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 α–AMYLASE
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.mesentericus, B. 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).
This material content is developed to serve as a GUIDE for students to conduct academic research
STUDIES ON THE ACTIVITY OF Α-AMYLASE PRODUCED FROM FUSARIUM SPP USING SWEET POTATO (IPOMOEA BATATAS) STARCH
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