ABSTRACT
The activity of α-amylase and protein concentration in African yam bean seeds increases as germination progresses up to day 8 of germination where it exhibited its highest level, followed by sequenced decreased activity and protein concentration till day 12. Starch from African yam bean seeds, corn and cassava were used for the hydrolysis experiment. There was a significant activity of α-amylase in each of the substrate used, however, the starch from African yam bean seeds had higher α-amylase activity (369.55µmol/min at pH 5.5 and 369.55µmol/min at pH 9.0) followed by starch from corn (367.08µmol/min and 360.49µmol/min at pH 5.5 and 9.0 respectively), while cassava had the least with activity of 353.50µmol/min and 351.03µmol/min at pH 5.5 and 9.0 respectively. The crude enzyme was purified to the level of gel filtration (sephadex G-25) via 80% ammonium sulphate precipitation. The purification fold of 1.36 with specific activity 226.44μmol/min/mg protein and 1.62 with specific activity 367.65μmol/min /mg protein were observed for 80% ammonium sulphate precipitation and gel filtration, respectively. The enzyme displayed optimum activity at pH 5.5 and temperature 45°C in all the three substrates (African yam bean, corn and cassava). The Michaelis menten constant (Km) and maximum velocity (Vmax) obtained from Lineweaver-Burk plot of initial velocity data at different concentrations of starch from African yam bean seeds as substrate were found to be 0.588mg/ml and 588.24μmol/min, respectively. Similarly, 0.625 mg/ml and 625μmol/min were obtained using starch from corn, respectively, while 0.733mg/ml and 666.7μmol/min were also observed using starch from cassava, respectively. The enzyme activities were enhanced in the presence of some metal ions like Ca2+, Co2+ and Fe2+. Zn2+ and Na2+ neither increased nor decreased enzyme activity while Pb2+ completely inactivated the enzyme.
CHAPTER ONE
INTRODUCTION
African yam bean (Sphenostylis stenocarpa), belongs to the legume family. It originated in Ethiopia, both wild and cultivated types now occur in tropical Africa as far north as Egypt and also throughout West Africa from Guinea to Southern Africa (Assefa and kliener, 1997). It is cultivated in Nigeria mainly for its seed. The African yam bean (AYB) is a climbing legume adapted to lowland tropical conditions. There are seven species in the genus Sphenostylis (Potter and Doyle 1994). African yam bean is the most valuable. Some species in the genus Sphenostylis provide two consumable products, the tuber which grows as the root source and the actual yam beans which develop in pods above ground (Daniel, 2010). African yam beans seed is classified as a neglected under-utilized species (Mentreddy, 2007) due to its low esteem and lack of detailed information on its compositional analysis (Adewale et al., 2010).
Starch is a storage polysaccharide present in seeds and it consist of two components; a linear glucose polymer, amylose, which contain α-1,4 linkage chains and a branch polymer, amylopectin in which linear chains of α-1,4 glucose residue are inter-linked by α-1,6 linkages. Starch is hydrolyzed into smaller oligossaccharides by α-amylase, which is one of the most important commercial enzyme processes.
α-Amylases catalyze the hydrolysis of α-1,4-glycosidic linkages in starch to produce low molecular weight products, such as glucose, maltose and maltotriose units (Tangphatsornruang et al., 2005). Amylases can be obtained from several sources, such as plants, animals and microorganisms. Funke and Melzing, (2006) reported that amylases of plant origin have the highest hydrolytic potential followed by that of fungi, while amylases from bacteria have relatively less hydrolytic potential. A number of plant amylases have been identified and plants are one of the abundant sources of α-amylase (Conforti et al., 2005). The enzyme has been extracted from plants sources like barley, millets, wheat, sorghum and maize among others. However, no study was carried out on activity of α-amylase from germinated African yam bean seeds. α-amylases from beans have gained importance due to their suitability for biotechnological applications in supplementary foods, breweries and starch saccharification (Muralikrishna and Nirmala, 2005).
Recently, interest and demand for enzymes with novel properties are very high in various
industries and it leads to the discovery of various types of α-amylase with unique properties. Therefore, the present investigation was initiated to African yam bean seeds for α-amylase activity, to determine the relative abundance and activity of this enzyme during germination and to establish some characteristics for their action
1.1 African Yam Beans
African yam bean is an underutilized tropical African tuberous legume (figure 1A). It belongs to the class Magnoliopsida; order Fabales; family Fabaceae; subfamily Papilionoideaea; and genus Sphenostylis. There are seven species in the genus Sphenostylis (Potter and Doyle, 1994). African yam bean (AYB) is the most valuable. It is a vigorously climbing herbaceous vine whose height can reach 1.5–3 m or more. The main vine/stem produces many branches which also twine strongly on available stakes. The vegetative growing stage is characterized with the profuse production of trifoliate leaves (Utter, 2007).
From four to ten flowers are arranged in racemes on long peduncles, usually on the primary and secondary branches. The large and attractive flowers blend pink with purple; the standard
petals twist slightly backwards on themselves at anthesis. The flowers seem to exhibit self- pollination; up to six pods/peduncle may result after fertilization
Figure 1: African yam bean plant showing mature pods ready for harvest (Daniel, 2010).
They are usually linear and long unicarpel pods turn brown when mature (Hutchinson and Dalziel, 1958; Dukes, 1981). The pods (figure 1) which may sometimes be flat or raised in a ridge-like form on both margins are usually prone to shattering; they dehisce along the dorsal and ventral sutures when dry. Each pod can yield up to 20 seeds which may be round, oval, oblong or rhomboid (Figure 2B). There are varieties of seed colour (Oshodi et al., 1995) and size (Adewale et al., 2010) with mono-coloured or mosaic types. Mono-coloured seeds are white, grey, cream, light or dark brown purple, or black. Sphenostylis sternocarpa is native to tropical west and central Africa and is cultivated in southern and eastern Africa. It thrives on deep, loose sandy and loamy soils with good organic content and good drainage. It grows better in regions where annual rainfalls range between 800-1400 mm and where temperatures are comprised between 19-27°C. The plant flowers after 90 days and the pods mature in 140 to 210 days while the tubers can be harvested 150 to 240 days after sowing (Utter, 2007). African yam bean is usually grown in mixtures with yam and cassava. Protein content is up to 19% in the tubers and 29% in the seed grain.
Figure 2: (A) Tuber yield per stand of AYB (Daniel, 2010) and (B) raw seeds of African yam bean
1.1.2 Seed germination
Seed germination is a very important phase in the growth of any plant. Seed coat which may be thick and hard or thin and soft is the outer covering of seed which protects embryo from mechanical injury , entry of parasites and prevents it from drying. Endosperm is a temporary food supply which is packed around the embryo in the form of cotyledons or seed leaves. Processes involved in germinating occur in different stages:
Absorption of water and bursting of seed coat is the first sign of germination. In this stage, there is an activation of enzymes, increase in respiration, and plant cells get duplicated. A chain of chemical changes starts which leads to development of plant embryo.
Chemical energy stored in the form of starch is converted to sugar which is used during germination process. This leads to enlargement and bursting of seed coat.
Growing plant emerges out tip of root first emerges and help to anchor the seed in a place. It also allows embryo to absorb mineral and water from the soil.
During germination, the principal enzyme involved in carbohydrate breakdown is α-amylase which hydrolyses α(1-4) bond in amylose and amylopectin releasing fragments that can be further broken down by β-amylase, α-glucosidase and debranching enzymes. α-amylase is synthesized de novo in two specific tissue of seeds, the scutella epithelium of the embryo and the aleurone layer of the endosperm (Ball et al., 2003). In the seed, enzyme syntheses begins initially in the scutellum after imbibition and then in the aleurone layer after few days (Okano et al., 2009). Secretion of amylase from the cells of the aleurone layers is well established in cereal grains and there is evidence that a similar process takes place in at least some dicotyledonous seeds (Niittyla et al., 2004).
Starch in the endosperm of cereals is the most abundant reserve synthesized during seed development. Degradation of starch into soluble sugar is important to support seedling growth
during seed germination. Starch can be degraded either by hydrolysis with amylase or phospholysis with starch phosphorylase. In germinating seeds, hydrolysis but not phospholysis is the major process to breakdown starch molecules. α-amylase and β-amylase are the major amylolytic enzymes found during seed germination and it was suggested that both enzymes are involved in the degradation of endospermic starch (Marc et al., 2002). However, β-amylase is synthesized and accumulated as a latent form in the starchy endosperm during seed development (Hang et al., 1996).
1.2 Starch
In the green leaves, carbon dioxide and water are transformed into glucose and oxygen under the influence of sunlight and with the help of chlorophyll. This process is known as photosynthesis. During the day this starch is deposited as grains in the leaf, the so-called leaf- transition starch. During the night this starch is partially broken down again into sugars which are transported to other areas of the plant. From these sugars the starch arises which is won in the familiar grain shape. The forming of starch is a process which has by far not been clarified yet and during which a number of enzymes play a role.
Starch or amylum is a carbohydrate consisting of a large number of glucose units joined by glycosidic bonds. 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 (Van der Maarel et a.l. 2002., Goyal et al,.2005).With the help of a microscope the grain shape reveals from which plant species the starch derives. Native starch, the starch as it occurs in the plant, cannot be dissolved in cold water. When we scatter starch, while stirring, into water we get a milky white suspension which can be stirred without much difficulty. When the stirring is stopped the starch sinks to the bottom (sedimentation), during which a transparent upper layer is formed. When the suspension is heated the white colour disappears at a temperature characteristic for starch. The starch dissolves into an almost transparent solution. This is what we call gelatinized starch. In comparison with the ungelatinized suspension, stirring takes considerably more difficulty. The temperature at which the resistance during stirring noticeably increases, is called the gelatinization temperature. Gelatinizing starch into viscous substances is one of the most, if not the most important characteristic(s) of starch. This phenomenon lies at the basis of the successful application of starch in a large number of sectors. 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 (Fig. 3). Amylose and amylopectin have different structures and properties. Amylose is a linear polymer consisting of up to 6000 glucose units with α-1,4glycosidic 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- 1,4glycosidic bonds present in the inner part of the amylose or amylopectin chain (Muralikrishna and Nirmala, 2005; Van der Maarel et al., 2002).
A. Structure of amylose
B. Structure of amylopectin
Figure 3: Two types of glucose polymers are present in starch: amylose (A) is a linear polymer consisting of up to 6000 glucose units with α-1,4glycosidic bonds (56) 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).
Endoamylases are able to cleave α,1-4 glycosidic bonds present in the inner part (endo-) of the amylose or amylopectin chain. α-Amylase (EC3.2.1.1) is a well-known endoamylase. It is found in a wide variety of microorganisms, plant and animal (Pandey et al., 2000). The end products of α-amylase action are oligosaccharides with varying length with an α-configuration and α-limit dextrins, which constitute branched oligosaccharides, which is one of the most important commercial enzyme processes. Saccharide composition obtained after hydrolyze 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). Exoamylases act on the external glucose residues of amylose or amylopectin and thus produce only glucose (glucoamylase and α-glucosidase), or maltose and β-limit dextrin (β-amylase).
1.2.1 Sources and utilization of starch
Starch occurs mainly in the seeds, roots and tubers of higher plants. Some algae produce a similar reserve polysaccharide called phytoglycogen. Plants synthesize starch via photosynthesis. The shape and diameter of these granules depend on the botanical origin. Regarding to commercial starch sources, the granule sizes range from 2–30 μm (maize starch) to 5–100 μm (potato starch) (Robyt and Whelan, 1998). A variety of different enzymes are involved in the synthesis of starch. Sucrose is the starting point of starch synthesis. It is converted into the nucleotide sugar ADP-glucose that forms the actual starter molecule for starch formation. Subsequently, enzymes such as soluble starch synthase and branching enzyme synthesize the amylopectin and amylose molecules (Smith, 2001). Starch-containing crops form an important constituent of the human diet. Besides the direct use of starch- containing plant parts as a food source, starch is harvested and chemically or enzymatically processed into a variety of different products such as starch hydrolysates, glucose syrups, fructose, starch or maltodextrin derivatives, or cyclodextrins. 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.
1.2.2 Biosynthesis of Starch
Plants produce starch by first converting glucose 1-phosphate to ADP-glucose using the enzyme glucose-1-phosphate adenylyltransferase. This step requires energy in the form of ATP. The enzyme starch synthase then adds the ADP-glucose via a 1,4-alpha glycosidic bond to a growing chain of glucose residues, liberating ADP and creating amylose. Starch branching enzyme introduces 1,6-alpha glycosidic bonds between these chains, creating the branched amylopectin. The starch debranching enzyme isoamylase removes some of these branches. Several isoforms of these enzymes exist, leading to a highly complex synthesis process (Smith, 2001). Glycogen and amylopectin have the same structure, but the former has about one branch point per ten 1,4-alpha bonds, compared to about one branch point per thirty 1,4-alpha bonds in amylopectin (Shinke et al., 1974). Amylopectin is synthesized from ADP- glucose while mammals and fungi synthesize glycogen from UDP-glucose; for most cases, bacteria synthesize glycogen from ADP-glucose (analogous to starch) (Ball et al., 2003).
1.2.3 Enzymatic degradation of starch
The effective hydrolysis of starch demands the action of many enzymes due to its complexity, although a prolonged incubation with one particular enzyme can lead to (almost) complete hydrolysis. There are basically four groups of starch-converting enzymes: (i) endoamylases; (ii) exoamylases; (iii) debranching enzymes; and (iv) transferase. Endoamylases are able to cleave α,1-4 glycosidic bonds present in the inner part (endo-) of the amylose or amylopectin chain. Exoamylases act on the external glucose residues of amylose or amylopectin and thus produce only glucose (glucoamylase and α-glucosidase), or maltose and β-limit dextrin (β-amylase). The third group of starch-converting enzymes is the debranching enzymes that exclusively hydrolyze α,1-6 glycosidic bonds: isoamylase (EC 3.2.1.68) and pullulanase type I (EC 3.2.1.41). These enzymes exclusively degrade amylopectin, thus leaving long linear polysaccharides. There are also a number of pullulanase type enzymes that hydrolyze both α, 1- 4 and α,1-6 glycosidic bonds. These belong to the group II pullulanase and are referred to as α- amylase–pullulanase or amylopullulanase. The main degradation products are maltose and maltotriose. The fourth group of starch-converting enzymes are transferases that cleave an α,1- 4 glycosidic bond of the donor molecule and transfer part of the donor to a glycosidic acceptor with the formation of a new glycosidic bond. Enzymes such as amylomaltase (EC 2.4.1.25) and cyclodextringlycosyltransferase (EC 2.4.1.19) form a new α, 1-4 glycosidic bond while branching enzyme (EC 2.4.1.18) forms a new α,1-6 glycosidic bond. Cyclodextringlycosyltransferases have a very low hydrolytic activity and make cyclic oligosaccharides with 6, 7, or 8 glucose residues and highly branched high molecular weight dextrins. Amylomaltases are very similar to cyclodextringlycosyltransferases with respect to the type of enzymatic reaction. The major difference is that amylomaltase performs a transglycosylation reaction resulting in a linear product while cyclodextringlycosyltransferase gives a cyclic product. Depending on the relative location of the bond under attack as counted from the end of the chain, the products of this digestive process are dextrin, maltotriose, maltose, and glucose, etc. Most of the enzymes that convert starch belong to one family based on the amino acid sequence homology: the α-amylase family or family 13 glycosyl hydrolases according to the classification by Henrissat (1991). Other little enzymes that convert starch don’t belong to family 13 glycosyl hydrolases like β-amylases that belong to family 14 glycosyl hydrolases (Henrissat and Bairoch, 1993); and glucoamylases which belong to family 15 glycosyl hydrolases (Aleshin et al., 1994).
This material content is developed to serve as a GUIDE for students to conduct academic research
PARTIAL PURIFICATION AND CHARACTERIZATION OF AMYLASE FROM GERMINATED AFRICAN YAM BEAN SEEDS (SPHENOSTYLIS STENOCARPA)
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