28

General Introduction

29

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).

30

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

31

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

32

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).

33

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

34

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

35

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.

36

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

37

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).

38

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.

39

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

40

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

41

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,

42

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).

43

Figure 1: A model phase diagram

44

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

45

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

46

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

47

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).

48

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,

49

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.

50

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-

51

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

52

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.