Post on 16-Mar-2020
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General Introduction
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1. Downstream Processing
Downstream processing (DSP) is an integral part of any biological product
development and the final cost of the product depends largely on the cost incurred
during DSP for its recovery. Scale-up problems are considerable during DSP using
conventional methods like centrifugation, which are expensive at large scale, making
them uneconomical unless the product is of high value (Diamond and Hsu, 1992;
Raghavarao et al., 1998a). The downstream processing of biological materials
requires purification techniques which are both economically feasible and delicate
enough to preserve their biological activity. The problems traditionally faced by the
biotechnologist in conventional separation operations pale in comparison with the
requirements often placed on separation of biomolecules and recombinant proteins. A
series of unit operations are required for the downstream processing of biomolecules.
While developing a large scale isolation procedure, it is mandatory to consider
processing time, energy, manpower, good manufacturing practices, recycling of
chemicals, sterilization and cleaning-in-place (CIP) of equipment, apart from
separation efficiency (Diamond and Hsu, 1992; Raghavarao et al., 1995; Rito-
palomares, 2004). Thus, scaling-up of the laboratory processes is crucial for the
industrial exploitation. Hence, there is a need to develop simple, efficient, economical,
environmentally benign downstream processing methods for the recovery of
biomolecules with flexibility for continuous operation. Liquid-liquid extraction using
aqueous two-phase systems (ATPSs) is one such method, popularly known as
aqueous two-phase extraction (ATPE).
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Unlike conventional liquid-liquid extraction involving organic/aqueous phases,
ATPE employs two aqueous phases. ATPE has been successful to a large extent in
overcoming the drawbacks of conventional extraction processes such as low
solubility and denaturation of biomolecules in organic solvents. The important step in
ATPE is the selection of suitable ATPS which gives the desired partitioning of the
biomolecules (cells, bacteria, protein/enzymes, etc.) under consideration. After
identification, the appropriate conditions must be arrived at depending on the
objective of the given partition step. If ATPE is used as the primary purification step
for the removal of cell debris from the fermentation broth containing the desired
product, the aim is to partition the debris and the product into opposite phases. Then
in subsequent partition steps the desired/required degree of purity of the product is
achieved. In all of these extraction steps, while standardizing the system conditions,
attention should be given to factors such as partition coefficient of the target
protein/enzyme, contaminating materials, volume ratio of the system employed. It
should be noted that the cell debris, itself being a biopolymer, contributes to the
formation of phases and affects the phase volume ratio as its concentration
increases. For example, if the desired protein/enzyme is to be partitioned to the top
phase, system conditions must be designed/selected in such a way that its partition
coefficient is relatively much higher than that of cell debris as well as contaminating
materials. In order to achieve this, the knowledge of the factors that affect the
partitioning should be exploited.
The productivity of a given bioprocess can be considerably improved by a
relatively new strategy, namely, process integration. For example, it could be by design of
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system conditions in such a way that various unit operations such as solid separation,
purification, concentration occur in a single step of extraction itself. It also could be
integration of one unit operation with the other, say extraction with membrane processes
or different membrane processes with each other, for achieving desired selectivity and
purity of the biomolecule (Patil et al., 2006; Azevedoa et al., 2008).
2. Aqueous Two-phase Extraction
Most biotechnological products, soluble molecules and particles are obtained
in very dilute solutions. Aqueous two-phase extraction has the ability to achieve their
concentration simultaneously during the extraction, provided it is designed in such a
way that most of the desired substances are partitioned to a phase with a small
volume compared to the original solution (Albertson, 1986). The particles may get
concentrated at the interface. During ATPE, impurities may be concentrated to a
certain extent while a concomitant purification is also achieved. The concentration
and purification of viruses was also reported using aqueous two-phase extraction. A
one-step or multi-step procedure may be applied depending on the partitioning of the
product and the contaminants. Compared to traditionally used techniques, the main
advantage of the aqueous two-phase extraction is the decrease in the process time
resulting in considerable savings in energy input and manpower (Kroner et al., 1982).
2.1 Phase systems
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Liquid-liquid extraction using aqueous two-phase systems (ATPSs) has been
recognized as a superior and versatile technique for the downstream processing of
biomolecules (Albertsson, 1986).
An aqueous two-phase system will form on mixing a pair of water soluble
polymers or a polymer and low molecular weight salt with water above a critical
concentration. Thus, ATPSs are of two types, polymer/polymer type and polymer/salt
type. Some of the commonly used phase systems are listed in Table 1. Both
components of these systems are separately miscible in water in all proportions and also
with each other at low concentrations. As the concentration of these phase components
in a common solvent (water) increases above a certain critical value, phase separation
occurs. Each ATPS is characterized by an exclusive phase diagram that indicates the
equilibrium composition for that particular system and constitutes the most fundamental
data for the biomolecule extraction involving that phase system. A model phase
diagram is shown in Figure 1. Bamberger et al., (1984) discussed in detail the
methods for the construction of these phase diagrams. Albertsson (1971), Diamond
and Hsu (1989) and Zaslavasky (1982) compiled phase diagrams for a number of systems.
Among these, systems formed by PEG–polymer–water and PEG–salt–water systems
are widely used for separation and purification of biomolecules. However, PEG–salt
two-phase systems have certain advantages over PEG–polymer systems such as
lower viscosity and cost. Recently new phase systems comprising of alcohol, detergent
and ionic liquid based aqueous two-phase systems are also reported for the recovery of
bioactive components (Madhusudhan et al., 2011).
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2.2 Factors affecting the formation of the phase systems
The formation of aqueous two-phase systems are influenced by many factors
such as concentration of phase forming polymer as well as salt, polymer molecular
weight, temperature, hydrophobicity, salt and pH (Albertsson, 1986). Phase systems
are significantly affected by the addition of neutral salts (univalent or multivalent) and
their concentration. Zaslavsky et al., (1982) demonstrated that increasing the
concentration of the univalent salts (up to 0.1 Molarity) in PEG/dextran system will
alter the composition of the phases without significant effect upon the position of the
binodial. However, multivalent salts such as phosphate, sulfate and tartarate in the
same system show an increasing tendency to partition to the bottom (dextran-rich)
phase with increasing salt concentration and distance from the critical point
(Zaslavsky et al., 1995). However, the effects and mechanisms by which they
influence phase formation are still not completely understood.
The physical properties of ATPSs such as density, viscosity, and interfacial
tension determine the phase demixing and indirectly contribute to the biomolecule
partitioning behavior in a given extraction. The measurement of these physical
properties is very important for designing and analyzing the results of extraction
employing these phase systems. The information related to the extent of variation of
these properties with the tie line length is of prime importance during optimization of
given extraction step. Methods of measuring some of these properties were given by
Walter et al., (1985), Albertsson (1986) and Zaslavsky (1995). Researchers measure
these properties of the systems they used but most of them do not report this data.
However, in the case of polymer/polymer type ATPSs such compilation of data still
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appears to be scarce. Recently, Zuniga et al., (2006) reported physical properties of
PEG-maltodextrin. There is a need for the systematic measurement and reporting of
the physical properties of these aqueous two-phase systems
2.3 Factors affecting the partitioning of biomolecules
The protein partitioning in ATPS is influenced by many environmental
conditions such as size and concentration of biomolecules, choice of polymers and
their molecular weight, composition of the phases, biomaterial surface properties,
system pH, temperature, etc. Influence of these parameters on partitioning was
collectively explained in terms of relative free volume (Grossman and Gainer, 1988;
Eiteman and Gainer, 1989).
2.4 Concentration and molecular weight of phase forming polymer
The molecular weight of phase forming polymer has significant effect on
partitioning of biomolecules because it alters the phase composition itself. Generally,
increasing the molecular weight of a phase forming polymer will cause biomolecule to
partition more towards the opposite phase. Similarly, when a phase forming
polymer‘s molecular weight is decreased, a biomolecule will tend to partition into the
polymer-rich (top) phase. The extent of this effect depends on the molecular weight of
the biomolecules also. Albertsson et al., (1986) reported that the effect of polymer
molecular weight was more prominent for the biomolecules of higher molecular
weight (up to 250 kDa). Near the plait point of the binodial curve, the partition
coefficient of biomolecule is ‗unity‘ (equal partitioning in both the phases). As polymer
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concentration is increased (moving away from the plate point) protein partitioning will
be one sided and consequently the partition coefficient becomes exceedingly higher
or lower than unity.
2.4 System temperature
The effect of system temperature on protein partitioning has not yet been
thoroughly investigated. The change in temperature causes sharp change of binodial
curve which in turn affects the partition behavior of the biomolecule during aqueous
two-phase extraction (Albertsson, 1986). Hence, the change in system temperature
has an indirect effect on partitioning of biomolecules.
2.6 Biomolecule size
The size of the biomolecule has a significant role on its partitioning behavior
during aqueous two-phase extraction. Generally, the small molecules tend to partition
themselves evenly between two phases, where as large molecules tend to distribute
in an uneven manner, while very large biomolecules partition themselves at interface
of the phase systems (Albertsson, 1986).
2.7 System pH
The change in system pH has an indirect effect on partitioning of
biomolecules. The partitioning of proteins/enzymes in ATPS is affected by net charge
on the biomaterial which in turn depends on the pH of the solution. The net charge on
the biomolecules can be varied by changing the pH of the solution. This is due to the
increased surface area of the biomolecules which causes more hydrophobic
interactions.
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2.8 Protein concentration
In general, with in certain range partitioning is not dependent on the
concentration of the protein/enzyme. However, at very high concentration of the
protein there could be a possibility of the formation of third phase by protein itself.
2.9 Chemical modification of phase polymers
The chemical modifications to PEG, like covalent bonding of fatty acid chains,
charged groups, hydrophobic derivatives and biospecific affinity ligands, have
considerable effect on partition behavior of proteins in the PEG-rich phase
(Raghavarao et al., 1995). Charged PEG derivatives such as trimethylamino-PEG
(TMA-PEG) and sulfonate-PEG (S-PEG) gives the information about the net charges
and isoelectric point of proteins as well as particles. Johansson and Shanbhag (1984)
observed that the increase in concentration of the derivatized PEG resulted in 10
times increase in partition coefficient compared to that of normal PEG. Many
researchers employed hydrophobic polymer derivatives during the partitioning study
of the biomolecules in ATPS.
In recent years metal ion and dye binding affinity partitioning was widely used
to enhance the partitioning of various biomolecules such as human hemoglobin,
bovine hemoglobin, whale and horse myoglobins, lactate dehydrogenase, glucose-6-
phosphate dehydrogenase and hexokinase (Wuenschell et al., 1990; Chung and
Arnold, 1990; Fernandes et al.,2002; Xu et al., 2002).
2.10 Equipments for extraction of biomolecules
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Transport phenomenon (mass, momentum and heat transfer as well as their interaction
with each other) is having considerable importance in ATPE and plays a major role in the
efficient design of extraction equipment. Mechanical agitated contactors and column
contactors (in which the phase separation occurs by gravity), which are commonly used in
the chemical industry for organic/aqueous phase extraction, can be conveniently adopted
for ATPE. The later by eliminates the use of expensive centrifuges.
For carrying out aqueous two-phase extraction of biomolecules, spray column
is one of the simple equipment, which is easy to operate even in continuous mode.
Use of spray columns for the extraction of various biomolecules such as bovine
serum albumin, horse radish peroxidase was demonstrated well in the literature
(Raghavarao et al., 1991; Srinivas et al., 2002b). Packed column, Perforated rotating
disc contactor, Graesser raining bucket contactor, York-Sheibel column are a few
equipments for continuous extraction of biomolecules employing ATPE (Jafarabad et
al., 1992; Coimbra et al., 1994; Porto et al., 2000; Sarubbo et al., 2003; Igarashi et
al., 2004; Zuniga et al., 2005; Zuniga et al., 2006).
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2.11 Extraction of biological products
Aqueous two-phase extraction has been employed for the extraction and
purification of biological materials such as proteins/enzymes, nucleic acids, viruses,
cell organelles etc. A large number of applications along with thermodynamic
properties of phase systems are reported in the literature. (Kula et al., 1982; Walter et
al., 1985; Albertsson, 1986; Diamond and Hsu, 1992; Zaslavasky, 1995; Raghavarao
et al., 1998; Hatti-Kaul, 2000). Furthermore, ATPE has been recognized as energy
efficient and a mild separation technique for product recovery in biotechnology. In
some cases ATPE has potential to achieve the desired purification and concentration
of the product even in a single step. In others, it is achieved in multiple steps. It
reduces the volume of the crude extract after partial purification in order to employ
more selective and expensive purification methods for final purification, depending on
the need. Thus it is recognized more as a primary purification step.
The basis of separation in the aqueous two-phase system is the selective
distribution of different substances in the two phases. The partitioning of small
molecules is even in both the phases and that of macromolecule is variable, whereas
the partitioning of particles being relatively one-sided. The distribution of
biomolecules is governed by various parameters relating to the properties of the
phase system and the partitioning substance as well as the interaction between the
two. Hence, the prediction of partitioning becomes a difficult task especially for the
large molecules. The partitioning of proteins/enzymes in ATPS is affected by net
charge on the biomolecule and the interaction of water with phase forming
components (polymers and salts) and proteins plays a key role in protein partitioning.
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The partitioning of biomolecules can be made selective by changing the system
properties to make a particular kind of interaction predominant. The multiplicity of
factors contributing to partitioning also makes the system very versatile, in contrast to
the other conventional separation techniques like centrifugation, electrophoresis etc.,
allowing the fractionation of molecular or particulate species differing very slightly
from each other. Thus, fractionation by partition in ATPS may often be used to
substitute other separation procedures.
2.11.1 Extraction of enzymes
Generally extraction and purification of enzymes involve a number of steps
such as filtration, centrifugation, precipitation, chromatography, electrophoretic
techniques, crystallization, etc. causing loss of yield at each step, there by affecting
adversely the overall productivity. ATPE has been employed as an alternative for the
large scale purification of several products, including recombinant products. In the
past three decades a wealth of information has been reported in the literature on
various aspects of ATPE for the isolation of many biological products. A few
examples are listed in Table 2.
2.11.2 Extraction of natural colorants
In recent years, interest in natural colorants has increased considerably,
mainly because of the apparent lack of toxicity and eco-friendliness. Natural and
synthetic colorants are used in medicines, foods, clothes and in other products.
However, the natural colorants that are permitted for human foods are very limited,
and the approval of new sources is often difficult. This is mainly because the US
Food and Drug Administration (FDA) considers the pigments as additives and hence
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are under strict regulations. Consumers are aware of the toxicological effects
associated with synthetic colors and hence the use of natural colorants has been
increasing. Some of the mainly used natural colorants are carotenoids, anthocyanins,
betalains, chlorophylls, phycobiliproteins, turmeric etc. The feasibility of ATPE for the
purification of natural colorants and synthetic dyes were demonstrated elsewhere
(Tong et al., 1999; Wang et al., 1992). Natural colorants such as betalains, C-
phycocyanin, Carmine etc. were successfully purified using ATPE comprising PEG
and inorganic salts (Patil and Raghavarao, 2007a; Chethana et al., 2008; Magestea
et al., 2009).
2.12 Advances in aqueous two-phase extraction
2.12.1 Affinity partitioning
Affinity based separations include precipitation, membrane based purification
and two-phase/three-phase extractions. Affinity partitioning (AP) in aqueous two-
phase systems is based on the preferential/biospecific interaction between the
molecule and affinity polymer derivative. The interaction results in a biomolecule-
polymer derivative complex which selectively partitions to one of the phases leaving
the contaminating substances or proteins in the other phase. Most of the reported
investigations regarding affinity partitioning pertain to polymer/polymer type ATPSs
(Diamond and Tsu, 1992). Only a few reports are available on polymer/salt type
ATPSs (Xu et al., 2002) mainly due to the interference of high salt concentrations
with the biospecific interactions (Raghavarao et al., 1995). AP is influenced by many
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factors such as the ligand concentration and its binding characteristics, concentration
and molecular weight of polymers, pH, temperature, salt type and concentration,
number of thionyl chloride, covalently linked to iminodiacetic acid (IDA), and the
specific metal ligand Cu2+ was attached to the PEG molecule, etc. (Johansson and
Joelsson, 1987; Teotia et al., 2004; Antov et al., 2006). AP reported use of
derivatives of polymer (PEG) for the purification of enzymes such as glucose
isomerase, peniciline acylase (Gavasane and Gaikar, 2003; Dolia and Gaikar, 2006).
2.12.2 Extractive bioconversion
‗Extractive fermentation‘ involves the use of ATPS-based in situ fermentation
processes. Integration of bioconversion and downstream processing steps not only
increases the productivity of the bioprocesses but also provides the possibility of
running the bioconversion in a continuous mode (Mattiasson, 1988). Extractive
bioconversion employing aqueous two-phase systems can improve certain existing
bio-processes to make them economically viable (Banik et al., 2003). Simultaneous
production and purification of a bio-product obtained through the use of enzymes or
microorganisms is the interesting feature of this technique. The advantages of such
technique include rapid mass transfer due to low-interfacial tension, ease of operation
in continuous mode, rapid and selective separation, biocompatibility, separation at
room temperature, easy and reliable scale-up of bench scale results to production
scale, eco-friendliness, suitability for systems with product inhibition and high yield of
biomolecules (Sinha et al., 2000).
Simultaneous extraction and purification of some biomolecules/enzymes such
β-galactosidases, Penciline G, amylase and serine proteinase, endoglucanase,
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cellulose, glyceraldehyde 3-phosphate dehydrogenase from various sources were
reported with better purity and yield. (Chen and Wang et al., 1991; Liao et al., 1999;
Ivanova et al., 2001; Ülger and Sağlam, 2001; Ülger and Cerakoglu, 2001; Rito-
Palomares and Lyddiatt, 2002).
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Figure 1: A model phase diagram
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Table 1: Components for the formation of aqueous two-phase systems (Madhusudhan et al., 2011)
Phase
systems
Component 1 Component 2
Polymer
/Polymer
phase
systems
Polyethylene glycol,
Polypropylene glycol, Polyvinyl
alcohol, Polyvinyl pyrrolidone,
Methyl cellulose, Ethyl
hydroxyethyl cellulose, Hydroxy
propyl dextran, Ficoll, Vinyl-2-
pyrrolidone-Guar gum
Dextran, Ficoll, Pullulan, Polyvinyl
alcohol, Reppal PES 100 (hydroxyl
propyl starch), Maltodexrin, Xanthan,
sodium polyacrylate (NaPA),
Gemini/SDS, Cashew-nut tree gum,
Mixture of 2-(dimethylamino) ethyl
methacrylate, t-butyl methacrylate, and
methyl methacrylate.
Thermo
separating
polymer
based phase
systems
Breox (Ethylene oxide–
propylene oxide), Ucon 50-
HB-5100, Poly(ethylene oxide-
co-maleic anhydride)
Dextran, Reppal PES 100, Potassium
phosphate, Ammonium sulphate,
polyvinyl alcohol,
Detergent
based phase
systems
Triton X114, Agrimul NRE
1205 (C12–18E5), Cetyl
trimethyl ammonium bromide
(CTAB), Triton X-114, Dodecyl
trimethyl ammonium Bromide
(DTAB),
Water, Sodium dodecyl sulfonate (AS),
Polyethylene glycol
Polymer /Salt
phase
systems
Polyethylene glycol,
Polypropylene glycol, Methoxy
polyethylene glycol, Polyvinyl
pyrrolidone, Derivatives of
PEG (PEG-benzoate (PEG-
Bz) PEG-phosphate (PEG-
PO4), PEG-trimethylamine
(PEG-tma), PEG-palmitate
(PEG-pal), and PEG-phenyl
acetamide (PEG-paa))
Potassium phosphate, Sodium sulphate,
Sodium formate, Sodium potassium
tortrate, Magnesium sulphate, Sodium
citrate, Ammonium sulphate, Ammonium
carbamate,
Alcohol based
phase
systems
Ethanol
2-propanol
1-propanol
Methanol
Acetone
Di-potassium hydrogen phosphate, Sodium
thiosulphate, Magnesium sulphate,
Ammonium sulphate, Sodium di-hydrogen
phosphate, Cesium Carbonate, Sodium
chloride, Tri-potassium phosphate, Calcium
chloride. Sodium carbonate
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Table 2: Enzymes purified by aqueous two-phase extraction (Madhusudhan et al., 2011)
Enzyme References
Peroxidase Miranda and Cascone, 1995, Srinivas et
al., 1999, 2002, Silva and Franco, 2000
Alcohol dehydrogenase Madhusudhan et al., 2008
Bromelain Babu et al., 2008
Lipoxigenase Lakshmi et al., 2009
Intracellular glyceraldehyde 3-
phosphate dehydrogenase
Rito-Palomares and Lyddiatt, 2002
Lipase Ooi et al., 2009
-glucosidase Gautam and Simon, 2006, Hemavathi and
Raghavarao, 2011
Amyloglucosidase Tanuja et al., 2000
Lysozyme Dembczynski et al., 2010
Plant-esterase Yanga et al., 2010
Papain Nitsawang et al., 2006
α-amylase Li et al., 2004
Endo-polygalacturonase Pereira et al., 2003
Phospholipase D Teotia and Gupta, 2004
Alpha galactosidase Gautam and Simon, 2008
Pepsin Imelio et.al., 2008
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3. Process Integration
When developing a downstream processing procedure at a production scale, it
is mandatory to consider processing time, energy, manpower, good manufacturing
practices (GMPs), recycling of chemicals, sterilization and cleaning of equipment
apart from separation efficiency.
Membrane processes such as microfiltration, ultrafiltration and reverse
osmosis are gaining importance in biotechnology for the purification and
concentration of biomolecules. Membrane processes are well suited for the
processing of biological molecules since they operate at relatively low (ambient)
temperature and pressure. Further, they do not involve phase changes or chemical
additives, thereby minimizing the extent of denaturation or degradation of
biomolecules. Current research and development efforts are directed towards
improvements in selectivity while maintaining the inherent high throughput
characteristics of membrane.
In recent years researchers in the area of downstream processing (i.e. the
recovery and purification of the product), are faced with a strong demand for
intensification and integration of process steps to increase yield, to reduce of process
time and to cut down the running costs and capital expenditure (Schugerl and
Hubbuch, 2005). Process integration, wherein two unit operations are combined into
one in order to achieve specific goals not effectively met by individual processes,
offers considerable potential benefits for the recovery and purification of biological
products (Rito-Palomares, 2004). Process integration is one of the most effective
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ways to increase the overall productivity of the process. However, not many
successful attempts are reported in this regard. The productivity of a given
bioprocess can be considerably improved by relatively new strategy of process
integration. It could be by design of extraction in such a way to integrate in itself,
various unit operations such as solid separation, purification, concentration etc. It also
could be integration of aqueous two-phase extraction with other processes (such as
fermentation, cell disruption and membrane processes) is one such approach which
gaining considerable attention by many researchers, in recent years.
Application of ATPS for extractive fermentation is a meaningful approach to
overcome low product yield in a conventional fermentation process having product
inhibition problems and by proper design of the two-phase systems it is feasible to
obtain the product in a cell-free stream. Recently, extractive fermentation using ATPS
have been developed for the recovery of different protein products that resulted in an
increase in the productivity (Guan et al., 1996; Li et al., 2000; Sinha et al., 2000).
Rito-Palomares and Lyddiatt (2002) have reported the integration of cell disruption
and aqueous two-phase systems for the recovery and purification of intracellular
proteins. There are a few research articles available on integration of ATPE with
membrane processes for the purification and concentration of various biological
products (Tanuja et al., 2000; Srinivas et al., 2002; Patil and Raghavarao, 2007b).
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4. Biomolecules (Enzymes)
Enzymes have played an important role in many aspects of life since the dawn
of time. In fact they are vitally important to the existence of life itself. Civilizations
have used enzymes over thousands of years without understanding what they are or
how they work. Over the past several generations, science has unlocked the mystery
of enzymes and has applied this knowledge to make better use of these amazing
substances in an ever-growing number of applications. Enzymes play crucial role in
producing the food we eat, the clothes we wear, even in producing fuel. Enzymes are
also important in reducing both energy consumption and environmental pollution.
Enzymes are biocatalysts produced by living cells to bring about specific
biochemical reactions generally forming parts of the metabolic processes of the cells.
Enzymes are highly specific in their action on substrates and often many different
enzymes are required to bring about, by concerted action, the sequence of metabolic
reactions performed by the living cell. All enzymes which have been purified are
basically proteins in nature and may or may not possess a nonprotein prosthetic
group. Enzymes belong to the most interesting natural products from both scientific
and industrial perspectives. They are enjoying increasing popularity in the chemical
and pharmaceutical industries as environmental friendly, economical and clean
catalysts (Wahler and Reymond, 2001).
From the ancient times, enzymes played an important role in food production.
Today, nearly all commercially prepared foods contain at least one ingredient that
has been made involving enzymes. Some of the typical enzyme applications include
their use in the production of sweeteners, chocolate syrups, bakery products,
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alcoholic beverages, precooked cereals, infant foods, fish meal, cheese and dairy
products, egg products, fruit juice, soft drinks, vegetable oil and puree, candy, spice
and flavor extracts and liquid coffee as well as for dough conditioning, chill proofing of
beer, flavor development and meat tenderizing.
Enzymes play a significant role also in non-food applications. Industrial
enzymes are used in laundry and dishwashing detergents, stonewashing jeans, pulp
and paper manufacture, leather dehairing and tanning, desizing of textiles, deinking
of paper and degreasing of hides.
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a) Alcohol dehydrogenase
In recent years, enzymes belonging to the oxidoreductase group such as
alcohol dehydrogenases (ADH) have caught much scientific attention in the
pharmaceutical industries. They are highly desirable as biological catalysts in the
production of chiral pharmaceutical intermediates or building blocks in the organic
synthesis of drugs (Clark and Ensign, 2002; D‘Auria, 2003; Patel, 2004; Uria et al.,
2005). Use of such enzymes as biocatalysts offers many advantages over chemical
catalysts, including their high enantioselectivity and regioselectivity, their high
catalytic activity, the easy and reproducible up-scaling of the process, lower energy
consumption due to reduced process temperature, utilization of low cost raw
materials (such as aldehydes and ketones) and reduced application of organic
solvents (Julich Chiral Solutions-Germany). In addition, enzymes (including ADH) can
be immobilized and reused for many enzymatic cycles and they can be over-
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produced to make catalytic processes more economically efficient (Patel, 2004). This
trend plays an important role in the use of chiral drugs (D‘Auria, 2003).
Reaction and classification of ADH
Alcohol dehydrogenases (ADH; EC 1.1.1) are enzymes capable of catalyzing
the reversible conversion of alcohols to their corresponding aldehydes or ketones by
using a variety of cofactors. They can be found both in prokaryotes and eukaryotes.
They vary with respect to their structural organization and their catalytic mechanism
(Reid and Fewson, 1994). ADHs are oxidoreductases that are dependent on a variety
of different electron-mediating cofactors. Therefore, they can be classified on the
basis of their cofactor specificity (Reid and Fewson, 1994; Radianingtyas and Wright,
2003): (i) NAD(P)-dependent alcohol dehydrogenases (ADH), (ii) F420-/Zn-/PQQ-
dependent alcohol dehydrogenases and (iii) FAD-dependent alcohol oxidases. Of the
three ADH classes, the NAD (P)-dependent ADHs appear very abundant in the
Thermococcales and yeasts. This group uses NAD (P)+ and NAD (P)H as the
electron donor and acceptor for catalyzing the inter conversion of alcohols with the
corresponding aldehydes or ketones. In particular, it converts reversibly primary (1o)
alcohols into aldehydes and secondary (2o) alcohols into ketones (Figure 2). In
methanogenic archaea such as Methanoculleus thermophilicus an ADH has been
identified that uses the F420 cofactor (Radianingtyas and Wright, 2003).
The NAD(P)-dependent ADHs can be further subdivided according to the size
of their amino acid sequences into three distinct classes (Reid and Fewson, 1994;
Antoine, et al., 1999; Van der Oost et al., 2001; Klimacek, et al., 2003; Guy, et al.,
2003), namely: (i) type I, medium-chain dehydrogenase/reductases (MDR, ~350
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amino acids per subunit); (ii) type II, short-chain dehydrogenase/reductases (SDR,
~250 amino acids); and (ii) type III, long-chain dehydrogenases (LDH, ~ 360-550
amino acids, but often as many as 900 residues). The ADHs belonging to type I may
be dimeric in higher eukaryotes (Guy, et al., 2003) or tetrameric forms in bacteria and
yeast (Esposito, et al., 2002), represented by horse liver ADH and Saccharomyces
cerevisiae ADH I, II, and III (Antoine, et al., 1999). This type mostly contains zinc
(Esposito, et al., 2002; Guy, et al., 2003); so it is widely known with the name, zinc-
dependent mediate chain ADHs.
The ADH of type II have been charaterized from both prokaryotes and
eukaryotes (Reid and Fewson, 1994). Since the type II rarely contain metals,
including zinc (Guy, et al., 2003), it is commonly called zinc-independent short chain
ADH. The type III ADH seems to be exclusively of microbial origin. Since they show
little sequence homology with type I or II and are frequently activated by iron, they are
also referred to as iron activated long chain ADHs (Reid and Fewson, 1994).
Alcohol production
Alcohol production by fermentation occurs due to enzyme-catalysed
conversion of sugars or sugar-containing polymers, by micro-organisms.
Saccharomyces cerevisiae is the most commonly used yeast but Kluyveromyces
have also been employed. Among the bacteria, Zymomonas mobilis has been the
preferred organism for ethanol production. Sugars in these organisms are broken
down to pyruvic acid by one of the three pathways—the Embden-Mayerhoff-Parnas
(EMP) Pathway, the Hexose Mono Phosphate (HMP) pathway and the Entner-
Doudoroff (ED) pathway. The pyruvic acid formed, under anaerobic conditions is split
53
by pyruvate decarboxylase into acetaldehyde and CO2. Ethanol is then produced
from the acelaldehyde by reduction due to the enzyme alcohol dehydrogenase.
(Figure 3). Yeasts follow the EMP pathway and theoretically from 1 g of glucose, 0.51
g of ethanol can be obtained. When pure substrates are fermented, the yield is
generally about 95% and usually drops down to 91% when industrial raw materials
are used. Thus 100g of pure glucose will in practice yield 48.4g of ethanol, 46.6 g of
CO2, 3.3 g of glycerol and 1.2 g of yeast biomass (Dhamija and Sangwan, 2007).
ADHs in biosensors
The control of food quality and freshness is of growing interest for both
consumer and food industry. In the food industry, the quality of a product is checked
using conventionally techniques such as chromatography, spectrophotometry and
others. These methods are expensive, slow, need well trained operators and in some
cases, require steps of extraction or sample pretreatment, increasing the time of
analysis. The food and drink industries need rapid methods to determine compounds
of interest (Wagner and Guilbault, 1994). An alternative to facilitate the analysis in
routine of industrial products is the biosensors development.
The determination of alcoholic compounds, particularly of ethanol, is relevant
to the food industry, especially in alcoholic beverages such as beer, wines and spirits.
In the case of ethanol a number of enzyme-based electrochemical devices have been
developed. For this purpose two enzymes namely, alcohol oxidase and alcohol
dehydrogenase were employed. The alcohol dehydrogenase is a NADH depending
enzyme and the biosensors for ethanol based on this enzyme require the co-
immobilization of both enzyme and co-enzyme. In addition this coenzyme requires an
54
over potential of about 1 V for oxidation and at this potential a number of other
substances present in food samples, are also oxidized and can interfere in the
measurement. Different chemical mediators could be used successfully to decrease
the over potential as well as to prevent the electrode pasivation (Bala et al., 2002;
Svensson et al., 2005).
55
Immobilized ADH for the regeneration of cofactors (NAD)
Enzyme technology has dealt mainly with simple reactions which do not
require coenzymes. One of the major challenges for enzyme technologists is in the
application of cofactor-dependent enzymes. Over 25% of the known enzymes require
cofactors like NAD/NADH, ATP/ADP, etc. which, unlike the enzymes, undergo
stoichiometric changes during a biochemical reaction. The living cells have
circumvented this problem either by introducing certain cofactor-recycling enzyme
systems in their metabolic pathways or through their recycling via the electron
transport system in the case of aerobic metabolism. In vitro applications of such
enzymes will largely depend on developing efficient in vitro cofactor recycling
systems. These can be obtained by co-immobilizing another enzyme so that it can
recycle the cofactors, e.g. alcohol dehydrogenase in the presence of ethanol can be
used for the recycling of NAD to NADH in a bioprocess catalysed by a NADH-
dependent enzyme. Choice of the coupling enzyme for such application has been
made based on its ability to use an economical substrate like ethanol or formate
(formate dehydrogenase) as well as one which results preferably in a volatile
byproduct like CO2 or acetaldehyde so as to minimize the downstream processing
problems (Mosbach, 1974; Kolot, 1981; Tampion and Tampion, 1987; Hartmier,
1988; Uria et al., 2005)
Pharmaceutical potency of ADHs
ADHs have a promising future in the pharmaceutical industry since they can
be applied for the preparation of single enantiomers, which are chiral intermediates
required in drug synthesis. Varieties of ADHs have shown promising features and are
56
future catalysts to be used in the biocatalytic synthesis of various pharmaceutical
valuable chiral intermediates. Two examples of commercially available ADHs which
are often used by organic chemists for laboratory-scale synthesis are ADHs from
yeast (YADH) and from horse liver (HLADH). HLADH, for instance, can be applied in
the preparation of (1S, 2R)-cis-2-carboxymethyl-3-cyclopenten-l-ol lactone, an
interesting starting material for prostaglandin synthesis (Uria et al., 2005). However,
preparative applications using these two mesophilic enzymes are limited by their
instability at temperatures above 30oC, their rather narrow substrate specificity, their
high sensitivity to organic solvents and their tendency to lose activity during
immobilization (Hummel and Kula, 1989). Some other mesophilic ADHs such as
those from Candida parapsilosis, Pseudomonas sp., and Lactobacillus kefir, have
also been used in the preparative applications, but they also suffer the disadvantages
of a narrow substrate specificity, insufficient stereospecificity or sensitivity to organic
solvents (Inoue, et al., 2005).
The great potency of hyperthermophilic ADHs is also shown in the preparation of (S)-
2-pentanol, a key intermediate required in the organic synthesis of some potential
anti-Alzheimer's drugs (Patel, 2004). So far production of (S)-2-pentanol has been
demonstrated with Gluconobacter oxydans using 2-pentanone as the substrate
(Patel, 2004). However, a recent report has shown that the thermostable ADHs from
the hyperthermophile, P. furiosus is also capable of reducing 2-pentanone to the
corresponding (S)-2-pentanol (Figure 4a), which was determined by gas
chromatography analysis (Machielsen et al., 2006). In particular, its enantioselectivity
was observed when NAD+ was used as the cofactor. Then the enantiomeric excess
57
(‗ee‘) value of 89.4% was obtained, while the ‗ee‘ value was 84.8% when NADP+ was
used (Machielsen, et al., 2006). Properties such as high thermal stability, enantio-
selectivity and a broad substrate specificity (Uria, 2004; Machielsen et al., 2006)
make this enzyme an interesting candidate for biocatalysts.
Another example of the potential of hyperthermophilic ADHs to the
pharmaceutical industry is in the biocatalytic preparation of S-(-)-4-chloro-3-
hydroxybutanoic acid or (S)-4-chloro-3- hydroxybutonoate, a key intermediate
required for the total chemical synthesis of the cholesterol lowering agent, HMG-CoA
reductase inhibitor (Figure 4b). So far, preparation of such key intermediate has been
achieved by a secondary ADH from Pichia finlandica through the asymmetric
reduction of ethyl 4-chloroacetoacetate to (S)-4-chloro-3-hydroxybutonoate. (Patel,
2004).
The enzymatic preparation of the alcohol, (1S,2R)-[3-chloro-2-oxo-1-
(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester, a key intermediate
required for the synthesis of the potent HIV inhibitor Atazanavir is an example of the
potential of alcohol dehydrogenases in the pharmaceutical industry. The
diastereoselective reduction of the (S)-ketone, (1S)-[3-chloro-2-oxo-1-
(phenylmethyl)propyl] carbamic acid,1,1-dimethyl-ethyl ester to such alcohol by using
specific hyperthermophilic ADHs seems to be an attractive alternative way when
considering the general advantages offered by the ADHs (Figure 4c). So far
preparation of this chiral alcohol has already been demonstrated with Rhodococcus,
Brevibacterium and Hansenula strains through the enantiomeric reduction of (S)-2
ketone (Patel, 2004; Uria et al., 2005).
58
Two other examples of pharmaceutically important chiral compounds are (S)-
2-Octanol and Ethyl-(S)-4-chloro-3-hydroxybutirate. Here there also seems to be an
interesting possibility for their production by using hyperthermophilic ADHs (Figure 5).
Preparation of these two compounds have succesfully been demonstrated with ADH
from Candida parapsilosis (Zelinski, et al., 1999) and ADH from Rhodococcus
erythropolis (Wolberg, et al., 2000). Hyperthermophilic alcohol dehydrogenases could
also play a prominent role in the enantioselective reduction of phenyl trifluoromethyl
ketone (Figure 5), acetophenone, and 2-heptanone into their coressponding alcohols.
So far this was reported by Inoue, et al., (2005) for ADH from Leifsonia sp.
59
Figure 2: The interconversion of alcohols into the corresponding aldehysdes or ketones catalyszed by NAD(P)- dependent ADH (Benach, 1999)
60
Figure 3: Biosynthesis of ethanol.
61
Figure 4: Examples of biocatalystic synthesis of chiral intermediates;
a) Reduction of 2-pentanone into (S)-2-pentanol, a key intermediate in the synthesis of potential anti-Alzheimer,s desease.
b) 4-chloro-3-oxobutanoic acid methyl ester to S-(-)-4-chloro-3-hydroxy butanoic acid methyl ester and
c) Enzymatic prparation of a (1S,2R)-alcohol, a key intermediate in the sysnthesis of potential HIV inhibitor, Aatazanavir drug through the reduction of (S)-ketone.
62
Figure 5: Some examples of the potential of hyperthermophilic ADHs in the organic sysnthesis of chiral building blocks in the pharmaceutical industry.
a) Enzymatic preparation of (S)-2-Octanol.
b) Enzymatic preparation of Ethyl-(S)-4-chloro-3-hydroxybutirate,
c) Enzymatic preparation of phenyl trifluromethyl alcohol.
63
B) Invertase
Invertase from Saccharomyces cerevisiae was discovered by Bertholet in
1980. Invertase (β-D-fructofuranoside fructohydrolase, β-fructofuranosidase, sucrase,
invertin, saccharase; EC 3.2.1.26) of yeast is a glycoenzyme which catalyses the
hydrolysis of terminal non-reducing β- fructofuranoside residues in β-
fructofuranosides (Boyer, 1971).
As result of invertase action, the effective net rotation of the plane polarized
light changes from right to left due to higher levorotatory action of fructose(-92). Since
the rotation of the plane-polarized light in inverted from right to left, this enzyme is
64
called invertase. The equimolar amount of glucose and fructose is known as ‗invert
sugar‘.
Several bacteria, yeast and fungi have been shown to produce invertase.
Though there is ubiquitous distribution, the enzyme of commercial interest originates
from yeast species. However, commercially invertase is usually obtained by the yeast
strains of Saccharomyces cerevisiae or S. carlsbergensis. The name ―Invertase‖
includes heterogeneous invertase forms and, even within the same yeast culture,
invertase exits in more than one form carrying different amounts of glycosylated
groups (Boyer, 1971; Chu et al., 1983). The yeast S. cerevisiae provides both internal
and external invertase. The external invertase is in the highly glycosylated form and
found in the periplasmic space (Burger et al., 1961; Zech and Gorisch, 1995).
Periplasmic invertase is found mainly in the form of dimers, tetramers and hexamers
of molecular weights of approximately 260, 360, and 560 kDa, respectively (Gascon,
1968; Chavez, 1997). Internal invertase having molecular weight of 135 kDa is found
in the cell membrane and less glycosylated form with respect to the external
65
invertase (Chu et al., 1978; Tammi et al., 1987). The non-glycosylated invertase form
having molecular weight of 60 kDa is located intracellularly (Kern et al., 1992).
At high substrate concentrations (1M), invertase exhibits transferase activity,
transferring the β-D- fructofuranosyl residue to primary alcohols such as methanol,
ethanol and n-propanol.
Beta-D-fructofuranoside
Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated
enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-
glycosylation is necessary for oligomerization to tetramers and octamers.
Dimerization is required and sufficient to generate enzymatic activity; further
association does not alter the specific activity of core-glycosylated invertase,
suggesting that the active sites of invertase are not affected by the association of the
dimeric units.
Applications
The hydrolysis of sucrose which yields an equimolar mixture of glucose and
fructose (invert sugar syrup) is sweeter than sucrose due to high degree of
66
sweetness of fructose. Consequently the sugar content can be increased
considerably without crystallization of the material. Hence, one of the important
applications of invertase lies in the production of non-crystallizable sugar syrup from
sucrose. Due to its hygroscopic nature, invert syrup is used as a humectant in the
manufacture of soft centered candies and fondants. Enzymatic hydrolysis of sucrose
is preferable to acid hydrolysis as it does not result in the formation of undesirable
flavoring agents as well as coloured impurities. Additionally, the use of immobilized
invertase for the continuous hydrolysis of sucrose can be advantageous because the
shifts in the pH brought about as a result of immobilization can be exploited to
prevent the formation of oligosaccharides by the transferase activity associated with
the soluble enzyme (Wiseman, 1978).
Invertase is used also whenever sucrose containing substrates are subjected
to fermentation viz. production of alcoholic beverages, lactic acid, glycerol etc. Due to
the associated inulinase activity, it is also used for the hydrolysis of inulin (poly
fructose) to fructose.
Invertase, the most widely used enzyme for hydrolysis of sucrose in
membrane reactor (Tomotani and Vitolo, 2010) and is used for the preparation of
high fructose syrup (HFS) (Tomotani and Vitolo, 2007). The high-fructose syrup
(HFS) is largely employed as a sweetener in food and pharmaceutical industries as
well as the source for attaining crystalline fructose. Moreover, more than the sucrose
syrup, it has more desirable functional properties such as high osmotic pressure, high
solubility, a source of instant energy as well as preventing crystallization of sugar in
food products (Kurup, et al., 2005; Aranda, et al., 2006).
67
Other uses of invertase include manufacture of artificial honey, plasticizing
agents used in cosmetics, drug and paper industries, its use as an analytical probe in
determination of glucose in molasses, sucrose in serum/urine and detection of
mercury in very low concentrations. Production of lactic acid, glycerol, and ethanol
using molasses a by-product of the sugar industry
In view of the high commercial potential of the enzyme, several attempts have
been made to purify and obtain a stable enzyme preparation suitable for commercial
application.
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5. Aim and scope of the present work
Recent developments in biotechnology have opened up new avenues towards
the production of many biomolecules of importance for research, pharmaceutical/
clinical and industrial usage. In view of the recognized fact that product recovery
costs become critical in the overall economics of modern biotechnological processes,
there has been an increased interest in the development of efficient downstream
processing methods for the separation, concentration and purification of biomolecules
from fermentation and cell culture media. The conventional method of filtration for
solid-liquid separation depends on particle size and hence is unsuitable for
bioseparation in case of small microorganisms especially if the product is
intracellular. Conventional downstream processing methods involve multiple steps
which result in low yield. Further, scale-up problems are considerable making them
uneconomical unless the product is of high value. Therefore, there is a need for
alternative approaches to the problem. Aqueous two-phase extraction (ATPE) is one
such method. It is a technique that has been known for quite some time but its
importance and applications are being realized only in recent years.
During this investigation, effect of various process conditions on partitioning of
ADH and invertase in aqueous two-phase systems were investigated. Integration of
ATPE and membrane process were employed for the selective separation and
purification of target biomolecules. Incorporation of nanoparticles in order to favorably
alter the partitioning of target biomolecules has been explored. Electroextraction
studies were carried out for enhancing the selectivity of extraction and also the phase
demixing rate of the equilibrated phases. The results obtained were consolidated in
69
the form of a Ph.D. thesis, spread over four different chapters. Each chapter is
systematically divided into introduction, materials and methods, results and
discussion and conclusions followed by the tables and figures.
Complete study on partitioning and purification for ADH and invertase
employing ATPE was demonstrated and explained in Chapters 1 and 2. The
influence of various process parameters on performance of the given membrane
process (feed rate, membrane pore size, operation pressure) was evaluated.
Similarly, the effect of different process parameters such as phase forming salt, pH,
polymer molecular weight, phase composition, phase volume ratio on the extraction
efficiency were analyzed. Incorporation of nanoparticles (such as
clay/alumina/gold/silver) was attempted for enhancing the partitioning and selectivity
in the extractions.
Chapter 3 explains the efforts made in order to improve the overall productivity
of the process by integrating two unit operations. ATPE followed by membrane
process was employed for the selective separation and purification of target
biomolecules (ADH and Invertase).
Electroextraction of model protein (BSA) and real system (ADH) was also
explored for enhancing the selectivity of extraction and also the phase demixing rate
of the equilibrated phases as described in Chapter 4.
Overall results and conclusions were given at the end followed by supporting
references.