript Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum José Manoel Wanderley Duarte Neto, Maria Carolina de Albuquerque Wanderley, Carolina de Albuquerque Lima, Ana Lúcia Figueiredo Porto PII: S1046-5928(17)30496-5 DOI: 10.1016/j.pep.2018.01.016 Reference: YPREP 5222 To appear in: Protein Expression and Purification Received Date: 10 August 2017 Revised Date: 31 January 2018 Accepted Date: 31 January 2018 Please cite this article as: José.Manoel.Wanderley. Duarte Neto, M.C.d.A. Wanderley, C.d.A. Lima, Ana.Lú.Figueiredo. Porto, Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum, Protein Expression and Purification (2018), doi: 10.1016/ j.pep.2018.01.016. 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. ACCEPTED MANUSCRIPT Single step purification via magnetic nanoparticles of new broad pH active protease from Penicillium aurantiogriseum José Manoel Wanderley Duarte Neto1; Maria Carolina de Albuquerque 1 RI PT Wanderley1, Carolina de Albuquerque Lima2; Ana Lúcia Figueiredo Porto1,3(*). Laboratorio de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco - UFPE, Av. Prof. Moraes Lins do Rego, s/n, 50670-901, Recife, PE, Brasil 2 Faculdade de Ciência, Educação e Tecnologia de Garanhuns, Universidade de M AN US C Pernambuco - UPE, Av. Capitão Pedro Rodrigues, n° 105, Garanhuns, PE, Brasil 3 Departmento de Morfologia e Fisiologia Animal, Universidade Federal Rural de Pernambuco - UFRPE, Av. Dom Manoel de Medeiros, s/n, 52171-900, Recife, PE, Brasil (*) Corresponding author: Address: Departmento de Morfologia e Fisiologia Animal, Universidade Federal Rural D de Pernambuco - UFRPE, Av. Dom Manoel de Medeiros, s/n, 52171-900, Recife, PE, TE Brasil. Tel.: +55 81 21012504 – Fax: +55 81 21268485 AC C EP E-mail address: analuporto@yahoo.com.br ACCEPTED MANUSCRIPT Abstract A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a RI PT single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1 M NaCl solution) and eluted 3 times (1 M NaCl M AN US C solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40 kDa on SDS-PAGE and optimum activity at 45 °C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55 °C, did not lose any activity over 48 h at 25 °C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative D purification method and novel broad pH tolerant protease. Key-words: Wide pH active protease, Polyaniline, Magnetite, Proteolytic enzyme, AC C EP TE Fungi. ACCEPTED MANUSCRIPT 1. Introduction Proteases have many industrial applications, they constitute the most important group of industrial enzymes and are capable of catalyzing peptide bond hydrolysis. Extracellular proteases are fundamentally related to catalyze the protein hydrolysis into peptides or amino acids for cellular absorption. Proteases are responsible for over 65% applicable, as constituent in detergents, food RI PT of the total world enzyme market [1,2]. They are versatile and largely industrially processing, leather industry, biotechnological and pharmaceutical products. This broad set of use and requirement in industries have motivated research on novel proteases with different properties, which may favor the development of its products preparation, storage and employment [3]. A M AN US C new set of applications can be achieved by applying proteases with high activity over a wide range of pHs and temperatures in a variety of industrial, medical and environmental areas. Thus, there is a growing industrial requirement for potential pH and temperature tolerant proteases [4]. The preference for microbial sources of proteases are based on its biochemical diversity and genetic manipulation possibility. Among microorganisms, filamentous fungi present several advantages such as high productivity at low cost, fast development, and the enzyme may be modified and recovered more easily. In addition, D enzyme production by filamentous fungi occurs extracellularly, which facilitates TE recovery [5]. The Penicillium aurantiogriseum is considered one of the most common fungal specie on cereals with worldwide occurrence, besides is not a classic human EP pathogenic fungi [6]. This filamentous fungus can be commonly found in soils of Pernambuco – Brazil and belong to a very studied taxa with saprophytic habitus and few nutritional requirements [7]. AC C In an industrial scale, the cost of purification is critical [8]. Traditional purification methods eventually involve steps of centrifugation, saline precipitation, ultrafiltration, successive chromatographic steps, dialysis and final product concentration. These many steps makes the downstream process time-consuming, difficult to scale up and require some expensive reagents and equipment, resulting in high costs and often loss in the process yield [9]. The mainly used purification strategies using affinity chromatography resins can make the process difficult to scale up due to its relative high price [8]. As an alternative to traditional chromatography, magnetic nanoparticles have been successfully used for rapid and scalable purification, particularly for biological samples [10]. ACCEPTED MANUSCRIPT Magnetic separation techniques may replace filtration and centrifugation by applying a magnetic field which carries out solid-liquid separation in less than 20 s [11]. Different nanoparticles compositions, coating and functionalization strategies can be used for efficient purification technics, using polymers, biomolecules, silica, metals, etc. [12]. Casein is the main group of proteins in milk, accounting for 80% of total milk RI PT proteins [13]. Caseins have many applications, including coating and binding agent, frequently been used as biopolymer for coating materials, presenting great potential in many applications including biocompatibility, multi-functional surfaces, anti-fouling and biodegradability [14]. In this work, magnetite nanoparticles coated with polyaniline were synthesized M AN US C which served as support for azocasein immobilization. This conjugate was used in the single step purification of the protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. 2. Material and Methods 2.1.Chemicals Azocasein protease substrate, Ammonium sulphate, TCA (trichloroacetic acid), D 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 6- TE hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), Glutaraldehyde, Iron (II) chloride tetrahydrate, Iron (III) chloride hexahydrate, were purchased from Sigma EP Chemicals Co. (St. Louis, MO, USA). All other chemicals were reagent grade and purchased from Merck (Darmstadt, Germany). AC C 2.2.Microorganism and storage conditions The Penicillium aurantiogriseum Dierchx was obtained from the University Recife Mycology (URM 4622 strain), inscribed in the Commonwealth Mycological Institute (CMI) and affiliated to World Federation for Culture Collections (WFCC). The strain was stored at 4 °C in a malt extract agar medium, consisting of 0.5% (w/v) malt extract, 0.1% (w/v) peptone, 0.5% (w/v) glucose and 1.5% (w/v) agar. 2.3.Protease production ACCEPTED MANUSCRIPT The inoculum of spores was produced in agar plates containing a cell culture grown for 5 days at 28 °C and then suspended in 3 mL 0.9% (w/v) NaCl and 0.01% (v/v) Tween 80 solution, previously sterilized at 121 °C for 20 min. The medium used for protease production was soybean flour medium [15] composed of 1.65% (w/v) filtered soybean flour, 0.1% (w/v) NH4Cl, 0.06% (w/v) RI PT MgSO4.7H2O, 0.435% (w/v) K2HPO4, 0.01% (w/v) glucose and 1.0% (v/v) mineral solution, pH 7.21. Mineral solution was prepared by adding in 100 mL of distilled water: 100 mg FeSO4·7H2O; 100 mg MnCl2·4H2O; 100 mg ZnSO4·H2O and 100 mg CaCl2·H2O. These mediums were sterilized in an autoclave at 121 °C for 20 min. After spores inoculation (106 spores/mL) in soybean flour medium (50 mL), M AN US C fermentations were carried out at 24 °C and 200 rpm in 250 mL Erlenmeyer flasks. The broth obtained after fermentation (72 h) was vacuum filtered through 0.45 µm pore diameter nitrocellulose membranes (Toyo Ink) to remove mycelia. Protein concentration and protease activity were established in the filtrated (from now on called as crude extract) since protease target was extracellular. 2.4.Sample Preparation The crude extract was concentrated by ammonium sulphate precipitation at 0-20, D 20-40, 40-60, 60-80, 80-100% saturation under 4 °C, over 2 h, and centrifuged at 11000 TE g for 15 min. The obtained precipitate was dissolved in 3 mL 0.05 M Tris-HCl buffer (pH 7.5), dialyzed against distilled water at 4 °C and the fraction with higher specific EP activity was diluted and stored for further use in purification experiment. 2.5.Magnetic Nanoparticles Synthesis AC C The nanoparticles synthesis methodology [16] was modified and optimized for the used protease. 5 mL of 1.1 M FeCl3 .6H2O and of 0.6 M FeCl2 .4H2O were added to 50 mL of distilled water. Slowly, 5.0 M NaOH was added dropwise, under agitation, until pH 10.0 was reached and black particles precipitates were produced. The mixture, under vigorous stirring, was kept on 50 °C for 30 min. Then the magnetic particles obtained were thoroughly washed with distilled water until pH 7.0 was reached. The nanoparticles recovering occurred always through magnetic field throughout this work. After dried up at 50 °C, the material was powdered and stored at room temperature (approximately 25 °C). ACCEPTED MANUSCRIPT The coating of magnetic particles was performed by adding 0.5 g of nanoparticles to 50 mL of 0.1 M KMnO4 solution at 25°C for 1 h of gently stirring. It was continuously washed with distilled water until clear. Magnetic-KMnO4 nanoparticles were immersed into 50 mL of 0.5 M aniline solution prepared in 1.0 M HNO3. Polymerization was led to happen at 4 °C for 1 h of gently stirring and magnetic RI PT nanoparticles coated with polyaniline (mPANI) were extensively washed with distilled water, 0.1 M citric acid and distilled water. Finally, the mPANI was dried up at 50 °C and stored at room temperature (approximately 25 °C) until used. 2.6.Azocasein Immobilization M AN US C Azocasein was used for its higher solubility and the coloring radical presence. In each tube, 10 mg of mPANI nanoparticles were activated with 1 mL of 2% (v/v) glutaraldehyde solution for 7 hours and washed 10 times with distilled water. Activated magnetic nanoparticles were previously washed three times with 0.2 M Tris-HCl buffer (pH 9.0) and mixed with 1% (w/v) azocasein solution for immobilization over 2 hours of gentle stirring. Then, nanoparticles were incubated with 0.1 M glycine solution for 30 2.7.Protease Purification D min and washed 2 times with distilled water. TE After sample preparation, it was incubated with the azocasein immobilized mPANI nanoparticles at room temperature (25 °C) on an orbital shaker. After 30 min of EP incubation, nanoparticles were separated using a magnet and the adsorption supernatant were stored. Then the adsorbents were washed three 3 times with 0.2 M Tris-HCl buffer (pH 9.0) and eluted 7 times using 0.2 M Tris-HCl buffer (pH 9.0) containing 0.1 M AC C NaCl. Then, the adsorbents were further eluted 3 times using 0.2 M Tris-HCl buffer (pH 9.0) containing 1 M NaCl, these final elutions were considered purified protease. All eluates were collected in every elution step dialyzed and analyzed by SDS-PAGE, and had its protein and protease activity measured. The nanoparticles were used alone, without the azocasein immobilization, on the purification process as negative control. 2.8.Derivative purification reuse After purification, the azocasein immobilized nanoparticles were repeatedly washed using the 1 M NaCl solution, rewashed using 0.2 M Tris-HCl buffer (pH 9.0) ACCEPTED MANUSCRIPT and reapplied in purification process. The first protease activity was compared with the subsequent ones to estimate the amount of protease purified [17]. 2.9.Protein concentration and protease activity Protein concentration was determined by bicinchoninic acid method, using RI PT bovine serum albumin as standard, using PierceTM BCA Protein Assay Kit [18]. In purification process measurements, the enhanced protocol was chosen due to low concentration samples, following manufactures instructions. Protease activity was measured using 1% (w/v) azocasein as substrate in 0.2 M Tris-HCl buffer (pH 7.5) [19]. One unit of protease activity (U) was defined as the amount of enzyme producing an 2.10. M AN US C increase of one unit in the optical density at 440 nm in 1 h. Optimal pH and stability Optimum pH for purified protease activity was established measuring its activity in the pH range of 5.0-11.0 using 1% (w/v) azocasein in buffer systems of 0.05 M citrate (pH 3.0 ~ 6.0), 0.05 M Tris-HCl (pH 7.0 ~ 9.0), and 0.05 M carbonatebicarbonate (pH 10.0 ~ 11.0). pH stability studies were performed by pre-incubating the purified protease in selected pH buffer (pH 6, 9 and 11) at 45 °C for 1, 3, 6, 12 and 24 h. D After each time interval, one aliquot was withdrawn and subjected to enzyme activity Optimal temperature and stability EP 2.11. TE analysis using substrate diluted in optimum buffer pH 9, determined by previous assay. Optimum protease temperature was determined by incubating the reaction mixture at different temperatures (15-65 °C) in 0.2 M Tris-HCl buffer (pH 9) and AC C measuring protease activity. Thermal stability was monitored after enzyme incubation for 12 and 24 h at 25 and 55 °C and proceed to residual activity analysis under optimum experimental conditions (pH 9, temperature 45 °C). 2.12. Effect of protease inhibitors Influence of five different protease inhibitors was investigated: ethylenediaminetetraacetic acid (EDTA), Pepstatin A, phenylmethylsulphonyl fluoride (PMSF), iodoacetic acid (IAA) and 2-Mercaptoethanol. The procedures of manufacturer’s guide of inhibitors were followed. The protease was incubated for 30 ACCEPTED MANUSCRIPT min at 45 °C in 50% (v/v) solution with respective inhibitor at 10 mM concentration and residual activity was determined as the percentage of proteolytic activity of an inhibitorfree control sample. 2.13. SDS-PAGE RI PT Purity of the enzyme was analyzed by 15% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) [20]. Electrophoresis gel was loaded with 10 µL of concentrated enzyme by lyophilization, and subject to electrophoresis at a constant current of 100 V. The molecular mass of the enzyme was determined using a M AN US C known standard protein molecular weight marker. The used molecular markers were phosphorylase b (97.0 kDa), bovine serum albumin (66.0 kDa), ovalbumin (54.0 kDa), carbonic anhydrase (30.0 kDa), trypsin inhibitor (20.1 kDa) and α- lactalbumin (14.4 kDa). Gel was stained with silver nitrate, adapted method [21]. 3. Results and Discussion 3.1.Sample Preparation D P. aurantiogriseum protease production was done in cost-effective medium, increasing industrial applicability of this process. Considering that, enzyme production TE should use inexpensive medium to be more economically viable, like soybean flour [15]. The crude extract was submitted to protein precipitation using different EP concentrations of ammonium sulphate. Approximately 3 mL was obtained from each 50 mL of Crude Extract to each saturation faction. Table 1 shows the results for precipitation fractions and the 60-80% fraction was the one with higher activity and AC C protein concentrations, almost 6 folds more concentrated than crude extract. After precipitation, the 3 mL 60-80% fraction was dialyzed over water and then diluted up to 10 mL for application on protease purification system. 3.2.Protease purification After applying the samples through the purification procedure, the results of volumetric protease activity and protein concentration (using enhanced protocol) of every elution step were summarized in Figure 1. Through this procedure a yield of approximately 785 µg of purified protease from 50 mL of crude extract was obtained, mixing the first two elutions using NaCl 1 M solution. The sample applied in this ACCEPTED MANUSCRIPT procedure, presented a specific activity of 19.02 U/mg and the first 1 M NaCl elution presented 1059.18 U/mg. The produced protease was purified 55.68-fold with an activity recovery of 46% through this process, high purification results when compared to other similar processes and other purifications strategies described in literature [22,23]. In addition, the process does not require any complex machine, skilled labor, RI PT expensive column or resin, making it easier, faster, cheaper and as efficient or even more than traditional methods of chromatography and HPLC [4,24–27]. The control nanoparticles, without azocasein immobilized, when used in purification process, could recover a purified protease activity of 9% (±3.2) of activity obtained using azocasein immobilized nanoparticles. This small activity could be M AN US C associated to small rate of protease adsorption to the mPANI nanoparticles surface that stayed through the washing process and/or protease unleash from glutaraldehyde unattached. Although, the azocasein immobilization is crucial to the purification process. Figure 2 shows the silver stained SDS-PAGE pattern of purification after elution steps. In the first line is visible a large number of bands that corresponds to proteins available in the soybean flower medium and produced by the P. aurantiogriseum. The next 3 lines shows fewer bands over successive elutions using 0.1 M NaCl solution and D the last two lines corresponds to the 2 first elutions using 1 M NaCl solution. That TE single strong band, with approximately 40 kDa correspond to the step 10 of the Figure 1, indicating that it is the molecular weight of targeted protease. This result is similar to EP the alkaline protease produces by Penicillium nalgiovense, with 45.2 kDa [26]. Results found in literature about protease purification technics are vast and diversified. Although comparative studies between chromatography and magnetic AC C particles technics are rare, some studies show several advantages in using magnetic particles over chromatography, beyond cost and practicality, its efficiency [8]. The expressive purification results achieved in this work corroborate with the effectiveness of enzyme purification methods using magnetic particles, in view of its quality and advantages when compared to traditional methods like chromatography. Besides, there is few, or none, reports of protein substrate immobilization for purification purposes. 3.3.Derivative Purification Reuse ACCEPTED MANUSCRIPT Azocasein presents 23.6 kDa of molecular weight. The absence of this band on the purified protease lines (5 and 6) of Figure 2 confirms that the substrate remained bounded to the support after the protease elution with NaCl 1 M solution. Nevertheless, Figure 3 shows the purified protease activity obtained after 3 consecutive cycles of purification using the same particles. In the first reuse, the purified protease activity RI PT obtained is more than 70% of first purification cycle, but in the third cycle, the purified protease relative activity is little more than 20%. Although the derivative can be reused maintaining 70% of its purification potential, the significant loss of purification potential on the second reuse could be associated to degradation of the immobilized substrate by consecutive contact with the protease. In addition to the fact that the used M AN US C particles are inexpensive, enabling applicability and scaling up, the particles exhibited a high purification potential rate after the first reuse, contributing to cost reduction. 3.4.pH effect on protease activity and stability The effect of pH on protease activity on azocasein was investigated in the range of 5.0-11.0. Figure 4A shows the effect of pH on the protease activity. The protease showed a relative activity not less than 80% from pH 6.0 to 11.0 and showed highest activity at pH 9.0, presenting a high activity in a broad range of pH, especially at D alkaline pH. The protease still showed more than 40% relative activity on pH 5.0, but TE the loss of protease activity can be also associated with the less solubility of casein at acid pH. This protease activity on broad pH spectrum was not observed in previous EP work with collagenase from P. aurantiogriseum, but presented the same optimum pH [28]. Other Penicillium species presented alkaline active proteases, but did not present broad pH activity [1,29]. Besides the optimum pH at 9.0, the present protease presented AC C similar characteristics to those produced by Penicillium sp. on defatted soybean cake by solid state fermentation, stable in the pH range from 6.0 to 9.0. [30] The protease storage pH stability was investigated using optimum pH (pH 9.0) and the pH 6.0 and 11.0, in which the protease presented not less than 80% relative activity in the previous assay. Figure 4B shows the effect of pH on protease stability during 24 h of incubation at 4 °C, selected as a cold storage reference temperature. The protease presented a high stability in all studied pH, maintaining its original activity over 24 h. This remarkable results are comparable with results from Bacillus proteases, traditionally used for detergents [4]. This unusual stability for Penicillium proteases is ACCEPTED MANUSCRIPT useful for different storage conditions and composition of a variety of solutions, like soaps and detergents. 3.5.Effect of temperature on protease activity and stability The effect of temperature on protease activity on azocasein was investigated in RI PT the range of 15-65 °C. Figure 5A shows the effect of temperature on the protease activity. It presented a wide bell shape, with a relative activity over 48% from 15 to 55 °C. The protease presented highest activity at 45 °C. This results are similar to the ones found by Lima et al. (2013) with P. aurantiogriseum collagenase, which presented the same optimum temperature, at 45 °C, and a similar shape on the chart, using the same M AN US C production medium [31]. And yet again to those found by Germano et al. (2003) using soybean defatted soybean cake by solid state fermentation [30]. The protease stability to different storage temperatures was investigated using optimum temperature, 45 °C, and room temperature, 25 °C. Figure 5B shows the effect of temperature on protease stability during 48 h of incubation at optimum pH 9.0. Interestingly, the protease showed an activity loss of 77.56% in its optimum temperature, 45 °C, after 12 h. Although, it kept all its activity under room temperature D (25 °C) over 48h, indicating that it can support long hours unrefrigerated. TE 3.6.Effect of inhibitors on protease activity Proteases can be categorized based on amino acids involved in their active sites EP such as aspartic, serine and cysteine, or as metalloprotease if its catalytic activity require a metal ion [2]. Figure 6 shows protease relative activity submitted to each inhibitor compared with the control, where just buffer solution was added. The enzyme activity AC C was fully abolished by PMSF, indicating that the enzyme belongs to the serine protease class. PMSF blocks the active site of those proteases by sulfonating the essential serine residue, resulting in complete inhibition of protease activity. EDTA is chelating agent of metal ions and the as it had no inhibition on protease activity it suggests that this protease does not have metal ions requirement for its stability nor activity. The protease presented a small reduction of 10% of relative activity when 2-mercaptoethanol was added, indicating its stability in presence of reducing agents [32]. Withal, IAA is an irreversible inhibitor of all cysteine peptidases, the protease showed no inhibition by it, seeming to have no activity site related cysteine residues. ACCEPTED MANUSCRIPT Pepstatin A inhibited the enzyme activity by about 55%. The pepstatin usually inhibits acid proteases and its inhibition is associated with aspartic residues on protease active site [33]. The 55% relative activity inhibition suggests that aspartic residues are involved in active site of this protease and it can be associated with its acid pH activity, although it has alkaline optimum activity pH. It seems that it is an aspartic active site RI PT serine protease. 4. Conclusion The fungus P. aurantiogriseum URM4622 was able to produce large amount of extracellular protease that was functional active over a broad range of pH (6.0-11.0) and M AN US C stable at room temperature using an inexpensive soybean flour medium and purification process with magnetic nanoparticles. The alternative purification process, using azocasein immobilized PANI coated magnetic nanoparticles, was validated as costeffective and efficient, achieving a 55-fold purification degree from a full concentrated interfering protein medium. The protease presented an optimum activity temperature at 45 °C and maintained half of it activity from 15 to 55 °C. It did not lose any percentage of activity over 48 h at 25 °C. It presented optimum activity at pH 9.0 and kept more than 60% of activity from pH 6.0 to 11.0. Was 100% inhibited by PMSF and 55% D inhibited by Pepstatin A, suggesting a serine protease with aspartic residues on its active TE site, although several additional studies should be done. The protease has demonstrated significant proteolytic activities, stability, ease of purification, and low-cost production EP results, which is promising for industrial applications and report a novel fungal strain that produces a broad pH tolerant protease that contribute to current knowledge on the AC C biodiversity of protease producing fungi from Brazil. 5. Acknowledgments This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), process 306563/2011-8, and Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE). J.M.W. Duarte Neto is very grateful to CAPES for his PhD’s degree fellowship. ACCEPTED MANUSCRIPT 6. References [1] E.R. 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(Tokyo). 23 AC C (1970) 259–62. http://www.ncbi.nlm.nih.gov/pubmed/4912600. AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT 5 80 4 60 3 40 2 20 1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 D 0 0.15 40 0.1 30 20 0.05 10 0 7 0 8 9 10 11 12 13 Elution Step TE Elution Step 50 0.2 SC 100 60 Protease Activity (U/mL) Protease Activity (U/mL) 6 M AN U 7 120 70 B Protein Concentation (mg/mL) 8 140 Protein Concentation (mg/mL) A RI PT ACCEPTED MANUSCRIPT Figure 1: (A) Protease activity and protein concentration of eluates during Penicillium aurantiogriseum protease purification process. (B) Last EP steps of elution from 8 to 12 enlarged for clarity. (○) indicate protease activity and (●) indicate protein concentration. Step 1: Fraction 60-80% isolation from other proteins. AC C used for purification, Step 2: Adsorption supernatant, Steps 3-9: 0.1 M NaCl eluates, Steps 10-12: 1 M NaCl eluates. Evidencing target enzyme AC C EP TE D M AN US C RI PT ACCEPTED MANUSCRIPT Figure 2: Silver stained SDS-PAGE patterns of purification of Penicillium aurantioriseum protease elution steps. Line 1: Unpurified protease sample; Lines 2 - 4: 1st, 4th and 7th 0,1M NaCl eluates, respectively; Lines 5 - 6: 1st and 2nd 1M NaCl eluates, respectively. Purified protease bands highlighted by arrows (approximately 40 kDa), evidencing target enzyme isolation from other proteins. RI PT ACCEPTED MANUSCRIPT 100 SC 80 M AN U 70 60 50 40 D 30 TE Purified Protease Relative Activity (%) 90 20 1 AC C 0 EP 10 2 3 Purification Cycles Figure 3: Purified protease relative activity on consecutive purifications cycles using the same derivatives. First particles protease purification reuse (2nd Purification Cycle) presenting 71.35% its original purification potential and second reuse (3rd Purification Cycle) presenting 21.9%. 100 SC 80 B M AN U 100 60 D 40 0 5 6 7 8 pH 9 10 11 EP 4 TE 20 Relative Activity (%) A Relative Activity (%) RI PT ACCEPTED MANUSCRIPT 80 60 pH 6 40 pH 9 pH 11 20 0 12 0 3 6 9 12 15 18 21 24 Time (h) AC C Figure 4: (A) Effect of pH on protease activity. Highest protease activity was observed at pH 9.0. (B) Effect of pH on protease stability with time, expressed as percentage of the first activity obtained at pH 9.0 and time zero, evidencing protease high stability in all studied pH. B 100 80 Relative Activity (%) 80 SC 100 M AN U A Relative Activity (%) RI PT ACCEPTED MANUSCRIPT 60 40 D 20 10 15 20 25 30 35 40 45 50 55 60 EP Temperature (°C) TE 0 65 70 25 °C 60 45 °C 40 20 0 0 3 6 9 12 15 18 21 24 Time (h) Fig. 5: (A) Effect of temperature on protease activity. Highest protease activity was observed at 45 °C. (B) Effect of temperature on protease AC C stability through time, expressed as percentage of the maximum one obtained at 45 °C at time zero, evidencing highest protease stability in 25 ºC. RI PT ACCEPTED MANUSCRIPT 100 SC 60 M AN U Relative Activity (%) 80 40 TE D 20 0 EDTA Peps. A PMSF IAA B-Merc. EP Control Figure 6. Penicillium aurantiogriseum purified protease relative activity using different protease inhibitors as a percentage of control activity. AC C Ethylenediaminetetraacetic acid (EDTA) and iodoacetic acid (IAA) presented no inhibition, 2-Mercaptoethanol (B-Merc) presented 10.55 % inhibition, Pepstatin A (Peps A) presented 55.08 % inhibition and phenylmethylsulphonyl fluoride (PMSF) presented complete inhibition. ACCEPTED MANUSCRIPT Tables Table 1. Total protein concentration and protease activity of Penicillium Proteins (µg/mL) 215.5 (±11) 221.6 (±25) 128.7 (±7) 474.8 *(±3) 1943.2 (±13) 164.8 (±10) C.E. Fr. 0-20 % Fr. 20-40 % Fr. 40-60 % Fr. 60-80 % Fr. 80-100 % AC C EP TE D M AN US C *the best results are shown in boldface. Protease Activity (U/mL) 188.9 (±5) 43.2 (±2) 69.4 (±3) 157.78 (±1) 1125.5 (±7) 156.11 (±8) RI PT aurantiogriseum crude extract (C.E.) and ammonium sulphate precipitation fractions* ACCEPTED MANUSCRIPT Highlights Magnetic nanoparticles have been used for low cost, rapid and scalable purification. Azocasein was immobilized on nanoparticles and applied for protease purification. The protease was purified 55.68-fold, retaining 46% of original activity. Protease had optimum activity at 45 °C and pH 9.0 and was stable from pH 6.0 to 11.0. AC C EP TE D M AN US C RI PT Inhibitors assay suggested that is a serine protease with aspartic residues.