CATALYTIC EFFICIENCY OF AMYLASE PURIFIED FROM FRESH SWEET POTATO (IPOMOEA BATATAS) STEMS USING DIFFERENT SUBSTRATES

ABSTRACT

Beta-amylase was extracted from fresh sweet potato (Ipomoea batatas) stem and was partially purified  using  60%  ammonium  sulphate  saturation  and  gel  filtration  chromatography.  The specific activity for the crude enzyme was found to be 95.56±2.8 U/mg with the purification fold of 1, while the protein concentration was 0.5 mg/ml. After gel filtration chromatography, the specific activity obtained was 186.37±2.56 U/mg with purification fold of 2 at 13.3% recovery. The enzyme displayed maximum activity at pH 5.0 and temperature 55 °C in all the three substrates (sweet potato starch, corn starch and cassava starch). Studies on the effect of substrate concentration   on   the   β-amylase   activity   revealed   increase   in   activity   until   at   certain concentrations from 4 mg/ml upward, and then the activity began to drop. The Km and Vmax were found to be 0.14 mg/ml and 166.67 μmol/min for sweet potato starch, 0.18 mg/ml and

142.86 μmol/min for corn starch, while 0.22 mg/ml and 125 μmol/min was found for cassava starch respectively. The presence of metal ions influenced the activity of the partially purified β- amylase such that the activity of the enzyme was enhanced by Fe3+, Ca2+ while Co2+, Mn2+ and Pb2+ inactivated the enzyme. The presence of Zn2+ and Cu2+ did not show any significant change in activity of the enzyme. The Kcat and Kcat/Km ratio were also found to be 2.06 s-1 and 14.7 s-1 ml mg-1  for sweet potato starch, 1.76 s-1 and 9.8 s-1  ml mg-1  for corn starch, while 1.54 s-1 and

6.16 s-1  ml mg-1    was obtained for cassava starch respectively. The results revealed that the partially purified β-amylase had preference for sweet potato starch over corn starch and cassava starch.

CHAPTER ONE

INTRODUCTION

Enzymes are biological or organic catalysts. They act by lowering the activation energy of biochemical reactions. Different enzymes exhibit varying catalytic powers or efficiencies based on their  catalytic  functions,  measured  as  the  ratio,  Kcat/Km  (Berg,  2007).  They  are,  therefore, specific, occur in low concentrations and their activity can be affected by extreme temperature and pH as well as the presence of activators or inhibitors (Ukoha, 1998).

Amylase is an enzyme that catalyzes the hydrolysis of 1, 4-glycoside linkages found in polysaccharides (Hesam et al., 2015). It actually hydrolyzes starch to produce low molecular weight products such as glucose, maltose and maltotriose units (Tangphatsornruang et al., 2005). Amylases are commonly represented with Greek letters such as α, β and γ-amylase (Oyeleke et al., 2011). The two types of amylases that exist in some species of plants are α-amylase or       E.C:

3.2.1.1; 1-4-α-D-glucan glucohydrolase and β-amylase or E.C: 3.2.1.2; 1-4-β-D-glucan maltohydrolase (Thoma et al., 1971). α-amylase is found in plant tissues, animal tissues and in microbes as well (Saha et al., 1987). β-amylase, mostly of plant origin, has also being recorded from microbial source (Pandey et al., 2000). The practical interest of β-amylase lies in its capacity to produce maltose units from starch (Biovin, 1997; Dicko et al., 2000). It is the hydrolytic activity of this enzyme that has been the basis for various industrial processes like preparation of glucose and   maltose   syrups   like   in   the   supplementary   foods   industries,   breweries   and   starch saccharification (Muralikrishna and Nirmala, 2005 and Prakash et al., 2011). The enzyme is also useful in structural studies of starch and glycogen (Priest, 1984).

In starch-enriched tissues such as seeds or tubers of plants, β-amylase may play a role in the mobilization of starch during the seed germination or sprouting of tubers (Greenwood and Milne,

1968). The characterization of β-amylase has been carried out on enzymes purified from organs enriched in starch from higher plants such as sweet potato tubers (Balls et al., 1948,), leaves (Vikso-Nelson et al., 1997), bulbs (Dicko et al., 2000), seeds of various cereal species such as barley (Shinke et al., 1971), wheat (Trachuk et al., 1966), rice (Okamato and Akazawa, 1978) and other higher plants  such as  soybean  (Gertler and Birk,  1965).  The enzyme was  successfully immobilized on agarose and sepharose gels in the late 1970s (Caldwell et al., 1976). By 1960 and

1970, there was much improvement in the techniques used in the purification of sweet potato β- amylase and this enhanced the catalytic potential of the enzyme (Nakayama and Amagase, 1963; Takeda and Hizukuri, 1969).

In  the  past,  the  production  of  amylases  has  focused  on  microbial  amylases  owing  to  their advantages over plant and animal amylases (Gopinath et al., 2003). The search for alternative sources or substitutes is now on the increase and has been largely linked to the increasing demand for enzymes in industrial processes and the high costs of imported microbial amylases (commercial amylases). This has lead to the study of some plants thought to contain substantial amounts of amylases such as rice, wheat, barley, corn, sweet potato, cassava, Curculigo pilosa, soya bean seeds, acha, onoins and petiole, node or leaf (Dunn, 1974). But less information is known about β- amylase from plant tissues containing transitory starch such as green leaves (Lizotte et al., 1990). An indication of β-amylase activity in plant stems has, however, been reported. Dicko et al. (1999) suggested that the degradation of raw starch in vegetative tissues occurs in the absence of α- amylase and α-glucosidase activities in C. pilosa tuber (before germination) but suggests a role for β-amylase in starch splitting in the vegetative tissues. Sweet potato plant has high levels of β- amylase. Of about 15% of the plant protein, 5% is β-amylase in the tuberous root (Nakamura et al.,

1991). In addition, most reports have shown that green plants are good sources of amylases and their presence show high hydrolytic activity. For instance, Hoyt (2017) opined that amylases are almost always present in the green parts of plants; hence, plants which contain high amounts of starch also contain reasonable amounts of amylases. Funke and Melzing, (2006) also reported that amylases of plant origin have the highest hydrolytic potential followed by fungi while amylases from bacteria have relatively less hydrolytic potential. Zhang and Wang (2002), however, said that plants and bacterial β-amylases are the most heat-stable compared to fungal β-amylase. Sweet potato is thought to be a promising source of β-amylase since β-amylase is one of the major proteins in sweet potato plant (Okon and Uwaifo, 1984). β-amylase from sweet potato, soybean, barley and wheat has been used industrially (Priest, 1984). However, sweet potato β-amylase has shown to  have advantages  over  β-amylases  from  the other  plants  (Hesam  et  al.,  2015).  For example, soybean β-amylase is relatively expensive; and barley and wheat β-amylases have been shown to be lacking in thermo-stability (Hesam et al., (2015). Moreover, sweet potato as a crop is more resistant to unfavourable environments such as typhoons, drought, pests and diseases compared to soybean, wheat and barley (Hesam et al., 2015).

The characterization of β-amylase from sweet potato has been underutilized. Hagemmana et al. (1992) characterized β-amylase from root tissue of sweet potato; Caldwell (1931) and Hesam et al. (2015) characterized β-amylase from sweet potato tuber, while Vikso-Nelson et al. (1997) characterized β-amylase from potato (Solanum tuberosum) leaves. Therefore, this investigation was initiated to characterize β-amylase from fresh sweet potato stem to bridge the gap and add to knowledge on previous works done by the other researchers. This will also lead to full utilization of sweet potato in the production of β-amylase for industrial applications.

1.1 Sweet Potato

Sweet   potato is   a starchy   tuberous crop   from   the   morning   glory   plant   family   called Convolvulaceae. It is a plant commonly classified as a runner, that is, a plant with weak green stems that creeps on soil surface and possess roots at the nodes and also has broad green trifoliate leaves. Sweet potato (Ipomoea batatas) is totally different from ordinary potato (Solanum tuberosum) as they are entirely unrelated, though; their uses can be similar (Eneji et al., 2000). The word “potato” may refer either to the plant itself or to the edible tuber (United Nations Food and Agricultural Organisation, 2009). Potato is a staple food in many parts of the world and an integral part   of   much   of   the   world’s food   supply.   It   is   the   world’s   fourth-largest   food   crop following maize, wheat, and rice (Miller et al., 2008). In Nigeria, only the tubers serve as stable food while the stems have been a popular staple vegetable in Korea, both fresh and dried just as the tubers. The leaves and stems are often prepared as namul (seasoned vegetables) and served as banchan (small side dishes) in Korea and China. Sweet potato plants are herbaceous perennials that grow  about  60 cm  (24 inches)  high,  depending  on  variety,  with  the  leaves dying  back after flowering, fruiting and tuber formation. Sweet potatoes occur in different varieties. The American sweet potato occur as white-skin variety and the orange-skin variety; the Nigerian sweet potato occur as white-skin variety and the red-skin variety; while Korean sweet potatoes have red skins,

but bright yellow flesh, not orange. Tubers form in response to decreasing day length, although this tendency has been minimized in commercial varieties. However, the local importance of the potato is variable and changing rapidly. It remains an essential crop in Europe (especially eastern and central Europe) where per capita production is still the highest in the world, but the most rapid expansion over the past few decades has occurred in southern and eastern Asia. As of 2014, China led the world in potato production, and, together with India, produced 37% of the world’s potatoes. Sweet potato production is determined primarily by seasonal rains. The best crop yields generally occur in areas of 750 to 1,000 millimetres (mm) annual precipitation, with at least 500 mm falling during the growing season (Tewe et  al., 2001). Very often, planting generally takes place from February through July in the central to southern regions, where rainfall is heavier.  However, planting along riverbanks in the central zone, or in swampy areas (fadama) in the north can extend the season to permit planting from September to December.

In Nigeria, the planting of sweet potato follows seasonal patterns and this also depends on the zone and states (Tewe et. al., 2001):

➢  Northwest: This includes Sokoto, Kano, Kebbi, Katsina and Kaduna. Planting is from July

to August and possibly,  a second  crop irrigated from November to December, where feasible;

➢  Northeast: This includes Jigawa, Yobe, Borno, Adamawa, Bauchi. Planting is from May to

July;

➢  North-central: This includes Niger, Kwara, Kogi, Benue, Plateau, Taraba. Ridges are made from May to July, and vines or stems are planted July through August.

➢  Southwest and Southeast: Oyo, Osun, Ondo, Ogun, Lagos, Delta, Edo. Planting is as early

as March, as late as August in Anambra and Enugu States.

Elsewhere in Africa, sweet potato is generally cultivated by independent smallholders, on plots of less than one hectare.  It is often intercropped, in the southern and central areas generally with other root crops (yam, cassava, and cocoyam), and in the north with cereals (maize and millet). However the case, sweet potato can provide soil cover and leaves a large quantity of vegetative residue which incorporates into the soil after harvest (Eneji et al., 1997).

Data pertaining to the marketing of fresh sweet potato in Nigeria are not readily available. Nigeria can provide opportunities for agro-processing of sweet potato, for example, as livestock feed (especially for broiler chickens) and for alcohol production which currently utilizes cassava and other locally made beverage drinks.  For this to be feasible, a large and consistent supply of sweet potato would have to be available at prices considerably below the current market (Tewe et. al., 2001).  However,  given  the  size  of  the  country  and  the  potential  for  greater  sweet potato production if the “yield gap” between optimal and current practices can be narrowed, a sweet potato industry might be feasible in Nigeria.

Sweet potato contains about 20 % carbohydrates particularly starch as the main constituent, 77% water and 1.6% protein (USDA National Nutrient database).  And foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth.  These amylases breakdown tasteless and chalklike starch in sweet potato into simple sugars particularly maltose and it is found that maltose is one-third as sweet as table sugar (Pandey et al., 2000).

Different  varieties  of  sweet  potato  have  been  shown  to  exhibit  varying  levels  of  β-amylase activities (Hesam et al., 2015). Also, all varieties of sweet potato have been good sources of vitamins C and E as well as dietary fibre (3%), potassium and iron, and they are low in fat and cholesterol (USDA National Nutrient database). Sweet potatoes of the orange-and-red-fleshed forms are particularly high in beta-carotene, the vitamin A precursor (Pandey et al., 2000).

The proteins present in sweet potato are mostly in the form of sporamin and β-amylase. According to Nakamura et al. (1991), sporamin and β-amylase are the two most abundant proteins in storage roots of sweet potato (Ipomoea batatas). Besides the developmentally regulated expression in storage roots, expression of genes for sporamin (Spo) and β-amylase (β-Amy) is inducible in vegetative tissues such as stem, leaf and petiole by high levels of sucrose or other metabolizable sugars (Hattori et al., 1990, 1991; Nakamura et al., 1991). Also, studies have shown that the major starch hydrolyzing enzyme is believed to be ⍺-amylase but in the leaves and stems, the amylolytic activity of β-amylase has been shown to be more substantial (Dreier et al., 1995). Expression of a gene for β-amylase in sweet potato is developmentally regulated in its tuberous roots (Yoshida et al., 1992); and it is inducible in typical tissues in response to high levels of sucrose or other metabolizable sugars (Nakamura et al., 1991; Takeda et al., 1994). Studies have also revealed that carbon assimilates synthesized in photosynthetic source tissues such as mature leaves are transported to sink tissues such as roots, fruits, seeds and tubers generally in the form of sucrose,

and utilized as a source of energy and carbon skeleton (Sheen et al., 2000; Smeekens, 2000). Caspar et al. (1989) also suggested that β-amylase may be induced in response to the high levels of soluble sugars that accumulate during photoperiod in mutants of Arabidopsis plants. Satoru et al. (1995) reported extracts of various organs of Arabidopsis plants that had been treated with 5% Sucrose in the presence or absence of light for 3 days and were assayed for levels of amylolytic activity and β-amylase protein. Extracts from stems, bracts (leaves attached to the stem), and roots of non-treated mature plants yielded a band of amylolytic activity that migrated at the same position as that of the β-amylase from rosette leaves. In addition to the results in rosette leaves, the levels of amylolytic activity and β-amylase protein in these organs increased significantly when plants were cultured with a solution of sucrose under continuous light. When sucrose was supplied to plants in darkness, there was no significant increase in the β-amylase activity in these organs. Glucose also induced increases in β-amylase activity in the various organs. This indicated the expression of a single-copy gene for β-amylase in the various vegetative tissues by an exogenous supply of high levels of sucrose or other metabolizable sugars, a condition that also induces the accumulation of starch (Caspar et al., 1989).

1.2 Starch

In the green leaves or tissues of plants, 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 or stem- the so-called leaf-transition starch or stem-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 as the familiar grain or tuber shape. The forming of starch is a process which has by far not been clarified yet and it is a process 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 limits its use in some industrial food applications (Van der Maarel et al., 2002; Goyal et al., 2005). 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 (sediment) 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 called “gelatinized starch”. In comparison with the “ungelatinized suspension”, stirring is considerably more difficult. The temperature at which the resistance during stirring noticeably increases is called the gelatinization temperature. Gelatinizing starch into viscous substances is one, if not the most, of 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 (Figure 3). 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 also considered to be responsible for the synthesis of unit chains of amylopectin.

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