ISOLATION PARTIAL PURIFICATION AND CHARACTERIZATION OF Α-AMYLASE FROM BACILLUS ALCALOPHILUS

ABSTRACT

The aim of this study was to isolate, partially purify and characterize α-Amylase from Bacillus alcalophilus. The enzyme (α-amylase) was isolated from Bacillus alcalophilus using cassava as only carbon source. 20 g of soil sample were weighed out and dissolved in 40 ml of distilled water in a clean conical flask, mixed vigorously and heated at 60oC for 60 min in water bath and taken as the stock culture. From the stock preparation, ten folds serial dilution were carried out and the 10-4 to 10-6 dilutions were plated out in a media plate. Percentage ammonium sulphate saturation, ammonium sulphate precipitation and gel filtrations were carried out to partially purify α-amylase from Bacillus alcalophilus. The α-amylase was then characterised by studying the effect of pH, change in temperature, substrate concentration and metal ion on the enzyme`s activity. The specific activity of the crude enzyme was 160.26 U/mg proteins. After ammonium sulphate  precipitation  and  gel  filtration,  the  specific  activity  were  found  to  be  88.9  and 285.9U/mg protein, respectively. The optimum pH was found to be 7.5 and 70°C respectively. The α-amylase activity was found to be enhanced by Ca2+, Mg2+, Mn2+and Co2+, whereas Fe2+ was found to have inhibitory effect. The enzyme retained more than 80% of its activity at 60 min in the presence of Ca2+, Mg2+ , Mn2+and Co2+,  and  lost up to 90% of its activity in the presence of Fe2+. In this study, Ca2+ maintained more stability of the enzyme than all other metal ions. The Michaelis-Mentens constant (Km) and maximum velocity (Vmax) obtained from the Line Weaver Burk plot of initial velocity data of different substrate concentrations were 1.159 mg/mL and 16.24 μmol/min respectively. In conclusion, this study revealed the potentials Bacillus alcalophilus to serve as other source of α-amylase, especially for industrial purposes.

CHAPTER ONE

INTRODUCTION

Enzymes are produced by plants, animals and microorganisms. Microbial enzyme production is of great importance as they are more economical to produce, calculable, tractable and stable (Burhan et al., 2003). Amylases are very important family of enzymes that hydrolyze starch into dextrins and small polymers of glucose. Two major classes of amylase, mostly identified amongst microorganisms are α-amylase and glucoamylase (Vijayabaskar et al., 2012). The β-Amylase, mostly from plant origin has also being recorded from microbial source (Pandey et al., 2000). The use of  α-amylase in  some industries especially in food, beverage,  textiles, leather and  paper industries is increasing. There is a need for other source of the enzyme to be discovered as Nigeria is a country which is rich in natural resources, particularly the microbes as enzyme producers. Over the years, amylases have been isolated from bacteria, fungi and actinomycetes. Bacterial amylases are mostly reported from thermophilic, acidophilic and alkalophilic bacteria (Kim et al.,

1995). They are commercially available and have replaced chemical hydrolysis of starch to a great extent in industries (Pandey et al., 2000). Bacillus alcalophilus, B. substilis, B. licheniformis, B. amyloliquifaciens and B. stearothermophilis are most prominent among the bacterial sources of amylase (Carlos et al., 2002).

α-Amylase is a ubiquitous enzyme produced by plants, animals and microbes, and they play an important role in carbohydrate metabolism (Swetha et al., 2006). Amylase (1, 4-α-D-glucano hydrolase; E.C 3.2.1.1) is applied in food, paper and clothing industries. They are also applied idustrially in fermentation such as brewing, baking, digestive acid production, fruit juice, starch syrups and chocolate cake’s production (Pandey et al., 2000). Several reports on starch degrading microorganisms from different source and respective amylase activity have been reported (Nguyan et al., 2002; Balkan and Ertan, 2005). The soil is one of the richest sources of starch degrading microorganisms as it contains starchy substances required for the continuous microbial growth and reproductive life. pH and thermal stability are very important factors considered in industrial enzymatic bio-reactions, most limitations in the applications of enzymes industrially could be attributed to these factors. Most industrial application of α-amylase (e.g. in starch liquefaction industries) takes place at high temperature ranges and during the liquefaction-saccharification

process, bye-products are given off which can lower the pH of the reaction medium. Stabilization of these parameters (pH and temperature) in α-amylase is of great importance due to the high industrial utilization of the enzyme. Most of the α-amylases reported till dates are metal ion- dependent enzymes and these metal ions are known to be stabilizers for amylases isolated from various microorganisms (Sudha, 2013). By various ways, these metal ions affect enzyme catalysis. They can act by modifying the electron flow in the enzyme substrate reaction or by changing the orientation of the substrate with reference to specific functional groups at active site (Singh et al.,

2014). Also, these metal ions can accept or donate electrons and act as electrophiles, mask nucleophiles to prevent unwanted side reactions, bind enzyme and substrate by coordinate bonds, hold the reacting groups in the required orientation, and simply stabilize a catalytically active conformation of the enzyme (Sudha, 2013; Singh et al., 2014). These stabilizing effects generally simplify industrial procedures during the downstream processing and help reduce the production of compounds that increase pH of the reaction medium.

1.1       Amylase Family

1.1.1     α-Amylases

The α-amylases (EC 3.2.1.1 ) (alternative names: 1,4-α-D-glucan glucanohydrolase; glycogenase) are calcium activated enzymes. By acting at random locations along the starch chain, α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and “limit dextrin” from amylopectin. α-amylase can act anywhere on its substrate, as a result, it tends to be faster in action than β-amylase. In animals, it is a major digestive enzyme, and its optimum pH is 6.7–7.0 (Ramasubbu et al., 1996). In human physiology, both the salivary and pancreatic amylases are α-amylases. α-amylases are also found in plants, fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus)

1.1.2    β-Amylase

Another form of amylase, β-amylase (E.C. 3.2.1.2 ) (α-1,4-D-glucan maltohydrolase; glycogenase; saccharogen amylase) can also be synthesized by bacteria, fungi, and plants. Working from the non-reducing  end,  β-amylase  catalyzes  the  hydrolysis  of  the  second  α-1,4  glycosidic  bond, cleaving off two glucose units (maltose) at a time. During the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet flavor of ripe fruit (Rejzek et al., 2011). Both α-amylase and β-amylase are present in seeds; β-amylase is present in an inactive form prior to germination, whereas  α-amylase  and  proteases  appear  once  germination  has  begun.  Many  microbes  also produce β-amylase to degrade extracellular starches. Animal tissues do not contain β-amylase, although it may be present in microorganisms contained within their digestive tract. The optimum pH for β-amylase is 4.0-5.0 (Rejzek et al., 2011).

1.1.3    γ-Amylase

γ-amylase (EC 3.2.1.3 ) (alternative names: Glucan α-1,4-glucosidase; amyloglucosidase; Exo-1,4- α-glucosidase;  glucoamylase;  lysosomal  α-glucosidase;  α-1,4-D-glucan  glucohydrolase)  will cleave α(1–6) glycosidic linkages,  as well as the last  α(1–4) glycosidic linkages  at the non- reducing end of amylose and amylopectin, yielding glucose. The γ-amylase has the most acidic optimum pH of all amylases because it is most active around pH 3.0 (Maton et al., 1993).

1.1.1.4 The α-Amylase Family

The α-amylase family i.e. clan glycoside hydrolase (GH-H) is the largest family of the glycoside hydrolases, transferases and isomerases comprising almost thirty different enzyme specificities (Henrisaat, 1991; Swetha et al., 2006). A variety of enzymes are able to act on starch. These enzymes can be basically classified into four groups:

        Endo amylase

        Exo amylase

        Debranching amylase

        Transferase amylases (Vander Maarel et al., 2002).

1.1.1.4.1 Endo Amylase

The endo amylase just like other known as α-amylase group are those amylase that catalyze the internal hydrolysis of α- 1,4 -o- glycosidic bonds which results in the formation of alpha anomeric products (Swetha et al., 2006).

1.1.1.4.2 Exo Amylases

The exo amylases are those amylases that catalyze the cleavage of either α- 1,4 or α- 1,6 linkages of external glucose residues resulting in  either alpha or beta anomeric products (Van der maarel et al., 2002).

1.1.1.4.3 Debranching Amylase

The debranching amylase catalyzes the exclusive hydrolysis of α- 1, 6 bonds, leaving long chain polysaccharides (Van der Maarel et al., 2002).

1.1.1.4.4 The Transferase Amylases

These are a group of amylases that catalysis the cleavage of α- 1,4 glycosidic bond of the donor molecule or compound and still catalysis its transfer to a glycosidic acceptor forming new glycosidic  bonds  mostly 1,6  glycosidic  bond  (Swetha  et  al.,  2006).  Generally,  the  glycoside hydrolases are able to metabolize large varieties of saccharides. They have been classified into classes based on their mode of action, families based on their amino acids sequence compositions and similarities with membered families. Most of the starch converting enzymes belong to the glycoside hydrolase thirteen family (GH13). The GH13 family can be further classified based on a larger or higher unit called clan, which is the three dimensional structure of catalytic module. A clan consists of two or more families with the same three dimensional structures of catalytic domain but with usually limited sequence similarities, thus indicating that protein structure is best conserved by evolution than amino acid sequences. Among the known fourteen clans ranging from A-N defined for glycosidase and trans glycosidase, α- amylase family belongs to the eight clan i.e GH-H (MacGregor, 2005). This idea was proposed by Takata et al. (1992). Members of this family satisfy the following requirements such as:

    They must act on α- 1,4 glycoside linkages and hydrolyze them to form an α-anomeric monosaccharide and oligosaccharide or produce α-glucosidic linkages by trans glycosylation.

    They  have  four  strongly  conserved  regions  in  their  primary  structures  consisting  of catalytic and substrate binding sites (Allosteric).

        Comprises of the following amino acids: Aspartate (Asp 206), Aspartate (Asp 297) and

Glutamate (Glu 230) at corresponding positions in their catalytic domain.

        They should possess an (alpha/ Beta)8 or Tim barrel catalytic domain.

1.2       Sources of α- Amylase.

α-amylase are produced by plants, animals and microbes, they play vital roles in carbohydrate metabolisms. It is widely known that amylase from microbes (bacteria and fungi) have been widely and greatly exploited and those from plants are less exploited. Amylases from plants and microbes have been applied for centuries as food additives. Barely amylase (malted barley) have been used in brewing industries, fungal amylases have been used for preparation of oriental foods (Burhan et al., 2003). Among the fungal classes, strains of fungi from the zygomycete class,

Ascomycete class and the Deutromycete class greatly produced extracellular amylase with unique properties of thermo stability and pH stability (Silva et al., 2005; Shen et al., 2012). Among the bacteria, strains of bacteria e.g B. alcalophilus, B. substilis, B. licheniformis, B. amyloliquefaciens and B. stearothermophilus are known to be good producers of alpha amylases with thermo stability properties and slight pH tolerant; these have been widely used for commercial production of enzymes for various applications (Swetha et al., 2006).

1.3       Uses of α-Amylase

1.3.1    Uses of Amylase in Fermentation

Alpha and beta amylases are important in brewing beer and liquor made from sugars derived from starch. In fermentation, yeast ingest sugars and excrete alcohol. In beer and some liquors, the sugars present at the beginning of fermentation have been produced by “mashing” grains or other starch sources (such as potatoes). In traditional beer brewing, malted barley is mixed with hot water to create a “mash,” which is held at a given temperature to allow the amylases in the malted grain to convert the barley’s starch into sugars (Sodhi et al., 2005). Different temperatures optimize the activity of alpha or beta amylase, resulting in different mixtures of fermentable and unfermentable sugars. In selecting mash temperature and grain-to-water ratio, a brewer can change the alcohol content, mouth feel, aroma, and flavor of the finished beer. In some historic methods of producing alcoholic beverages, the conversion of starch to sugar starts with the brewer chewing grain to mix it with saliva (Jochanan, 1998). This practice is no longer in use.

1.3.2    α-Amylase as Flour Additive

Amylases are used in bread making and to break down complex sugars, such as starch (found in flour), into simple sugars. Yeast then feeds on these simple sugars and converts it into the waste products of alcohol and CO2. This imparts flavour and causes the bread to rise. While amylases are found naturally in yeast cells, it takes time for the yeast to produce enough of these enzymes to break down significant quantities of starch in the bread. This is the reason for long fermented dough such as sour dough. Modern bread making techniques have included amylases (often in the form of malted barley) into bread improver, thereby making the process faster and more practical for commercial  use  (Maton  et  al.,  1993).  Alpha amylase  is  often  listed  as  an  ingredient  on

commercially package milled flour. Bakers with long exposure to amylase-enriched flour are at risk of developing dermatitis or asthma (Mapp, 2001).

1.3.3    Application of α-Amylase in Molecular Biology

In molecular biology, the presence of amylase can serve as an additional method of selecting for successful integration of a reporter construct in addition to antibiotic resistance. As reporter genes are flanked by homologous regions of the structural gene for amylase, successful integration will disrupt the amylase gene and prevent starch degradation, which is easily detectable through iodine staining.

1.3.4    Therapeutic Uses

Amylase also has medical applications in the use of Pancreatic Enzyme Replacement Therapy (PERT). It is one of the components in Sollpura (Liprotamase) to help in the breakdown of carbohydrates into simple sugars (Sollpura, 2015).

1.3.5    Other Uses

An inhibitor of alpha-amylase, called phaseolamin, has been tested as a potential diet aid (food additive). Amylase has E number E1100, and may be derived from swine pancreas or mould mushroom (Hardy and Madsen 2004). Bacilliary amylase is also used in clothing and dishwasher detergents to dissolve starches from fabrics and dishes. Factory workers who work with amylase for any of the above uses are at increased risk of occupational asthma. Five to nine percent of bakers have a positive skin test, and a fourth to a third of bakers with breathing problems are hypersensitive to amylase (Mapp, 2001).

1.3.6    Hyperamylasemia

Blood serum amylase may be measured for purposes of medical diagnosis. A higher than normal concentration of amylase may reflect one of several medical conditions, including acute inflammation of the pancreas (it may be measured concurrently with the more specific lipase), perforated  peptic  ulcer,  torsion  of  an  ovarian  cyst,  strangulation,  ileus,  mesenteric  ischemia, macro-amylasemia and mumps. Amylase may be measured in body fluids such as urine and

peritoneal fluid. A January 2007 study from Washington University in St. Louis suggests that saliva tests of the enzyme activity could be used to indicate sleep deficits, as the enzyme increases its activity in correlation with the length of time a subject has been deprived of sleep (St. Louis Record, 2007).

1.4       Methods of Production of α- Amylase from Microbial Sources.

Generally, enzyme from microbial sources are generally secreted and produced in a bioreactor otherwise known as fermenters, through fermentation. Basically there are two approaches to this method:

        Solid state fermentation

        Submerge fermentation (Swetha et al., 2006)

The two approaches, mentioned above are suitable for production of α- amylase.

1.4.1     Solid State Fermentation.

Just as the name suggests, solid state fermentation can generally be defined as the growth of micro- organisms on solid substrate with negligible free water; the solid substrate provides support or nutrients or both for the enzyme and the microbe from which the enzyme is secreted from (Pandey,

1992). This technique is mostly confined to processes involving fungi. In a wider sense several alpha amylase have been successfully isolated from bacterial growth. Solid state fermentation has been documented in many natural fermentation, this is limited to the genus Bacillus (Babu and Satyanarayana, 1995).

1.4.2    Submerge Fermentation

This technique has been long used in α- amylase production. This involves the growth of organism in a synthetic media as nutrient broth, soluble starch as well as other component which enhances the secretion and production of α- amylase from the microbe (Swetha et al., 2006).

1.5        Bacillus alcalophilus Scientific Classification Kingdom:       Bacteria Division:         Firmicutes Class:              Bacilli

Order:             Bacillales Family:           Bacillaceae Genus:            Bacillus

Type species:   B.alcalophilus (Vedder, 1934).

Bacillus alcalophilus is a gram positive rod shaped bacteria. Like strains have been isolated from highly alkaline waste water. It is a moderate halotolerant obligate alkaliphile that grows at 40oC and at pH 9-10.5 (and possibly higher than that). It has been isolated from soil, animal manure and waste waters (Ntougias et al., 2006; Lewis, 1982).

1.6       Process Optimization for Production of α- Amylase

Media manipulation and optimization of parameters for secretion of an enzyme is one of the most significant  techniques  used  for  the  mass  production  of  enzyme  especially  to  meet  industrial demands (Tanyildizi et al., 2005). α- Amylase produced from bacteria is known to depend on both the morphological and metabolic state of the culture. Microbial growth i.e the body mass of bacteria is crucial for extracellular enzymes like α- amylase production (Swetha et al., 2006). Various physical and chemical factors have been known to affect the production of α- amylase, these are:

        pH

        Temperature

        Metal ions concentrations

        Period of incubation

        Carbon sources acting as an inducers

    Surfactants, Moisture, Nitrogen sources, Phosphorus sources and agitation with respect to solid state fermentation and submerge fermentation.

Interactions of these parameters in the culture media are reported to have a significant influence on

the production of α- amylase (Vijayaraghavan et al., 2011).

1.6.1    pH

pH of a medium, is a measure of hydrogen ion concentrations present in that particular medium. It is an important factor that determines the growth and morphology of microorganism (which are vessels that house the enzyme) as they are sensitive to the concentrations of hydrogen ions present in their medium (Paula and Perola, 2010). Earlier studies have revealed that bacillus alcalophilus require an alkaline pH of about 9-10 for growth while other bacteria adapt well at a neutral pH range for optimum growth (Paula and Perola, 2010). Also Shen et al. (2012) reported that pH affects the secretion of α- amylase as well as its stabilities. Fungi of Aspergillus species such as A. oryzae, A. fucum and A. niger were found to give significant yield of alpha amylase at pH 5.0-6.0 using submerge fermentations (Carlsen et al., 1996b; Dakhmouche et al., 2005), while amylase produced  from  the  yeast  strains  such  as  Saccharomyces  cerevisiae  and  S.  Kluyveri  exhibit maximum enzyme production at pH 5.0 (Knox et al., 2004).

1.6.2    Temperature

The impact of temperature on α- amylase production is related to the growth of the microorganism. Generally, the class of Bacillus being an extremophilic bacteria, requires an extreme range of temperature for optimum enzyme production mostly temperature ranges of 50-80οC (Silva et al.,

2005; Shen et al., 2012). Meanwhile, starch metabolizing α- amylase from other bacteria like Bacillus  substantilis  species  and  Saccharomyces  species   yield  α-  amylase  maximally  at temperature range of 25-37οC (Francis et al., 2003; Moller et al., 2004) as was reviewed by Swetha et al. (2006).

Carbon, nitrogen and other essential nutrient (sulphur and phosphorus) sources in the culture media are very important for the maximum α- amylase production by microbes. Carbon sources such as starch, galactose, glycogen and inulin have been reported as suitable substrate for the production of α- amylase from microbes like bacteria and fungi (Vijayaraghavan et al., 2011; Shen et al., 2012). Nitrogen sources such as soya beans, peptone, Ammonium nitrate, yeast extract, casein are promising nitrogen sources for maximal yield of α- amylase (Swetha et al., 2006).

Comparing the impact of organic and inorganic nitrogen sources on the production of alpha amylase as reported by Aiyer, (2004) brought to our attention that organic nitrogen sources e.g Casein, yeast extracts, peptone are much required for the production of alpha amylase than inorganic nitrogen sources. Nwagu and Okolo, (2011) reported that a combination of organic and inorganic  nitrogen  sources  have  more  promising  effects  in  the  enzyme  production.  Other nutritional supplement like sulphur can be obtained from the Epsom salts (MgSO4), iron sulphates and manganese sulphates are included in the fermentation media for α- amylase production also with the phosphorus sources e.g the sodium or potassium hydrogen phosphates (Silva et al., 2005; Oziengbe and Onilude, 2012).

1.6.3    Metal Ions

Supplementation with salts of certain metals provides better growth of microorganism and good enzyme production. It is recorded that most α- amylase are metal activated i.e. its active sites are bound to metal ions which stimulate its activities and stabilities (Sudha, 2012). Calcium ions are reported to be present in majority of these α- amylases. Addition of salts of calcium chloride (CaCl2) and sodium chloride (NaCl) had positive effects on α- amylase production as recorded in solid state fermentation using Amaranthus grain as substrate (Vishwanathan and Surlikar, 2001).

1.6.4    Moisture

Moisture  is  one  of  the  most  important  parameters  in  the  growth  of  organisms  for  enzyme production especially in solid state fermentation (Shen et al., 2012). Low and high moisture levels of the substrate affect the growth of the microorganism resulting in lower enzyme production. High  moisture  content  results  to  reduction  in  substrate  porosity,  changes  in  the  structure  of substrate particles and reduction of gas volume (Swetha et al., 2006). The effects of moisture on α- amylase production as reported by Vijayaraghavan et al. (2011) revealed that fungal α-amylase has maximum production at moisture content of about 60-76%. Thus the production decreases as the moisture content decreases around 40-50%, implying that the production of α- amylase from microbe requires high moisture content.

1.6.5    Particle Size of Substrate

In solid state fermentation, particle size of the substrate affects growth of the organism and thereby influences the degree of enzyme production. The adherence and penetration of microorganisms as well as the enzyme action on the substrate depends clearly upon the physical properties of the substrate  such  as  its  crystalline  or  amorphous  nature,  accessible  area,  surface  area,  porosity, particle size, etc. In all the above parameters, particle size plays an important role because all these factors depend on it (Pandey, 1991). Smaller substrate particles have greater surface area for microbial growth but inter-particle porosity is lower. For larger particle sizes, the porosity is greater but the saturated surface area is smaller. Therefore, determination of particle size corresponding to optimum growth and enzyme production is necessary (Pandey, 1991). A mathematical model was described, which enables the quantification and prediction of the extent of metabolism  of the  substrate particles  with  fermentation  time  for any given  initial  size of substrate particles. Studies conducted on the release of α-amylase have been  recorded  and a gradual increase was noticed up to 72 h for A. niger and 36 h for B. coagulans. The reduction of particle size was about 60 % at the end of the fermentation time, 72 and 36 h for A. niger and B. coagulans, respectively. Wheat bran having a particle size between 500–1000 μm amounted to better enzyme yields by B. subtilis when compared with the larger particles (Baysal et al., 2003).

1.6.6    Oxidative Stress and Amylase Production

Increase in enzyme production must be reached without actually altering the economics of enzyme production. Oxidative stress is induced and used to enhance enzyme production (Swetha et al.,

2006). This oxidative stress occurs when the production rate of reactive oxygen species (ROS) goes beyond the capacity of the cell for disposal. Reactive oxygen species inducing agents such as hydrogen peroxide (H2O2) and hypochlorous acid showed increased specific levels of α- amylase production. This is reported to be due to the adaptive response of microorganism under oxidative stress (Mishra et al., 2005).

1.6.7    Purification

Downstream processing for the production of the pure α- amylase can generally constitute a major percentage of overall production cost, especially if end purity requirements are stringent. Purification  processes  in  downstream  processing  after  fermentation  strongly  depends  on  the market, processing cost, final quality, and available technology. Most enzymes are purified by chromatographic techniques after crude isolation by precipitation and membrane separations as shown in Table 1 below. The need for large-scale and lesser cost effective purification of proteins have resulted in evolution of techniques that provide fast, efficient and economical protocols in fewer processing steps (Amritkar et al., 2004). Table 1 shows a chart for purification techniques that produced homogeneous preparation of amylases in a single step. These techniques are briefly explained bellow;

1.6.7.1 Affinity Adsorption Chromatography

Life is based on specific interactions between biomolecules. The underlying affinities form the basis  for  molecular  recognition  events  that  make  up  the  complex  machinery  of  all  living organisms, including man. In fact, the genome project (Lander et al., 2001; Venter et al., 2001) has taught us that the number of protein-coding genes is probably as few as between 21,000 (Clamp et al., 2007) and 23,000 (Birney et al., 2007) and this reinforces the notion that cellular processes are built up by complex networks of specific interactions. The affinities between various molecules in biological systems range from low-affinity interactions to very high-affinity interactions in the picomolar range. The interactions can be transient, such as the molecules in signal pathways, or very stable, such as heterodimer-forming protein complexes or multicomponent organelles, such as ribosomes or proteosomes.  In life science, affinity has been used as  a tool to study cellular processes in normal and disease tissues, but it has also been used to develop products for diagnoses and therapeutics.

The use of affinity for purification of proteins through chromatography was first described in the late 60s (Cuatrecasas and Wilchek, 1968). The method relies on the use of an affinity ligand coupled to a matrix to allow specific capture of the product from a complex mixture. In this way, an essentially pure product can be obtained with a single operation. The most frequent use of affinity chromatography during  the last  decade  has  been  the  purification  of antibodies  using

recombinant protein A (Uhlén et al., 1984) or protein G (Guss et al., 1986). Most monoclonal antibodies used for research and diagnoses and essentially all therapeutic antibodies used to treat patients have been purified using affinity chromatography (Walsh, 2006). Recently, protein engineering and design have been used to create new affinity reagents more suitable for affinity chromatography, as exemplified by the protein A derivative engineered to be stable during industrial “cleaning-in-place” procedures involving 0.1 M NaOH (Linhult et al., 2004).

Another  application  of  affinity  capture  is  the  technique  most  commonly  called immunoprecipitation (Berggard et al., 2007), which is based on the use of a specific antibody coupled to a solid matrix to capture the protein targets, often in a complex with its interaction partners. This  technique  has  become very popular,  with  applications  ranging from  molecular profiling of protein modifications to pathway mapping and network analysis (Collura and Boissy,

2007). Affinity purification has also been used to facilitate analysis of plasma and serum samples based on affinity capture to remove the most abundant proteins in sera, such as albumin and IgG or transferrin (Gronwall et al., 2007). This affinity procedure allows, for some cases, a more sensitive analysis in proteomics efforts aimed at discovering biomarkers useful for distinguishing patients with a particular disease.

With recombinant DNA methods, it is possible to create fusion proteins consisting of a protein to be studied and various tags used for detection and purification (Nilsson et al., 1997). Such affinity tags have been used for the generation of purified fusion proteins in a multitude of applications, including structural genomics, antibody generation, and interaction analysis. The first affinity tag was described in 1983 (Uhlén et al., 1983) and during the last 25 years many alternative systems have been described, all having advantages and disadvantages depending on the application and the requirement for specificity, solubility, and the binding and elution conditions. The most often used affinity tag today is probably the His-tag, consisting of a short peptide of histidine residues, which allows a convenient affinity chromatography step using metal-chelating chromatography (Porath et al., 1992).

1.6.7.2 Countercurrent Chromatography

Countercurrent chromatography (CCC, also known as “counter-current” or “counter current” chromatography)  is  an  analytical  chemistry  technique  that  is  used  to  separate,  identify,  and

quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography  encompasses  a  collection  of  related  liquid  chromatography  techniques  that employ two  immiscible liquid  phases  without a solid  support  (Berthod  et  al.,  2009;  Ito  and Bowman, 1970). The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases. A wide variety of two phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation (Lui et al., 2015).

Countercurrent chromatography and related liquid-liquid separation techniques have been used on both industrial and laboratory scale to purify a wide variety of chemical substances. Separation realizations  include proteins,(  Mekaoui  et  al.,  2012) DNA,(Kendall  et  al.,  2001) antibiotics,( McAlpine et al., 2012) vitamins,(Kurumaya et al., 1988) natural products,( Friesen et al., 2015) pharmaceuticals, metal ions,(Sumner, 2011; Araki et al., 1988) pesticides,(Ito et al., 2008) enantiomers,(Berthod, 2010) polyaromatic hydrocarbons from environmental samples,(Cao et al.,

2012) active  enzymes,(Baldermann  et  al.,  2011) and  carbon  nanotubes  (Zhang  et  al.,  2014). Countercurrent chromatography is known for its high dynamic range of scalability: milligram to kilogram  quantities  purified  chemical  components  may  be  obtained  with  this  technique (Sutherland, 2007). It also has the advantage of accommodating chemically complex samples with undissolved particulates.

Some  types  of  countercurrent  chromatography,  such  as  dual  flow  CCC,  feature  a  true countercurrent process where the two immiscible phases flow past each other and exit at opposite ends of the column (Ito et al., 2006). More often, however, one liquid acts as the stationary phase and is retained in the column while the mobile phase is pumped through it. The liquid stationary phase is held in place by gravity or by centrifugal force. An example of a gravity method is called droplet counter current chromatography (DCCC) (Tanimura et al., 1970). There are two modes by which the stationary phase is retained by centrifugal force: hydrostatic and hydrodynamic. In the hydrostatic method, the column, a series of chambers connected by channels, is rotated about a central axis (Foucault, 1994). Hydrostatic instruments are marketed under the name centrifugal partition  chromatography  (CPC)  (Marchal  et  al.,  2003).  Hydrodynamic  instruments  are  often

marketed  as  high-speed  or  high-performance  countercurrent  chromatography  (HSCCC  and HPCCC respectively) instruments which rely on the Archimedes’ screw force in a helical coil to retain the stationary phase in the column (Ito, 2005).

The components of a CCC system are similar to most liquid chromatography configurations such as HPLC. One or more pumps deliver the phases to the column which is the CCC instrument itself. Samples are introduced into the column through a sample loop filled with as automated or manual syringe. The outflow is monitored with various detectors such as UV-vis or Mass Spectrometry. The operation of the pumps, CCC instrument, sample injection, and detection may be controlled manually or with a microprocessor.

1.6.7.3 Substitute Western Affinity Purification Assay (SWAP)

SWAP: A High Throughput Automated Microfluidic Alternative to Western Blotting Introduction Western  blotting,  which  provides  information  on  the  size  and  relative  abundance  of  a specific  protein,  is  widely  applied for  the  detection  of  target  proteins  in  cell  or  tissue extracts.    Unfortunately,  the  technique  is  not  well  suited  for  highthroughput  analysis  and  is nearly  impossible  to  automate.    In  addition,  western  blotting  results  can  vary  widely  based on  the  quality  of  the  gel  and  transfer  steps  and  the  detection  method  and/or  development time.  Caliper  life  sciences  (2010),  developed  an   automated  Substitute  Western  Affinity Purification  (SWAP)  assay for  high-throughput  detection  of specific  proteins  where  complex protein  samples  are  denatured  then  labeled  with  a  red  fluorescent  dye  via  a  succinimidyl ester linkage to  lysine residues (CLS, 2010).

From their method, the  labeled  samples  are  incubated  with  Protein  A  or  Protein  G  coated magnetic  beads  bound  to  target-specific antibody. After washing, the unlabeled Ab and labeled protein target are eluted into sample buffer and analyzed using a microfluidic chip and Caliper’s LabChip GXII Pico Protein assay. The sizes and relative concentrations of the labeled captured proteins  are then  reported  by the analysis  software.  The assay has  numerous  potential  high- throughput applications including protein expression analysis, monitoring post-translational modifications, and biomarker detection (CLS, 2010).

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