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PARTIAL PURIFICATION AND CHARACTERIZATION OF AMYLASE FROM GERMINATED AFRICAN YAM BEAN SEEDS (SPHENOSTYLIS STENOCARPA)

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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  Î±-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.   Î±-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,  Î±-glucosidase  and  debranching enzymes.  Î±-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  Î²-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.  Î±-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  Î±-glucosidase),  or maltose and β-limit dextrin (β-amylase). The third  group of  starch-converting  enzymes  is the debranching  enzymes  that exclusively hydrolyze  Î±,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  Î±,  1-4  glycosidic  bond  while branching     enzyme      (EC     2.4.1.18)     forms     a     new      Î±,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).


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PARTIAL PURIFICATION AND CHARACTERIZATION OF AMYLASE FROM GERMINATED AFRICAN YAM BEAN SEEDS (SPHENOSTYLIS STENOCARPA)

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