Campus Monterrey
School of Engineering and Sciences
Strategies for the primary recovery of bioactive molecules using Aqueous Two-Phase Systems
A thesis presented by
Daniela Enriquez Ochoa
Submitted to the
School of Engineering and Sciences
in partial fulfillment of the requirements for the degree of
Master of Science In Biotechnology
Monterrey Nuevo León, June 11th, 2020
This thesis was done in the FEMSA Biotechnology Center of Tecnologico de Monterrey, Monterrey Campus under the guidance of Dr. Karla Patricia Mayolo Deloisa with support of Translational Omics Focus Group from Tecnologico de Monterrey and Consejo Nacional de Ciencia y Tecnología (CONACyT) through scholarship 712327.
To my parents who always told me to dream big
The completion of this thesis would not have been possible without a great number of people.
Some of them, however, deserve special mention:
My supervisor Dr. Karla Mayolo Deloisa who has given me unfailing support and guided me through graduate life.
My co-advisor Dr. Judith Zavala Arcos who introduced me to research a couple of years ago.
My mom who has always encouraged me to further my horizons.
My dad who has been there unconditionally.
Calef because you taught me too many things.
Pedro and Alán for the good times and invaluable (hopefully long-life) friendship.
Clarisa because you have provided me unconditional friendship.
Cristina, Gabriela, Claudia, Alejandro and Martín for reminding me there is life outside the lab.
Aqueous Two-Phase Systems by
Daniela Enriquez Ochoa
Abstract
Nature has historically been a source of a wide variety of bioactive molecules. They play a relevant role in many fields, such as the pharmaceutical, agrochemical, biotechnological, cosmetic and food industries. Almost half of the products in the drug market are either bioactive natural molecules or derived from them. Appropriate extraction and isolation steps are crucial for downstream processing and sample preparation methods of natural products. There are several available methods for the extraction and isolation of bioactive molecules from different natural sources, however they are often associated with major drawbacks. To overcome some of these, aqueous two-phase systems (ATPS) are an attractive alternative.
This thesis presents the application of ATPS as an alternative method for the recovery of different bioactive molecules from complex natural sources: laccase produced by the fungus Pleurotus ostreatus, metalloproteases (MPs) and phospholipases A2 (PLA2) present in the venom of the snake Crotalus molossus nigrescens and phenolic compounds from the medicinal plant Sedum dendroideum. There is no information about the extraction and isolation of the majority of these molecules from the previously mentioned matrices using ATPS. Different types of systems were evaluated to optimize the recovery of such biomolecules. Also, the effect of different system parameters such as volume ratio (VR), tie line length (TLL), polymer molecular weight, pH and sodium chloride (NaCl) addition were explored. All of the bioactive molecules were efficiently recovered from their natural sources, demonstrating the versatility of ATPS. Furthermore, biological activity of these biomolecules was retained, or even increased, after ATPS extraction process. These findings contribute to the establishment of a simple, rapid and cost-effective alternative method for the extraction and isolation of the studied biomolecules and potentially aid their study and application in a wide range of fields.
Keywords
Aqueous two-phase systems; Crotalus molossus nigrescens; Extraction; Laccase; Protease;
Phospholipases A2; Recovery; Snake venom; Pleurotus ostreatus; Sedum dendroideum.
Figure 1.1. Effect of PEG molecular weight upon partition coefficient of proteases, laccase and total protein. ... 18 Figure 1.2. Effect of volume ratio upon partition coefficient of proteases, laccase and total protein ... 19 Figure 1.3. Effect of sodium chloride concentration upon partition coefficient of protease, laccase and total protein. ... 21 Figure 1.4. Effect of PEG molecular weight on partition coefficient of proteases, laccase and total protein on PEG-dextran systems... 23 Figure 1.5. Sequential PEG-salt and PEG-dextran ATPS ... 24 Figure 1.6. Optimal process for separation of laccase from proteases produced by Pleurotus ostreatus using ATPS. ... 25
Figure 2.1. Effect of PEG molecular weight on the recovery of MPs and PLA2 in PEG-salt systems ... 39 Figure 2.2. Effect of different concentrations of NaCl on the recovery of MPs and PLA2 in PEG- salt systems ... 42 Figure 2.3. Effect of pH on the recovery of MPs and PLA2 in PEG-salt systems. ... 45 Figure 2.4. Recovery of MPs and PLA2 in ethanol-salt systems. ... 48
Figure 3.1. Effect of VR and sample load on the partition coefficient and top phase recovery yield of phenolic compounds in ethanol-salt ATPS ... 63 Figure 3.2. Effect of TLL on the partition coefficient and top phase recovery yield of phenolic compounds in ethanol-salt ATPS.. ... 66 Figure 3.3. Van t Hoff plot of ethanol-salt ATPS ... 68 Figure 3.4. Antioxidant activity of top phase from selected ethanol-salt ATPS. ... 69 Figure 3.5. HPLC-DAD chromatograms of crude extract (50 mg/mL) and the top phase of ethanol- salt ATPS ... 71 Figure 3.6. Antiproliferative activity on cancer cell line MCF-7 from top phase of ethanol-salt ATPS ... 73
Table 1.1. Sources and characteristics of the reagents used. ... 12
Table 1.2. Composition of PEG-salt, UCON-salt and PEG-dextran ATPS used in this work. .... 16
Table 1.3. Effect of tie line length upon partition coefficient and enzymatic activity of proteases and laccase . ... .20
Table 1.4. Partition coefficient of proteases, laccase and total protein on UCON-salt systems. 22 Table 2.1. Effect of PEG molecular weight, VR and TLL on the recovery of PLA2 and MPs in PEG- salt systems. ... 36
Table 2.2. Effect of NaCl addition to PEG-salt systems on the recovery of MPs and PLA2. ...40
Table 2.3. Effect of pH on the recovery of MPs and PLA2 in PEG-salt systems.. ... 43
Table 2.4. Recovery of MPs and PLA2 using ethanol-salt systems. ... 46
Table 3.1. Composition of the ethanol-salt ATPS used in this work. ... 58
Table 3.2. Chemical composition of S. dendroideum extracts reported in previous studies. ... 64
Table 3.3. Effect of NaCl addition upon the partition coefficient and top phase recovery yield of phenolic compounds. ... 67
Table 3.4. Thermodynamic properties of phenolic compounds partitioning process. ... 68
Table 3.5. Phenolic compound concentration of the top phase of ethanol-salt ATPS ... 72
1. Introduction ... 1
1.1 Aqueous two-phase systems ... 1
1.2 Experimental models ... 2
1.3 Hypothesis ... 4
1.4 Objectives ... 5
1.5 Thesis structure ... 5
2. Strategies based on aqueous two-phase systems for the separation of laccase from protease produced by Pleurotus ostreatus ... 9
2.1 Introduction ... 10
2.2 Materials and methods ... 12
2.3 Results and discussion ... 17
2.4 Conclusions ... 26
3. Development of aqueous two-phase systems based approaches for the selective recovery of metalloproteases (MPs) and phospholipases A2 (PLA2) toxins from Crotalus molossus nigrescens venom ... 30
3.1 Introduction ... 31
3.2 Materials and methods ... 32
3.3 Results and discussion ... 35
3.4 Conclusions ... 49
4. Aqueous two-phase extraction of phenolic compounds from Sedum dendroideum with antioxidant activity and anti-proliferative properties against breast cancer cells ... 55
4.1 Introduction ... 56
4.2 Materials and methods ... 57
4.4 Conclusions ... 74
5. Concluding remarks ... 79
Annex 1. Published papers ... 81
Annex 2. Congress presentations ... 90
Curriculum vitae ... 96
For many years, humans have relied on nature as a source of a wide variety of bioactive molecules. They exhibit biological activities by triggering a response in organisms, and are highly diverse in their properties, structure and chemical nature [1]. Bioactive molecules can be found in several living organisms, including plants, bacteria, marine microorganisms, animals and fungi.
In addition to natural sources, these molecules can also have a synthetic origin. However, bioactive molecules from natural sources play a significant role in the discovery and development of new drugs [2]. An estimate of 42% of all approved therapeutic agents from 1981 to 2014 were either natural products or directly derived from them [3]. In the field of anticancer therapy, over this same time frame, more than 50% of approved drugs were of a natural source or based on natural products [3]. Besides contributing to drug development, bioactive molecules from natural sources provide us an extensive variety of products, which range from nutraceuticals to agrochemicals and biological probes. Thus, the extraction, isolation and analysis of these molecules are of great interest in several fields, such as the pharmaceutical, agrochemical, cosmetic and food industries, biotechnology research, biochemistry and nutrition [4].
The approaches used to extract and isolate bioactive molecules are determined by several factors including the nature of the matrix, the properties of the individual components and the method of analysis [4]. The development of optimized and economic methods for the extraction and isolation of bioactive molecules is crucial not only for downstream processing, but also as sample preparation methods that enable the development of faster, cheaper, reproducible and environmentally safe analytical studies [5]. Furthermore, time consuming, expensive and labor intensive extraction and isolation processes have been identified as the bottle neck of the application of natural products in drug development [6].
1.1 Aqueous two-phase systems
Aqueous two-phase systems (ATPS) have shown great potential for the recovery and isolation of a wide variety of bioproducts, including proteins, cells, genetic material, virus, organelles, and low molecular weight molecules [7]. In comparison to other extraction methods, these systems provide a gentle environment for biomolecules, since both phases are mainly composed of water and most of the polymers used have a stabilizing effect on the structure and biological activity [8].
ATPS have important advantages over other extraction methods, such as their biocompatibility;
combined with low cost, easy operation, and short processing time. Besides cost-effectiveness
and technological simplicity, this method can be considered as an integrated process, in which contaminants are removed while the target biomolecule is recovered [8].
ATPS result when two incompatible aqueous solutions are mixed at a certain critical concentration, which depends on the phase forming constituents, pH, ionic strength and temperature of the solution. Based on their phase forming constituents, ATPS can be classified in five main groups that include polymer-polymer, polymer-salt, alcohol-salt, micellar and ionic liquid-based systems [7]. The partitioning process of target biomolecules in the system depends on several parameters related to system design (phase constituents, tie-line length (TLL), volume ratio (VR), sample loading, pH and ionic strength) and the physicochemical properties of the biomolecules (charge, molecular weight, hydrophobicity, and conformational characteristics) [9]. The complex chemical and physical interactions involved in the partitioning process of the biomolecules make these systems a powerful method, since system parameters can be easily modified to achieve a high resolving power [8].
1.2 Experimental models
In this thesis focus is placed in different bioactive molecules present in complex natural matrices:
laccase produced by the fungi Pleurotus ostreatus, metalloproteases (MPs) and phospholipases A2 (PLA2) present in the venom of the snake Crotalus molossus nigrescens, and phenolic compounds from the plant Sedum dendroideum. Due to the multiple benefits of ATPS, the application of this technique for attending specific problems related to the extraction and isolation processes of these bioactive molecules is proposed. The physicochemical properties of phenolic compounds and enzymes are very different among each other, allowing to assess the versatility of ATPS. In addition, they have a potential pharmacological application or are already widely used in industrial settings. A brief description of the evaluated experimental models is presented in the following subsections.
1.2.1 Laccase produced by Pleurotus ostreatus
Laccase is a multicopper enzyme that catalyzes the oxidation of a broad range of substrates including phenols, polyphenols, aromatics amines, and even certain inorganic molecules [10,11].
Due to its ability to oxidize a broad variety of substrates, laccase has a strong potential application in biotechnological processes in the textile, pulp and paper, food, cosmetic and pharmaceutical industries [12]. This enzyme is found in fungi, plants, bacteria, and insects. Although it is present in several organisms, the most important source are fungi. The production of laccase by basidiomycetes has been widely studied as they are able to grow from cheap substrates, the
enzyme is secreted and exhibits high redox potential [11,13]. Pleurotus ostreatus is a well-known laccase producing basidiomycete considered a model organism in basic and applied laccase research. There are a lot of reports describing laccase production processes by Pleurotus ostreatus [11,13,14]. Particularly, efficient production of laccase by Pleurotus ostreatus has been reported using stirred tank bioreactors. However, laccase activity is affected by aeration due to simultaneous protease production on culture media [11]. Therefore, it is necessary to develop an effective method to separate proteases to laccase in order to enhance laccase production in stirred tanks.
1.2.2 Metalloproteases (MPs) and phospholipases A2 (PLA2) from Crotalus molossus nigrescens venom
The snake Crotalus molossus nigrescens has the largest distribution in Mexico among Crotalus molossus subspecies [18]. Its venom is rich in PLA2 and MPs, which play an important role in envenomation deleterious effects [19 21]. The study of these toxins is of growing interest for biotechnological and pharmaceutical applications, as they can be used for antivenom design, development of new drugs, and elucidation of the mechanism of action of venoms. A key aspect of the study of toxins is purity. The isolation of PLA2 and MPs usually involves two or more chromatographic separations steps [19 21]. Although proven to be effective, the use of a multistep chromatographic approach is costly, requires specialized equipment and long operating times. Hence, the study of simple and economic alternative methods for the recovery of these toxins to decrease the number of chromatographic steps is of great interest.
1.2.3 Phenolic compounds from Sedum dendroideum
Sedum is a genus that comprises about 100 plant species from the Crassulaceae family; many of them have been used for medicinal purposes [25]. Sedum dendroideum, known as Siempreviva , is a small shrub found from Mexico to Guatemala and Brazil [26,27]. In traditional medicine this specie is used for the treatment of eye conditions, mouth diseases, typhoid, dysentery, scurvy, headache, fever, venereal diseases, or as a contraceptive [27,28]. Previous studies have shown several pharmacological activities of Sedum dendroideum extracts including antinociceptive, antiulcer, anti-inflammatory, spermicidal, and antidiabetic [26,27,29 33]. Those medicinal properties have been mainly attributed to diverse phenolic compounds including phenols, tannins, and flavonoids [26,31 33].
The extraction of phenolic compounds is commonly performed by liquid-liquid extraction and solid-liquid extraction. Their use is associated to major challenges, including high solvent
consumption, long processing time and significant energy consumption. More advanced techniques have also been used to extract phenolic compounds, such as supercritical fluid extraction, microwave assisted extraction, ultrasonic assisted extraction and pressurized fluid extraction [31 34]. However, their application involves higher costs, requires specialized equipment and rigorous operating conditions [35]. To overcome some of these drawbacks, the development of cost-effective and simple methods for the extraction of phenolic compounds is needed. In addition, extraction methods must guarantee preservation of biological activity as well as ensuring that there are no traces of solvents that interfere with therapeutic applications.
1.3 Hypothesis
The bioactive molecules selected as experimental models have very different physicochemical properties and effects and come from diverse complex natural matrices. Their extraction and isolation processes are related to specific problems. The production of laccase by P. ostreatus using stirred tanks bioreactors exhibits a major drawback: the concomitant production of proteases. To avoid degradation of laccase in broths, the establishment of a suitable method that allows to separate laccase from proteases is needed. There are several well-established methods for the extraction of phenolic compounds, however they are associated to different problems, evidencing the need of alternative methods. Furthermore, since phenolic compounds from S.
dendroideum have potential medicinal application, extraction methods that have low toxicity and preserve biological activity are required. The isolation of MPs and PLA2 usually involves multiple chromatographic steps. Although proven to be effective, this approach involves high costs and long operating times. ATPS offer several advantages, such as easy scale up, short processing time, low costs and process integration capability, and higher purity for target molecules in a single step. The use of ATPS could contribute to overcome the problems related to the extraction and isolation processes of these bioactive molecules.
In this context, the experimental work presented throughout this thesis revolves around the following hypothesis:
Laccase can be separated from proteases in P. ostreatus culture media using ATPS by manipulating system parameters, such as phase constituents, TLL, polymer molecular weight, VR and addition of neutral salt.
MPs and PLA2 can be recovered from C. m. nigrescens venom using ATPS and their recovery can be optimized by altering system parameters such as phase constituents, VR, TLL, pH, addition of neutral salts and polymer molecular weight.
Alcohol-salt ATPS are a suitable technique for recovering phenolic compounds from S.
dendroideum and their affinity for either the top or bottom phase can be manipulated by altering system parameters such as VR, TLL, addition of neutral salts, and sample loading.
1.4 Objectives
From the previously mentioned hypothesis a series of objectives were determined, which will allow to establish extraction and isolation processes of different bioactive molecules from complex natural matrices based on ATPS.
To study the partition behavior of laccase and protease from P. ostreatus crude extract using different types of ATPS (polymer-salt and polymer-polymer).
To optimize the separation of laccase and protease by evaluating the effect of the TLL, polymer molecular weight, VR, and addition of neutral salt on their partition behavior.
To study the recovery of PLA2 and MPs from C. m. nigrescens venom using ethanol-salt and polymer-salt ATPS.
To evaluate the effect of the TLL, polymer molecular weight, VR and addition of neutral salt on PLA2 and MPs recovery.
To investigate the recovery of phenolic compounds from S. dendroideum using ethanol- salt ATPS.
To study the influence of sample loading, VR, TLL and neutral salt addition upon phenolic compounds partition behavior in ethanol-salt ATPS.
1.5 Thesis structure
This thesis is divided into 5 chapters. The first and current chapter, Chapter 1, briefly describes the relevance of extraction and isolation methods of bioactive molecules, gives context on the use of ATPS for the recovery and isolation of bioactive molecules and includes a summary of the experimental models used in this thesis. This chapter also incorporates the hypothesis and objectives to be tested and fulfilled. The experimental results are presented from Chapters 2 to 4.
Chapter 2 comprises a research article entitled Strategies based on aqueous two-phase systems for the separation of laccase from protease produced by Pleurotus ostreatus and published in Fluid Phase Equilibria journal. Chapter 3 presents a research article entitled Development of Aqueous Two-Phase Systems based approaches for the selective recovery of MPs and PLA2
toxins from Crotalus molossus nigrescens venom to be submitted to Separation and Purification Technology journal. Chapter 4 includes a research article entitled Aqueous two-phase extraction of phenolic compounds from Sedum dendroideum with antioxidant activity and anti-proliferative properties against brain cancer cells submitted to Separation and Purification Technology journal.
All the articles are presented in scientific manuscript format following guidelines of their respective journals. It should be mentioned that the figure and table numbers were edited for the purposes of this document. Finally, Chapter 5 states the concluding remarks of the work presented throughout this thesis.
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separation of laccase from protease produced by Pleurotus ostreatus
Abstract
Laccases are currently employed in several biotechnological applications such as pharmaceutical, environmental, textile and petrochemical. Their production in submerged bioreactors has been extensively reported, however, this approach exhibits a major drawback: the concomitant production of proteases, which reduces laccase activity in broths. In this work, aqueous two-phase systems (ATPS) composed by polyethylene glycol (PEG)-salt, UCON-salt and PEG-dextran were tested to separate laccase from proteases in culture media. Results showed that PEG of low molecular weight (400 g mol-1), volume ratio of 0.33 and tie line length of 45 % (w/w) in a PEG- salt system allow the separation of both enzymes. The extraction efficiency decreased when sodium chloride was added into the system, but increased with a second ATPS stage. A PEG 4600-dextran 500 000 system was also suitable to separate laccase from proteases with an enzymatic activity recovery of 95%. The UCON-salt systems tested were not adequate for the separation of both enzymes since they partitioned to the same phase. Therefore, two options for laccase separation from proteases are proposed: a two-step PEG 400-salt system for laccase separation after the fermentation process and PEG 4600-dextran 500 000 for in situ recovery of laccase due to its lower salt content, which is suitable for fungal growth. To our knowledge, this is the first time that conditions for the separation of laccase and proteases on ATPS different phases are reported. These findings open the opportunity to establish optimum process conditions for the efficient production and recovery of laccases produced by Pleurotus ostreatus.
Keywords
Aqueous Two-Phase Systems; Laccase; Pleurotus ostreatus; Protease; Recovery.
Published as
C. Sánchez-Trasviña*, D. Enriquez-Ochoa*, C. Arellano-Gurrola*, R. Tinoco-Valencia, M. Rito- Palomares, L. Serrano-Carreón, K. Mayolo-Deloisa. Strategies based on Aqueous Two-Phase Systems for the separation of laccase from protease produced by Pleurotus ostreatus. Fluid Phase Equilib. 502 (2019). https://doi.org/10.1016/j."uid.2019.112281. IF=2.515, Q1.
*The authors contributed equally to this work.
2.1 Introduction
Laccases (EC 1.10.3.2) are multicopper enzymes that catalyze the oxidation of a broad range of substrates including phenols, polyphenols, aromatics amines, and even certain inorganic compounds [1,2]. This feature has provoked that laccases have been used in several biotechnological applications. Such applications include textile, paper, food, and pharmaceutical industries, as well as organic synthesis, biosensors, and bioremediation [3]. Basidiomycetes have been broadly studied for laccase production, since they can grow on cheap substrates, the enzyme is secreted and exhibits high redox potential [2,4]. Some of the basidiomycetes which have been used for laccase production are Pheniophora sp., Funalia trogii, Trametes versicolor, Pleurotus ostreatus, and Agaricus bisporus [2,4 6]. The production of these fungi is usually carried out through submerged fermentation (stirred tank and air-lift) and solid state fermentation [4,5,7]. The influence of different factors, such as agitation and aeration, on laccase production by different fungal strains cultivated in stirred tanks has been studied in several reports.
Particularly, Tinoco-Valencia et al. [2] concluded that aeration was the main factor affecting negatively laccase activity in Pleurotus ostreatus cultures most likely due to simultaneous protease production on culture media. Then, if proteases are removed from the culture, laccase degradation could be avoided allowing an enzymatic activity increase. Therefore, it is necessary to develop an effective method to separate proteases from laccase in order to enhance laccase production in stirred tanks.
The most common Aqueous Two-Phase Systems (ATPS) are those formed by mixing a polymer and salt or aqueous solutions of two incompatible polymers above a certain critical concentration [8]. When a biological sample is loaded into the ATPS, its partition towards the bottom or top phase is driven by different interactions (between the chemical phase forming components and the biological sample), such as electrostatic, Van der Waals and hydrophobic, until thermodynamic equilibrium is achieved [9]. ATPS provide several advantages compared to traditional liquid-liquid separation methods, including low cost and easy scale-up [10].
Furthermore, ATPS are able to separate sensitive biological samples due to the high water content in both phases [6]. The biocompatibility of ATPS has permitted bioprocesses development for the separation and purification of different biological products including proteins, antibodies, genetic material, growth factors, cell, organelles, bionanoparticles and, low molecular weight compounds [10,11].
The use of ATPS to recover and separate enzymes, including laccase, has been extensively reported previously [8]. Polyethylene glycol-salt (PEG-salt) systems are the most commonly used ATPS for this purpose since PEG is a recommended polymer for biological products recovery due to its hydroxyl groups [12]. A wide range of PEG molecular weights in combination with different salts have been tested [12 18]. Polymer-salt and polymer-polymer systems composed of ficoll, ethylene oxide/propylene oxide copolymer (UCON), PEG, dextran and hydroxypropyl starch (PES) have been used to separate laccase from residual compost and culture media from different fungi species [6,18,19]. For example, Mayolo-Deloisa et al. [13] reached a yield up to 95% in laccase recovery from residual compost of Agaricus bisporus using PEG 1000 g mol-1 and potassium phosphate ATPS. Bertrand et al. [14] also achieved a laccase percentage recovery up to 95% from residual compost from P. ostreatus using UCON-salt systems. In another study, laccases were partitioned from the culture supernatant of Cerrena unicolor and Pleurotus sapudis using PEG-potassium phosphate ATPS and achieved a recovery percentage over 90% [12]. Prinz et al. [16] recovered lacasse using PEG 3000 g mol-1-potassium phosphate systems from the culture supernatant of Pleurotus sapudis and Trametes versicolor and reached 96% of enzymatic activity recovery. In all of these studies, laccase affinity for either the top or bottom phase varied depending on the fungi species being used, this is mainly attributed to laccase glycosylation grade [12]. In several cases the reported laccase recovery is higher than 95%, suggesting that PEG- potassium phosphate ATPS are a suitable option to recover laccases.
The partition of proteases in ATPS from different fungi species (Aspergillus niger, Streptomyces sp., Penicillum restrictum, Aspergillum tamari, etc.) has been studied using mainly PEG-salt systems. The effect of the type of salt (sulphate, citrate, phosphate) and its concentration, and PEG molecular weight and its concentration, as well as the effect of the addition of neutral salts such as sodium chloride in proteases partition using this type of systems has been thoroughly investigated [20 27]. Results have indicated that proteases partition preferently to the PEG-rich phase and that PEG increases proteases stability depending on PEG molecular weight [27]. Both enzymes, laccase and protease have been partitioned in ATPS, however, there are no reports regarding their simultaneous partition and separation. Especially if both enzymes are simultaneously produced from the fermentation process of the fungi, which may represent a challenge considering that both enzymes have affinity for the PEG-rich phase. In this work, the partition of protease and laccase from a Pleurotus ostreatus crude extract using different types of ATPS (PEG-potassium phosphate, UCON-potassium phosphate and PEG-dextran) was studied.
The effect of tie-line length (TLL), PEG molecular weight, volume ratio (VR) and the addition of
sodium chloride on the enzymes separation was evaluated. Such strategies were studied with the purpose to obtain suitable conditions to increase laccases volumetric activity and concentrate it at large scale by reducing its proteolytic degradation through the use of an extractive fermentation system or simply separating proteases from laccase after the fermentation process.
2.2 Materials and methods 2.2.1 Materials
The materials used in this work are listed in Table 1.1 PEG of nominal molecular mass of 400, 1000, 3350, 4600 and 6000 g mol 1, dextran of nominal molecular mass of 500 000 g mol-1, ABTS, L-tyrosine, TCA, Bradford reagent, Folin-Ciocalteu reagent and Na2CO3 were purchased from Sigma-Aldrich (St. Louis, MO, USA). UCON 50-HB 5100, a random copolymer with 50% ethylene oxide and 50% propylene oxide, was obtained from NuferPlus, S.A. de C.V. (Guanajuato, Mexico).
Sodium chloride (NaCl), dibasic potassium phosphate (K2HPO4) and monobasic potassium phosphate (KH2PO4) were purchased from J.T. Baker (Center Valley, PA, USA). Casein was donated kindly by Dr. Esther Pérez-Carrillo from Tecnologico de Monterrey. All these compounds were used without further purification. Any other reagents used were of analytical grade.
Table 1.1. Sources and characteristics of the reagents used.
NP: Data not provided by the supplier. NA: Not applicable. LN: Long name, the complete IUPAC name can be consulted in https://pubchem.ncbi.nlm.nih.gov.
Compound IUPAC name
Purity, mass based (%)
Purification
method Supplier
PEG 400 g mol-1 Polyethylene glycol NP None Sigma-Aldrich
PEG 1000 g mol-1 Polyethylene glycol NP None Sigma-Aldrich
PEG 3350 g mol-1 Polyethylene glycol NP None Sigma-Aldrich
PEG 4600 g mol-1 Polyethylene glycol NP None Sigma-Aldrich
PEG 6000 g mol-1 Polyethylene glycol NP None Sigma-Aldrich
Dextran 500 000 g mol-1 2,3,4,5-tetrahydroxy-6-[3,4,5-trihydroxy-6- [[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-
yl]oxymethyl]oxan-2-yl]oxyhexanal
NP None Sigma-Aldrich
UCON 50-HB 5100 Polyalkylene glycol monobutyl ether NP None NuferPlus, S.A. de C.V.
L-tyrosine (2S)-2-amino-3-(4-hydroxyphenyl)propanoic
acid 98% None Sigma-Aldrich
TCA Trichloroacetic acid 99% None Sigma-Aldrich
Bradford reagent NA NP None Sigma-Aldrich
Folin Ciocalteu reagent NA NP None Sigma-Aldrich
Na2CO3 Sodium carbonate 99.5% None Sigma-Aldrich
ABTS 2,2 -Azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) diammonium salt
99% None Sigma-Aldrich
NaCl Sodium chloride 99% None J.T. Baker
KH2PO4 Potassium dihydrogen phosphate 99.1% None J.T. Baker
K2HPO4 Dipotassium hydrogen phosphate 99% None J.T. Baker
Casein NA None
2.2.2 Production of laccase by submerged culture of Pleurotus ostreatus
P. ostreatus CP-50 was obtained from the Postgraduate College at Puebla (Colegio de Postgraduados, Unidad Puebla, Km 125.5 Carretera México-Puebla La Libertad, Cholula, 72130, México). The microorganism was maintained on 2% malt extract-agar plates at 4 C.
Inoculum was prepared as reported by Tinoco-Valencia et al. [2]. P. ostreatus was grown on plates containing 2 % malt extract-agar at 30 C. Inoculum was produced in 500 ml Erlenmeyer flasks containing 100 ml of medium: malt extract (20 g/L), glucose (10 g/L) and yeast extract (10 g/L), pH 6. The flasks were inoculated with 1 agar plug (1 cm2) covered by mycelia from a 7-day- old culture previously homogenized with 10 ml culture medium in a Sorvall Omni-mixer (model 17150; Ivan Sorvall Inc., Newtown, Conn.). After 4 days of incubation at 150 rev min-1 and 30 C, the mycelium from 3 flasks was collected and used to inoculate the 10 L fermenter.
Fermentations were conducted as described before [2]. A MicroFerm Model 14 L fermenter (New Brunswick, Edison, NJ) with a working volume of 10 L, vessel diameter (T) of 0.21 m and four baffles was used. Three Rushton turbines with an impeller-vessel diameter ratio (D/T) of 0.5 were used. Impeller spacing was 0.074 m. The culture medium contained the following in g/L: malt extract 20, yeast extract 10 and glucose 10. After the solubilization of the ingredients, liquid medium was poured in the vessel, the fermenter was prepared and autoclaved at 121°C, 40 min.
Culture was conducted at 30°C and pH was controlled at 6 by automatic H2PO4 (0.2 N) addition, 230 rpm during the first 72 h and 147 rpm to the final time. 0.5 vvm were set as the aeration rate.
After 156 h of culturing, biomass was separated by sieving and the culture broth was processed.
The cell-free broth was ultrafiltered using an Amicon DC10L (Danvers, MA, USA) equipped with a 10 kDa hollow fiber cartridge (H1LP10-20, Amicon, Danvers, MA, USA) the working pressure was controlled below 20 psi and the temperature of the sample was kept at 10 °C. The 10-fold concentrated sample (1 L) was then frozen for three days, defrozen and centrifuged in a Beckman Model J2-21 centrifuge with a JA-14 rotor at 15300 x g. Finally, the sample was concentrated to 70 mL in an Amicon stirred ultrafiltration cell using a 10 KDa membrane.
2.2.3 Enzymatic activity assays
The enzymatic activity of laccase was determined according to Sanchez-Trasviña et al. [28]
procedure. Briefly, the change in optical density at 436 nm during 5 minutes was done using ABTS as substrate and the molar extinction coefficient of 436 = 29,300 M 1 cm 1, using a Synergy-HT microplate spectrophotometer (Biotek, VT, USA). The assay mixture consisted of 222 µL of acetate buffer (0.1 M, pH 4.0), 25 µL of ABTS (1 mM; dissolved in ethanol) and 3 µL of sample.
One unit of enzymatic activity was defined as the amount of enzyme required to oxide 1.0 µmol of substrate per minute.
The protease activity was determined by the method of Anson [29] with modifications. To initiate the reaction, 0.25 mL of the sample were added to 1.25 mL of 0.65% (w/v) casein solution in potassium phosphate buffer 50 mM pH 7.5, and incubated at 37ºC for 10 minutes. To stop the reaction, 1.25 mL of trichloroacetic acid solution at 110 mM solution were added. Next, precipitates were removed by centrifugation at 10 000 rpm for 15 minutes at 25 ºC using an Allegra 64R Centrifuge (VWR, PA, USA) followed by recovery of 0.5 mL of supernatant. Then, 1.25 mL of sodium carbonate solution at 500 mM were added to the recovered supernatant, followed by addition of 0.25 mL of a 0.4 N Folin and Ciocalteu s phenol reagent solution and incubation at 37 ºC for 30 minutes. Finally, tubes were centrifuged at 14,000 rpm for 10 minutes at 25 ºC using a 5417R centrifuge (Eppendorf, Hamburg, Germany) and the supernatant was collected for its analysis. The proteases activity was determined by pouring 0.1 mL of the treated samples into a 96-well flat bottom microplate and using a microplate reader (Synergy-HT, Biotek) at 660 nm. The experiments were carried out in triplicate with one control. One unit of enzymatic activity was defined as the amount of enzyme necessary to produce one µM of tyrosine per minute at pH 7.5 and 37ºC. A calibration curve was done using L-tyrosine as standard (0-558 µM).
The protein determination was done by Bradford technique [14] in a microplate reader (Synergy HT, Biotek, VT, USA); using a calibration curve prepared with bovine serum albumin (BSA) solutions ranging from 0.0 to 2.0 mg/mL. Briefly, 10 µL of the sample was mixed with 250 µL of Bradford reagent and incubated 10 min. Then, absorbance at 595 nm was measured.
2.2.4 Laccase and proteases partition in ATPS
In order to evaluate the partition of both enzymes laccase and protease, 13 ATPS were tested, 7 systems of PEG-potassium phosphate, 3 UCON-potassium phosphate systems and 3 PEG- dextran systems. For all polymer-salt systems potassium buffer phosphate (K2HPO4 KH2PO4, ratio 18:7, 40% w/w, pH 7.0) was used and the total weight of ATPS was 2 g with 10% w/w of sample (laccase concentrate containing laccase and proteases). ATPS were selected based on previous reports [14,28,30]. All experiments were carried out by triplicate.
For PEG-salt systems, PEG stock solutions of nominal molecular weights of 400 (liquid at room temperature and therefore used at 100 %) and 1000 g mol-1 (50 w/w%) were prepared using Milli Q water [9]. The composition of the PEG-salt systems were reported previously by Sanchez- Trasviña et al. [28] (see Table 1.2). In order to evaluate the effect of PEG molecular weight, both PEG molecular sizes were tested using a tie line length (TLL; defined by the concentration of the chemicals forming the phases) of 15 % w/w and volume ratio (VR; volume of the top phase/volume of the bottom phase) of 0.33. The PEG molecular weight with best separation performance (400 g mol-1) was selected to evaluate the effect of TLL (15, 25, 35 and 45 % w/w) and VR (0.33 and 1). The improvement of separation of laccase and proteases was attempted through NaCl addition at different concentrations (0-360 mM) to the system with best separation performance (PEG 400, TLL 45 % w/w and VR 0.33). For UCON-potassium phosphate systems, UCON at 100 % (w/w) and ATPS compositions reported by Bertrand et al. [14] were used (see Table 1.2). In case of polymer-polymer systems (PEG-dextran), PEG stock solutions of nominal molecular weights of 3350 (50% w/w), 4600 (50% w/w) and 6000 g mol-1 (40 % w/w) and dextran stock solution of nominal molecular weight of 500 000 g mol-1 (15 % w/w) were prepared using Milli Q water. The compositions used (see Table 1.2) were reported previously [30].
Table 1.2. Composition of PEG-salt, UCON-salt and PEG-dextran ATPS used in this work.
NA: Data not available (protease activity was not detected on the bottom phase).
After the extract was loaded into the ATPS, systems were slightly mixed for 15 min. Complete phase separation was accomplished by batch centrifugation at 10,000 rpm at 25 ºC for 10 min.
Graduated tubes were used for clear visualization of the phase volumes and each phase was carefully separated for further analysis. Then, total protein concentration as well as enzymatic activity of both enzymes in each phase was estimated by the methods previously described. The partition coefficient (Kp; defined as the ratio of protein concentrations in the top and bottom phases), laccase purification factor and recovery percentage were further calculated using the following equations:
Kp = Ctop/Cbottom (1)
where Ctop and Cbottom are either concentrations of total protein or enzymatic units of laccase/proteases in the top and bottom phase, respectively.
PF = SAphase/SAextract (2)
Composition
ATPS VR TLL
(% w/w) PEG
(% w/w) Salt (% w/w)
PEG 400-Salt
0.33 15.00 9.70 22.30
0.33 25.00 10.20 23.00
0.33 35.00 10.00 27.00
0.33 45.00 10.00 29.00
1.00 15.00 16.50 16.00
PEG 1000-Salt 0.33 15.00 8.50 16.90
UCON
(% w/w) Salt (% w/w)
UCON-salt
1.00 21.50 13.01 5.30
1.00 27.00 14.01 5.70
1.00 31.00 15.02 6.10
PEG
(% w/w)
Dextran 500 000(% w/w)
PEG 3350-dex 1.80 NA 9.00 9.60
PEG 4600-dex 1.80 NA 7.00 6.50
PEG 6000-dex 1.00 NA 7.00 6.50
where SAphase and SAextract are the specific activities of laccase on each phase and crude extract, respectively.
% recovery = (Uphase/Uloaded)*100 (3)
where Uphase is the laccase enzymatic units in each phase and Uloaded are the total enzymatic units of laccase loaded into the system.
2.2.5 Sequential ATPS
The ATPS with best separation performance were selected for a two-step sequential separation (PEG 400 g mol-1-salt, TLL 45% w/w and VR 0.33 and PEG 4600 g mol-1 -dextran 500 000 g mol- 1). The bottom phase of the first step system was replaced for a fresh bottom phase (salt or dextran-rich phase with same composition from the previous step). This new second step system was slightly mixed for 15 minutes, and centrifuged at 14 000 rpm for 10 minutes at 25ºC using a 5417R Centrifuge (Eppendorf, Hamburg, Germany) to achieve proper phase separation. After, each phase of this new system was collected for enzymatic activity and total protein concentration determination as described above.
2.3 Results and discussion
2.3.1 Characterization of the crude extract
The laccase extract contained a total protein concentration of 3.57 mg mL-1, 33.25 U mL-1 of laccase activity and 2.98 U mL-1 of protease activity. The extract was centrifuged at 6000 rpm during 10 min at room temperature to eliminate solids prior loading into ATPS.
2.3.2 Partition of laccase and proteases in PEG-salt systems
The production and partition of laccase using ATPS have already been studied, however, there are no reports about its separation from proteases, which play an important role affecting laccase volumetric activity in bioreactors [31]. In this work, the partition of laccase and proteases was evaluated using different PEG-salt ATPS. Firstly, the effect of PEG molecular weight upon partition of both enzymes was analyzed through the logarithm of the partition coefficient (ln KP).
Fig. 1.1 shows that laccase and proteases prefer the top phase (PEG-rich phase). Laccases have an isoelectric point around of 4.0 [1], while proteases have it around of 4.5 [31], therefore at pH 7 (system pH), the surface of both enzymes is negatively charged, promoting their partition to the PEG rich-phase positive environment [10]. Furthermore, when PEG molecular weight is increased, laccase migrates gradually to the bottom phase and proteases increase their partition to the top
phase. This behavior has been reported previously [20,28]. An increase in PEG molecular weight modifies the free available volume in the top phase, which in consequence increases the phase hydrophobicity, therefore if a protein has hydrophobic affinity, its partition will be enhanced to the PEG phase [20]. Because proteases partition is enhanced when PEG hydrophobicity increases due to a change in its molecular weight, it can be assumed that proteases in the extract produced by Pleurotus ostreatus have hydrophobic affinity.
Figure 1.1. Effect of PEG molecular weight upon partition coefficient of proteases, laccase and total protein. ATPS volume ratio and tie line length were 0.33 and 15 % (w/w) respectively.
Proteases ( ), laccase ( ) and total protein ( ).
For the following experiments a PEG molecular weight of 400 g mol-1 was selected because at higher PEG molecular weights, laccase may partition to the salt-rich phase where is less stable than in the PEG-rich phase. Additionally, low PEG molecular weights are easier to use in bioreactors due to their lower viscosity. The effect of the VR in the enzymes partition (see Fig.
1.2) shows that the affinity of both enzymes to the top phase decreases when the VR increases.
Consequently, the recovery percentage of laccase in the top phase decreases from 99 to 90%
(VR 0.33 to 1). A similar effect was reported by Sánchez-Trasviña et al. [28] when laccase from Trametes versicolor was partitioned in PEG-salt systems. On the other hand, proteases became more active in both phases when the VR increased. The purification factor of laccase with PEG 400 g mol-1 at VR 0.33 was 2.54 and increased to 3.44 at VR 1. Whereas, the protease purification factor increased from 0.48 to 2.57.
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
400 1000
Ln Kp
PEG molecular weight (g mol-1)
Figure 1.2. Effect of volume ratio upon partition coefficient of proteases, laccase and total protein. PEG molecular weight and tie line length were 400 g mol-1 and 15 % (w/w) respectively.
Proteases ( ), laccase ( ) and total protein ( ).
Since laccase presented a higher Kp and proteases a lower Kp at VR 0.33 compared to VR 1, PEG 400 g mol-1 and VR 0.33 were selected to evaluate the effect of TLL upon separation of protease and laccase. Table 1.3 shows how the partition of both enzymes changes when TLL increases.
The ln (Kp) of proteases and laccase are lower when TLL increases. An increase of TLL is related to less free volume available in the top phase, which promotes that the desired proteins and other contaminants migrate to the bottom phase [13]. In this case, when a TLL of 45% (w/w) was used a better specific activity of laccase in the top phase (12.72 U mg-1 protein) was reached as well as the highest specific activity of proteases in the bottom phase (8.10 U mg-1 protein). Therefore, at these conditions (PEG 400 g mol-1, VR 0.33 and TLL 45 % w/w) undesired proteins are partitioned to the bottom phase together with proteases meanwhile laccase migrates to the top phase. This selective movement could be attributed to the surface properties (hydrophobicity and charge) of each enzyme and their interactions with the environment, since the presence of different proteins and molecules makes the partition more complex and results could be different from a single protein partition. The effects of free volume reduction in the top phase and salting- out in the bottom phase occur for both proteins, nevertheless both proteins have a similar molecular weight ( 68-74 kDa) and superficial charge, therefore it is more likely that the difference in their hydrophobicity plays a major role in the selective separation of the enzymes (proteases migrate towards the bottom phase meanwhile laccase towards the top phase).
-1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
0.33 1
Ln Kp
Volume ratio
Table 1.3. Effect of tie line length upon partition coefficient and enzymatic activity of proteases and laccase. PEG molecular weight and volume ratio were 400 g mol-1 and 0.33 respectively.
NA: Data not available (protease activity was not detected on the bottom phase). Bold numbers represent the best separation.
2.3.3 Effect of sodium chloride upon partition of laccase and proteases in PEG-salt systems
In order to improve the separation between laccase and proteases, different concentrations of NaCl (0 - 360 mM) were tested using PEG 400 g mol-1, VR 0.33 and TLL 45% (w/w). The addition of NaCl to ATPS has been used to separate efficiently hydrophobic proteins from contaminants since NaCl may alter the protein partition coefficient due to differential distribution of the salt ions between both phases [32]. This means that when the NaCl concentration increases, the PEG-rich top phase becomes more negative and therefore, positively charged proteins are attracted to it [10]. Results show that (see Fig. 1.3) ln Kp of laccase decreased from 5.55 (without NaCl) to 4.9 (360 mM NaCl). This could be due to a decrease in protein solubility as a consequence of the addition of NaCl at a concentration of 360 mM (2% w/v) [33]. On the other hand, proteases increased their affinity to the top phase from ln Kp 1.05 (without NaCl) to 2.66 (360 mM NaCl). As mentioned before, when using higher NaCl concentrations, solubility decreases by increasing hydrophobic interactions and the moisturizing effect of the salt ions enclosing the protein.
Therefore, most proteins migrate to the phase with a lower NaCl concentration, allowing better interactions between proteins and PEG, and finally improving the separation of proteins to the PEG-rich phase [20]. In this context, Yavari et al. [20] determined that NaCl concentration is statistically the most important factor for ln Kp partitioning alkaline protease from Bacillus licheniformis. It can be expected that the crude extract has a great number of compounds
TLL % (w/w) ln Kp Top specific activity (U/mg protein)
Bottom specific activity (U/mg protein)
Protease
15 1.66 ± 0.23 1.44 ± 0.04 4.25 ± 1.04 25 1.34 ± 0.04 2.07 ± 0.11 7.62 ± 0.49
35 NA 1.58 ± 0.14 NA
45 1.05 ± 0.07 1.62 ± 0.21 8.10 ± 1.49
Laccase
15 6.97 ± 0.34 9.93 ± 0.52 0.15 ± 0.04 25 5.03 ± 0.10 10.87 ± 0.19 1.00 ± 0.11 35 5.27 ± 0.27 9.61 ± 1.04 0.77 ± 0.15 45 5.55 ± 0.45 12.72 ± 0.10 0.79 ± 0.24
(pigments, sugars, fungal metabolites among others) that avoid the separation of laccase and proteases when NaCl is added.
Figure 1.3. Effect of sodium chloride concentration upon partition coefficient of protease, laccase and total protein. PEG molecular weight, tie line length and volume ratio were 400 g
mol-1,45 % (w/w) and 0.33 respectively. Protease ( ), laccase ( ) and total protein ( ).
2.3.4 Separation of laccase and proteases using UCON-salt systems
Another polymer-salt ATPS (UCON-potassium phosphate) was evaluated in order to separate laccase and proteases from the crude extract. UCON was selected because it is a temperature- responsive polymer, when heated above its critical temperature, it precipitates and allows the separation of proteins in the UCON-rich phase [33]. Our results (see Table 1.4) showed that both enzymes, laccase and proteases are partitioned to the bottom phase (salt-rich phase). In UCON- salt systems, the molecular weight of proteins is a factor that affects the partial purification of laccase, larger proteins prefer the top phase [14]. Since proteases and laccase have a similar molecular weight, this could have influenced the migration of both enzymes to the bottom phase.
Other studies involving laccase partition in UCON-salt systems reported that this type of ATPS are suitable for separating laccases produced by different fungi species (Trametes versicolor, Peniophora cinereal, Pleurotus ostreatus) from crude extracts (containing pigments, sugars and other contaminants) [6,14,18]. However, in this work it was not possible to separate laccase and proteases present in the crude extract, meaning that the differences of these two enzymes could not be exploited to achieve their separation using UCON-salt systems.
0.00 1.00 2.00 3.00 4.00 5.00 6.00
0 50 100 150 200 250 300 350 400
Ln Kp
NaCl (mM)
Table 1.4. Partition coefficient of proteases, laccase and total protein on UCON-salt systems.
2.3.5 PEG-dextran for separation of protease and laccases
Polymer-polymer aqueous two-phase systems are an alternative to conventional extraction steps for purifying molecules with biotechnological applications [34]. In this work PEG-dextran systems were used to evaluate laccase separation from proteases. The results demonstrated (see Fig.
1.3) that proteases preferentially partitioned to the bottom phase (dextran-rich phase). This behavior is enhanced when PEG molecular weight increases. Previous reports have shown that in PEG-dextran systems, proteases enzymatic activity is greater in the dextran phase than in the PEG phase [19]. On the other hand, laccase was preferentially partitioned to the top phase (PEG- rich phase) and reached a maximum ln (Kp) value (1.49) and activity recovery percentage (95.94%) using PEG 4600 g mol-1; meanwhile at PEG 6000 g mol-1 ln (Kp) decreased to -0.33.
The high hydrophobicity produced by PEG 6000 g mol-1 promoted the interaction between laccase and the opposite dextran phase resulting in a low partition coefficient. The laccase activity recovery percentage at the top phase obtained in this work was better than the one obtained by Bertrand et al. [14] (87.89 %) using PEG 400 g mol-1-dextran 100 000 g mol-1. Regarding total protein, it partitioned to the top phase and migrated to the bottom phase when PEG molecular weight increased. This behavior is possibly due to a reduction of free volume available in the top phase produced by high molecular weight PEG (6000 g mol-1). The obtained results suggest that PEG 4600 g mol-1-dextran 500 000 g mol-1 is the more adequate PEG-dextran system to separate proteases from laccase.
System Kp
Proteases Laccase Total protein
1 -1.73 ± 0.56 -5.58 ± 0.36 -4.66 ± 0.04 2 -4.13 ± 0.28 -6.90 ± 0.12 -4.34 ± 0.24 3 -2.84 ± 0.36 -7.72 ± 0.11 -4.52 ± 0.02
Figure 1.4. Effect of PEG molecular weight on partition coefficient of proteases, laccase and total protein on PEG-dextran systems. Dextran 500 000 was used. Proteases ( ), laccase ( )
and total protein ( ).
2.3.6 Sequential ATPS to improve separation
In some cases, a single ATPS stage is not enough to separate efficiently contaminants from the target molecule, in those cases a second ATPS step may be desirable to achieve better purification. For this reason, the systems with best laccase and proteases separation (PEG 400 g mol-1-potassium phosphate, VR 0.33, TLL 45 % (w/w) and PEG 4600 g mol-1-dextran 500 000 g mol-1) were selected to carry out two ATPS stages. In both cases the top phase (PEG-rich phase) from the first ATPS was mixed with fresh bottom phase (salt or dextran-rich phase). In case of PEG-salt systems, the second ATPS step changed the proteases partition towards the bottom phase. This resulted in an increment of both laccase partition coefficient and recovery percentage (from 92.42 to 96.53%) in the second stage (See Fig. 1.5). Also, the purification factor of laccase increased from 3.26 to 4.30-folds in the top phase of the second step. This behavior was related to contaminants in the top phase of the first ATPS, among them proteases, which migrated to the bottom phase of the second ATPS. The fresh new added bottom phase was unsaturated (available free volume), allowing biomolecules partition towards that phase. Therefore, in the tested PEG-salt system a second ATPS step to separate laccase from proteases is suitable. In the PEG-dextran systems, proteases increased their affinity to the bottom phase in the second
-2 -1.5 -1 -0.5 0 0.5 1 1.5 2
3350 4600 6000
Ln Kp
PEG molecular weigth (g mol-1)
