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STUDIES ON THE ACTIVITY OF Α-AMYLASE PRODUCED FROM FUSARIUM SPP USING SWEET POTATO (IPOMOEA BATATAS) STARCH

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ABSTRACT

After  a seven  day pilot  studies,  day 6  was  found  suitable  for  enzyme  production  from Fusarium  species using starch from Ipomoea  batatas (sweet potato)  tubers as  the carbon source. The specific activity of the crude enzyme was 55.45µ/mg. After ammonium sulphate precipitation  and  gel  filtration,  the  specific  activities  were  found  to  be  35.93µ/mg  and 119.61µ/mg, respectively which corresponds to 3.33 fold purification. The optimum pH and temperature of the partially purified enzyme were 6.0 and 50oC, respectively. The  enzyme activity was strongly activated by Mn2+, Ca2+, and Mg2+ but inhibited by Co2+. The Michaelis constant (Km) and maximum velocity (Vmax) obtained from the  Lineweaver-Burk  plot of initial velocity data at different substrate concentrations were 5.44mg/ml and 12.57µmol/min, respectively

CHAPTER ONE

1.0  INTRODUCTION

The  α-Amylase  (1,4-α-D-glucan  glucano  hydrolase,  EC  3.2.1.1)  is  the  most  important carbohydrate  degrading enzyme for all starch based industries  viz. food,  paper, detergent, pharmaceutical, textile, baking and brewing industries (Gupta et al., 2003). It belongs to the family  13  of  glycoside  hydrolases  (GH13)  which  randomly  cleaves  the  α-1,4  linkages between  adjacent  glucose  units  in starch  and  related  polysaccharides  to  produce  mainly maltodextrins and maltose retaining α-anomeric configuration in the products. According to the vast majority of known α-amylase  structure, it composed of a single polypeptide chain folded into two large domains: N-terminal (A and B) and C-terminal (C) domains. Catalytic domain A contains  (β⧸α)8   or TIM barrel structure,  domain  B consists of three-stranded antiparallel β-sheet structures and protrudes between β3 and α3 of domain A, whereas domain C with a β sheet structure is located in the C-terminal part of the polypeptide chain. Domain B is concerned for the substrate specificity and stability of the enzyme (Kuriki and Imanaka,

1999; Singh and Kayastha, 2014). Amylases are widespread in animals, fungi, plants and are also found in the unicellular eukaryotes, bacteria, and archaea (Da lagea et al., 2007). Though plants and animals produce amylases, enzymes from microbial sources are generally used in industrial  processes.  This  is  due  to  a  number  of  factors  including  productivity,  thermo stability of the enzyme as well as ease of cultivating microorganisms (Reddy et al., 1999). Amylases have been reported to be produced by a number of fungi, including Aspergillus, Rhizopus, Fusarium, Candida, Penicillium,  Thermomucor,  Basidiomycete,  Fomitopsis, and Thermomyces (Kunamneni et al., 2005;Balkan and Ertan 2005;Yoon et al., 2006; Kumar et al., 2007; Mohamed et al., 2007). Majority of the studies on fungal amylases are based on mesophiles, rarely on facultative thermophiles (Maheswari et al., 2000). Current researches focus on thermo  tolerant enzymes from thermophilic microbial strains. Bhatti et al. (2007) and  Figueira  and  Hirooka  (2000)  reported  amylases  from  mesophilic  Fusarium  species. Fusarium  is  a  large  genus  of  filamentousfungi,   part  of  a  group  often  referred  to  as hyphomycetes,  widely  distributed  in  soil  and  associated  with  plants.  Most  species  are harmless saprobes, and are relatively abundant members of the soil microbial  community. Agricultural wastes are being used for both liquid and solid fermentation to reduce the cost of fermentation  media.  These  wastes  consist  of  carbon  and  nitrogen  sources  necessary  for growth and metabolism  of organisms.  These  nutrient  sources  include orange waste, pearl millet starch, potato, corn, tapioca, wheat, and rice as  flours (Djekrif-Dakhmouche  et al.,

2005;  Haq  et al.,  2005).  In this  work,  the  tubers  of  sweet  potato  will be  used  for  the production  of alpha  amylase.  α-amylases  with characteristics  suitable  for the  industrially relevant   process   conditions   such   as  temperature,   pH,   nature  of   substrate,   substrate concentration, enzyme concentration, presence of metal salts, surfactants and other stabilizing agents should be appropriately selected as per the demand (Sivaramakrishnan et al., 2006).

1.1TYPES OF AMYLASE

1.1.1 αAMYLASE

Alpha amylase (E.C.3.2.1.1) is a hydrolase enzyme that catalyses the hydrolysis of internal α-

1, 4-glycosidic linkages in starch to yield products like glucose and maltose. It is a calcium dependent enzyme i.e. it depends on the presence of a metal co factor (Ca2+) for its activity. There are 2 types of hydrolases: endo-hydrolase and exo-hydrolase. Endo-hydrolases act on the  interior  of  the  substrate  molecule,  whereas  exo-hydrolases  act  on  the  terminal  non

reducing ends (Gupta et al., 2003). Hence, terminal  glucose  residues and α-1,  6-linkages cannot  be  cleaved  by α-amylase.  Starch  is  a  polysaccharide  composed  of  two  types  of polymers – amylose and amylopectin. Amylose constitutes 20-25% of the starch molecule. It is a linear chain consisting of repetitive glucose units linked by α-1,  4-glycosidic  linkage. Amylopectin constitutes 75-80% of starch and is characterized by branched chains of glucose units.  The  linear  successive  glucose  units  are  linked  by  α-1, 4-glycosidic  linkage  while branching occurs every 15-45 glucose units where α-1, 6 glycosidic bonds are present. The hydrolysate composition obtained after hydrolysis of starch is highly dependent on the effect of temperature, the conditions of hydrolysis and the origin of enzyme. The optimum pH for α-amylase activity is found to be 7.0. α-amylase has become an enzyme of crucial importance due  to  its starch  hydrolysis  activity.  The  use  of  enzymes  in detergent  formulations  has increased dramatically with growing awareness about environment protection. Enzymes are environmentally safe and enhance the detergents  ability to remove tough stains. They are biodegradable and work at milder conditions than chemical catalysts and hence preferred to the latter. There are many such applications of the enzyme which is the driving force behind the research to produce this enzyme in an optimum, safe and convenient manner (Gupta et al., 2003)

1.1.2 β – AMYLASE

Beta Amylase (EC 3.2.1.2) is an exo-hydrolase enzyme that acts from the non-reducing end of a polysaccharide chain by hydrolysis of α-1, 4-glucan linkages to yield successive maltose

units. Since it is unable to cleave branched  linkages  in branched  polysaccharides  such  as glycogen  or amylopectin,  the hydrolysis  is incomplete  and dextrin units remain.  Primary sources of β-amylase are the seeds of higher plants and sweet potatoes. During ripening of fruits, β-amylase breaks down starch into maltose resulting in the sweetness of ripened fruit. The optimal pH of the enzyme ranges from 4.0 to 5.5. βeta amylase can be used for different applications on the research as well as industrial front. It can be used for structural studies of starch and glycogen molecules produced by various methods. It is used during fermentation in  brewing  and  distilling  industry.  Also,  it  is  used  to  produce  high  maltose  syrups (Sivaramakrishnan et al., 2006).

1.1.3γ – AMYLASE

Gamma Amylase (EC 3.2.1.3 ) cleaves α(1-6)glycosidic linkages, in addition to cleaving the last α(1-4) glycosidic linkages at the non-reducing end of amylose and amylopectin, unlike the other forms of amylase,  yielding glucose. Gamma amylase  is most efficient  in acidic environments and has an optimum pH of 3 (Sivaramakrishnan et al., 2006).

1.2ALPHA AMYLASE FAMILY

The α-amylase family, i.e. the clan GH-H of glycoside hydrolyses, is the largest family of glycoside  hydrolases,  transferases  and isomerases  comprising  nearly 30 different  enzyme specificities (Van Der Maarel et al., 2002). A large variety of enzymes are  able  to act on starch. These enzymes can be divided basically into four groups: endoamylases, exoamylases, debranching enzymes, and transferases (Henrissat, 1991; Van Der Maarel etal.,2002).

1.2.1    Endo Amylase

The endo amylase otherwise known as α-amylase group are those amylase that catalyzes the internal hydrolysis of α- 1,4-o-glycosidic bonds resulting in the formation of alpha anomeric products (Swetha etal.,2006).

1.2.2    Exo Amylases

The exo amylase are those amylase that catalysis the cleavage of either α- 1, 4 or α-  1,6 linkages of external glucose residues resulting in alpha or beta anomeric products (Van Der Maarel etal.,2002).

1.2.3    Debranching Amylase

The debranching amylase catalysis the hydrolysis of α- 1, 6 bonds exclusively leaving long chain polysaccharides (Van Der Maarel etal.,2002).

1.2.4    The Transferases

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

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

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

    Comprises of the following amino acids: Aspartate (Asp 206), Glutamate (Glu 230)

and Aspartate (ASP 297) at corresponding positions in their catalytic domain.

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

1.3STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF   -AMYLASE

The α-amylase (α-1,4-glucan-4-glucanohydrolase)  can be found in microorganisms,  plants and higher organisms (Kandra, 2003). The α-amylase belongs to a family of endo-amylases that  catalyses  the  initial  hydrolysis  of  starch  into  shorter  oligosaccharides  through  the

cleavage of α-D-(1-4) glycosidic bonds (Brayer et al., 1995; Iulek et al., 2000; Kandra, 2003; Tangphatsornruang et al., 2005). Neither terminal glucose residues nor α-1,6-linkages can be cleaved by α-amylase (Whitcomb and Lowe, 2007). The end  products of α-amylase action are oligosaccharides with varying length with an α-configuration and α-limit dextrins (Van Der Maarel et al., 2002), which constitute a mixture of maltose, maltotriose, and branched oligosaccharides of 6–8 glucose units that  contain both α-1,4 and α-1,6 linkages (Whitcom and Lowe, 2007). Other amylolytic enzymes participate in the process of starch breakdown, but  the   contribution of α-amylase is the most important for the initiation of this process(Tangphatsornruang  et  al.,  2005).  The  amylase  has  a  three-dimensional  structure capable of binding to substrate and, by the action of highly specific catalytic groups, promote the breakage of the glycoside links (Iulek et al., 2000). The human α-amylase is a classical calcium-containing enzyme composed of 512 amino acids in a single oligosaccharide chain with a molecular weight of 57.6 kDa (Whitcomb and Lowe, 2007).

1.4STARCH

Starch is an important constituent of the human diet and, for this purpose, is used chemically and enzymatically processed into a variety of different products such as starch hydrolysates, glucose syrups, fructose, maltodextrin derivatives or cyclodextrins, used in food industry. In addition to that, the sugars produced can be fermented to  produce ethanol. In spite of the large number of plants able to produce starch, only a few plants are important for industrial starch processing.  The major industrial sources  are maize, tapioca, potato, and wheat, but limitations such as low shear resistance, thermal resistance, thermal decomposition and high tendency towards retro gradation limit its use in some industrial food applications (Agrawal etal., 2005;  Goyal  et al.,  2005;  Muralikrishna  and  Nirmala,  2005).  Among  carbohydrate polymers, starch is currently enjoying increased attention due to its usefulness in different food products.  Starch contributes  greatly to the textural  properties  of many foods and is widely used  in food and industrial applications  as a thickener,  colloidal stabilizer, gelling agent, bulking agent and water retention agent (Jaspreet et al., 2007). Starch is a polymer of glucose linked to another one through the glycosidic bond. Two types of glucose polymers are  present  in  starch:  amylose  and  amylopectin  (Figures  1a  and  1b).   Amylose  and amylopectin have different structures and properties. Amylose is a linear polymer consisting of up to 6000 glucose units with α-1,4 glycosidic bonds. Amylopectin consists of short α-1,4 linked to linear chains of 10–60 glucose units and α-1,6 linked  to  side chains with 15–45 glucose units. Granule bound starch synthase can elongate  malto-oligosaccharides  to form

amylose and is considered to be responsible for the synthesis of this polymer. Soluble starch synthase is considered to be responsible for the synthesis of unit chains of amylopectin. α- amylase is able to cleave α-1,4 glycosidic bonds present in the inner part of the amylose or amylopectin chain (Van Der Maarel et al., 2002; Sorensen et al., 2004; Tester et al., 2004; Muralikrishna and Nirmala, 2005).

Source:  (Van  Der  Maarel  et  al.,  2002;  Sorensen  et  al.,  2004;  Tester  et  al.,   2004; Muralikrishna and Nirmala, 2005).

B.  Structure of amylopectin

Fig: 1b. Structure of amylopectin

Source: (Muralikrishna and Nirmala, 2005).

Two  types  of glucose  polymers  are  present  in  starch:  amylose  (A)  is  a  linear  polymer consisting  of up  to  6000  glucose  units  with  α-1,4  glycosidic  bonds  (Muralikrishna  and Nirmala, 2005) and amylopectin (B) consists of short α-1,4 linked to linear chains of 10–60 glucose units and α-1,6 linked to side chains with 15–45 glucose units (Muralikrishna and Nirmala, 2005).

Starch is hydrolyzed into smaller oligosaccharides by α-amylase, which is one of the most important commercial enzyme processes. Amylases find application in all the industrial processes such as in food, detergents, textiles and in paper industry, for the  hydrolysis of starch (Gupta et al., 2003; Konsula  and Liakopoulou-Kyriakides,  2004;  Tanyildizi  et al.,

2005). Saccharide composition obtained after hydrolysis of starch is highly dependent on the

effect of temperature,  the conditions  of hydrolysis and the origin of enzyme.  Specificity, thermo stability, and pH response of the enzymes are critical properties  for  industrial use (Kandra, 2003).

1.5SOURCES OF α-AMYLASE

Alpha amylases are ubiquitous enzymes produced by plants, animals and microbes,  where they play a dominant role in carbohydrate metabolism. Amylases from plant and  microbial

sources have been employed for centuries as food additives. Barley amylases have been used in the brewing industry. Fungal amylases have been widely used in preparation of oriental foods. In spite of the wide distribution of amylases, microbial sources, namely fungal and bacterial amylases,  are used for the industrial production  due to  advantages such as cost effectiveness,  consistency, less time and space required for production and ease of process modification and optimization (Burhan et al., 2003). Among bacteria, Bacillus sp. is widely used   for   thermostable   α-amylase   production   to   meet   industrial   needs.   B.   subtilis, B.stearothermophilus,  B.  licheniformis,  and  B.  amyloliquefaciens  are  known  to  be  good producers of α-amylase and these have been widely used for commercial production of the enzyme for various applications. Similarly, filamentous fungi have been widely used for the production of amylases for centuries. As these moulds are known to be prolific producers of extracellular  proteins,  they are widely  exploited  for  the production  of different  enzymes including α-amylase. Fungi belonging to the genus Aspergillus have been most commonly employed for the production of α-amylase. Production of enzymes by solid-state fermentation (SSF) using these moulds turned a cost-effective production technique. Detailed literature is available  on  various  microbial  sources  for  the  production  of  amylases  (Vihinen  and Mantasala, 1989; Pandey et al., 2000).

1.5.1 BACTERIAL AMYLASES

Alpha Amylase can be produced by different species of microorganisms, but for commercial applications α-amylase is mainly derived from the genus Bacillus. α-amylases produced from Bacillus  licheniformis,  Bacillus  stearothermophilus,  and  Bacillus  amyloliquefaciens  find potential  application  in  a  number  of  industrial  processes  such  as  in  food,  fermentation, textiles,  and paper industries  (Pandey et al., 2000; Konsula  and Liakopoulou-Kyriakides,

2007). Thermostability is a desired characteristic of most of the industrial enzymes. Thermal stable enzymes isolated from thermophilic organisms have found a number of commercial applications  because  of their  stability.  As enzymatic  liquefaction  and  saccharification  of

starch are performed at high temperatures (100–110oC), thermal stable amylolytic enzymes

have been currently investigated to improve industrial processes of starch degradation and are of great interest for the production of valuable products like glucose, crystalline  dextrose, dextrose  syrup,  maltose,  and  maltodextrins  (Stamford  et al., 2001;  Gomes  et al.,  2003; Asgher et al., 2007). Bacillus subtilis, Bacillus stearothermophilus,  Bacillus licheniformis, and Bacillus amyloliquefaciens are known to be good producers of thermal stable α-amylase, and  these  have  been widely used  for  commercial  production  of the enzyme  for various

applications. Thermal stable α-amylases have been reported from several bacterial strains and have been produced using submerged fermentation(SmF) as well as solid state fermentation (SSF) (Teodoro and Martins, 2000). However, the use of SSF has been found to be more advantageous than SmF and allows a cheaper production of enzymes (Sodhi et al., 2005). The  production  of  α-amylase  by SSF  is  limited  to  the  genus  Bacillus,  and  B.  subtilis, B.polymyxia,B.mesentericusB. vulgarus, B. megaterium, and B.licheniformis have been used for α-amylase production in SSF (Baysal et al., 2003). Currently, thermal stable amylases of Bacillus  stearothermophilus  or Bacillus  licheniformis  are being used in starch processing industries (Gomes et al., 2003). Enzymes produced by some halophilic microorganisms have optimal  activity  at  high  salinities  and  could  therefore  be  used  in many  harsh  industrial processes where the concentrated salt solutions used would otherwise inhibit many enzymatic conversions (Amoozegar et al., 2003; Prakash et al., 2009). In addition, most halobacterial enzymes are considerably thermotolerant and remain stable at room temperature over long periods  (Mohapatra  et  al.,  1998).  Halophilic  amylases  have  been  characterized   from halophilic  bacteria such as Chromohalobacter  sp. (Prakash et al., 2009),  Halobacillus  sp. (Amoozegar  et  al.,  2003),  Haloarcula  hispanica  (Hutcheon  et  al.,  2005),  Halomonas meridiana (Coronado et al., 2000), and Bacillus dipsosauri (Deutch, 2002).

1.5.2 FUNGAL AMYLASES

Most reports  about  fungi that produce α-amylase  have been limited  to a few species  of mesophilic  fungi, and attempts have been made to specify the cultural conditions  and to select superior strains of the fungus to produce on a commercial scale (Gupta et al., 2003). Fungal sources  are confined  to terrestrial  isolates,  mostly to Aspergillus  and Penicillium (Kathiresan  and  Manivanna,  2006).  The  Aspergillus  species  produce  a  large  variety  of extracellular enzymes, and amylases are the ones with most significant industrial importance (Hernandez  et al., 2006).  Filamentous  fungi,  such as  Aspergillus  oryzae  and Aspergillus niger, produce considerable quantities of enzymes that are used extensively in the industry. A. oryzae  has  received   increased   attention  as   a  favourable   host  for  the  production  of heterologous proteins because of its ability to secrete a vast amount of high value proteins and industrial enzymes, e.g. α-amylase (Jin et al., 1998). Aspergillus oryzae has been largely used in the production of food such as soy sauce, organic acid such as citric and acetic acids and commercial enzymes including α-amylase (Kammoun et al., 2008). Aspergillus niger has important  hydrolytic  capacities  in the  α-amylase  production  and,  due to  its tolerance  of acidity (pH <3), it allows the avoidance of bacterial contamination (Djekrif-Dakhmouche et

al., 2005). Filamentous fungi are suitable microorganisms for solid-state fermentation (SSF), especially because their morphology allows them to colonize and penetrate the solid substrate (Rahardjo et al., 2005). The fungal α-amylases are preferred over other microbial sources due to their more accepted status GRAS (Generally Recognized  As Safe) (Gupta et al., 2003). The thermophilic  fungus  Thermomyces  lanuginosus  is an  excellent  producer  of amylase. Jensen  et  al.  (2002)  and  Kunamneni  et  al.  (2005)  purified  the  α-amylase,  proving  its thermostability.

1.6PRODUCTION METHODS FOR α-AMYLASE

There are mainly two methods which are used for production of α-amylase on a commercial scale. These are; Submerged and solid state fermentation.

The  latter  is  a  fairly  new  method  while  the  former  is  a  traditional  method  of  enzyme production from microbes which has been in use for a longer period of time.

1.6.1 SUBMERGED FERMENTATION (SmF)

Employs free flowing liquid substrates, such as molasses and broths. The products yielded in fermentation  are  secreted  into  the  fermentation  broth.  The  substrates  are  utilized  quite rapidly; hence the substrates need to be constantly replenished. This fermentation technique is suitable for microorganisms such as bacteria that require high moisture content for their growth. SmF is primarily used for the extraction of secondary metabolites that need to be used in liquid form (Couto and Sanroman, 2006). This method has several advantages. SmF allows the utilization of genetically modified  organisms to a greater extent than SSF. The sterilization of the medium and purification process of the end products can be done easily. Also the control of process parameters like temperature, pH, aeration, oxygen transfer and moisture can be done conveniently (Kunamneni et al., 2005).

1.6.2SOLID STATE FERMENTATION

This is a method which require less moisture content for their growth.The solid  substrates commonly used in this method are, bran, bagasse, and paper pulp. The main advantage is that nutrient-rich waste materials can be easily recycled and used as  substrates in this method. Unlike  SmF,  in this  fermentation  technique,  the  substrates  are utilized  very  slowly  and steadily. Hence the same substrate can be used for a longer duration, thereby eliminating the need to constantly supply substrate to the process (Kunamneni et al., 2005). Other advantages

that SSF offers over  SmF are simpler  equipment,  higher  volumetric  productivity,  higher concentration of products and lesser effluent generation (Couto and Sanroman,  2006). For several such reasons SSF is considered as a promising method for commercial production of enzymes.

Table 1: Microbial sources and the mode of fermentation used for production

Bacterial source                                                Method

Bacillus amyloliquefaciens                               Solid-state fermentation Bacillus licheniformis                                       Solid-state fermentation Halomonas meridian                                        Submerged fermentation Rhodothermus marinus                                     Submerged fermentation Bacillus cereus                                                  Solid-state fermentation Fungal source                                                  Method

Aspergillus oryzae                                            Solid-state fermentation

Penicillium Fellutanum                                    Submerged fermentation

Streptomyces rimosus                                       Solid-state fermentation, Submerged fermentation

Aspergillus kawachii                                        Solid-state fermentation, Submerged fermentation

Pycnoporus sanguineus                                    Solid-state fermentation

Source: (Kunamneni et al., 2005; Couto and    Sanroman, 2006).

Fungal sources  have been investigated  for α-amylase  production  through  submerged  and solid state fermentation. However, studies reveal that SSF is the most appropriate process in developing countries due to the advantages it offers which make it a cost effective production process (Kunamneni et al., 2005). Also SSF provides a medium that resembles the natural

habitat of fungal species, unlike Smf which is considered a violation of their habitat (Kenneth

et al., 1993).

1.7PURIFICATION OF α-AMYLASE

Industrial  enzymes  produced  in bulk  generally  require  little  downstream  processing  and hence are relatively crude preparations. The commercial use of α-amylase generally does not require purification of the enzyme, but enzyme applications in pharmaceutical and clinical sectors require high purity amylases. The enzyme in the purified form is also a prerequisite in studies of structure-function  relationships and biochemical properties  (Gupta et al., 2003). Different strategies for purification of enzymes have been investigated,  exploiting specific characteristics of the target biomolecule. Laboratory scale purification for α-amylase includes various combinations of ion exchange, gel filtration, hydrophobicity interactions, and reverse phase chromatography. Alternatively, α-amylase extraction protocols using organic solvents such as ethanol, acetone and ammonium sulphate precipitation (Glymph and Stutzenberger,

1977;  Hamilton  et al.,  1999;  Khoo  et al., 1994)  and  ultrafiltration  have  been  proposed (Moraes et al., 1999). These conventional multi-step methods requires expensive equipment at each step, making them laborious, time consuming, barely reproducible and may result in increasing  loss  of  the  desired  product  (Arauza  et   al.,  2009).  However,  liquid–liquid extractions consist of an interesting purification alternative since several features of the early processing steps can be combined  into a  single  operation.  Liquid–liquid  extraction  is the transfer of certain component from one phase to another when immiscible or partially soluble liquid phases are brought into contact with each other. This process is widely employed in the chemical  industry  due  to  its simplicity,  low costs,  and  ease of scale  up. Purification  of biomolecules using liquid–liquid extraction has been successfully carried out on a large scale for more than a decade. Advantages of using this system are lower viscosity, lower cost of chemicals and shorter phase separation time. The dynamic behaviour of these systems has to be  investigated  and understood  to enhance plant-wide  control of continuous  liquid–liquid extraction and to assess safety and environmental risks at the earliest possible design stage (Mazzola et al., 2008).


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STUDIES ON THE ACTIVITY OF Α-AMYLASE PRODUCED FROM FUSARIUM SPP USING SWEET POTATO (IPOMOEA BATATAS) STARCH

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