ISOLATION PARTIAL PURIFICATION AND CHARACTERIZATION OF GLUCOAMYLASE FROM ASPERGILLUS NIGER IN SUBMERGED FERMENTATION USING AMYLOPECTIN FROM TIGER NUT STARCH AS CARBON SOURCE

ABSTRACT

Glucoamylase  was  isolated  from  Aspergillus  niger  and  purified  using  ammonium sulphate precipitation and  gel  filtration respectively.  The  purified  enzyme was then characterized to determine the optimum conditions required by the produced enzyme. A fourteen day pilot study was carried out to determine the day of isolation of crude with highest glucoamylase activity. Day 5 and 12 had highest glucoamylase activity. The specific activities for the crude enzyme were found to be 757.5U/mg and 1223.88U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from tiger nut starch as the carbon source after five days of fermentation (GluAgTN5) and twelve days of fermentation (GluAgTN12). Ammonium sulphate (20% and 90%) saturation was found suitable to precipitate protein with highest glucoamylase activity for GluAgTN5 and GluAgTN12, respectively. Following ammonium sulphate precipitation and gel filtration, the specific activities were found to be 89.90U/mg and 276.03U/mg for GluAgTN5, While for GluAgTN12, the specific activities were 88.75U/mg and 80.95U/mg following ammonium sulphate precipitation and gel filtration, respectively. The optimum pH and temperature for GluAgTN5 were found to be 6.5, 7.0, 6.0 at 55°C and 8.5, 6.0, 7.5 at 50°C for GluAgTN12 using cassava, guinea corn and tiger nut starch as substrates. The enzyme activity in GluAgTN5 was enhanced by Ca2+  and Fe2+  while Zn2+  and Co2+  had inhibitory effects, Mn2+and Pb2+, however completely inactivated the enzyme. The enzyme activity in GluAgTN12 was enhanced  by  Ca2+   while  Co2+and  Zn2+   had  inhibitory effects,  Fe2+,  Mn2+   and  Pb2+ completely inactivated the enzyme. The Michealis-Menten constant, Km   and maximum velocity, Vmax obtained from Line-Weaver-Burk plot of initial velocity data at different substrate concentrations were found to be 222mg/ml and 500µmol/min using cassava starch,  291mg/ml  and  1000µmol/min  using  guinea  corn  starch,  137.5mg/ml  and 500µmol/min using tiger nut starch as substrate for GluAgTN5. While for GluAgTN12, Km  and Vmax  were found to be 176.6mg/ml and 100µmol/min using cassava starch, 491mg/ml and 1000µmol/min using guinea corn starch, 131.5mg/ml and 500µmol/min using tiger nut starch as substrate.

CHAPTER ONE

INTRODUCTION

Cyperus esculentus is an edible perennial sedge of the family Cyperacheae which is under-utilized but yet, has been reported to be highly beneficial to health (Oladele et al., 2010). The common names for which it is known by include; tiger nut, yellow nut grass (or nutsedge), earth almond, chufa (in Spanish), aya (in Hausa), aki hausa (in Igbo) and ofio  (in Yoruba) (Bamishaiye et  al., 2010; Bamishaiye and Bamishaiye, 2011). Native to most of the Western hemisphere as well as southern Europe, Middle East, Africa and India, C. esculentus is a temperate sedge that require moist sandy soils for survival. C. esculentus has been found to be rich in carbohydrates (33.8%), unsaturated oils (22.5%), proteins (8.37%), and fibre (23.3%) (Ekeanyanwu and Ononogbu, 2010).

Starch, a white granular organic substance produced by all green plants (Acharya et al., 2014). It is made up glucose units linked together by glycosidic linkages. It has two major components; amylose and amylopectin. While amylose is the straight unbranched component of starch linked together by α-1,4-glycosidic linkages, amylopectin occurs at branch points with the glucose units attached by α-1,6-glycosidic linkages (Nelson and Cox, 2008). Of the two components of starch, amylopectin presents the greatest challenge to hydrolytic enzyme systems (Bhatti et al., 2007; Lin et al., 2013).

Amylases are a group of hydrolases which can specifically cleave glycosidic bonds in starch (Imran et al., 2012; Acharya et al., 2014). They are calcium metalloenzymes. There are two important groups of amylases which include α-amylase and glucoamylase. Glucoamylase (exo-1,4-α-D-glucan glucanohydrolase, E.C. 3.2.1.3) hydrolysesz single glucose units from the non-reducing ends of amylose and amylopectin (Anto  et  al.,  2006; Nahid et  al.,  2012).  However, α-amylases (endo-1,4-α-D-glucan glucohydrolase, E.C. 3.2.1.1) are extracellular enzymes that can randomly cleave 1,4-α- D-glucosidic linkages between adjacent glucose units inside the linear amylose chain, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and “limit dextrin” from amylopectin (Pandey et al., 2000; Anto et al., 2006; Castro et al., 2010). A third form of amylase called β-amylase (E.C. 3.2.1.2) synthesized by bacteria, plants and

fungi, cleaves α-1,4 glycosidic bond successively removing maltose units from the non- reducing end of amylase (Olufunke, 2013).

Glucoamylase (GA) (exo-1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) is an exo- amylase that catalyses the hydrolysis of α-1,4glucosidic linkages and to a lesser extent α- 1,6 glucosidic linkages to release β-D-glucose from the non-reducing ends of starch (Svensson et al., 2000). They are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region (Horváthova et al., 2001; Eliasson, 2004). Glucoamylase is widely used in food industry to produce high glucose syrup, and also in fermentation processes for production of beer and ethanol (Shenoy et al., 1985; Pavezzi et al., 2008). This enzyme is produced industrially by a variety of microorganisms (Kumar et al., 2012). Aspergillus niger, Aspergillus awamori and Rhizopus oryzae have been considered the most important for industrial application (Coutinho and Reilly, 1997). Glucoamylase has been reported to occur mostly in animal and plants especially during the process of fruit ripening (Mertens and Skory, 2006). Conventionally glucoamylase is produced by submerged fermentation (SmF) using fungi. The commercial use of fungal glucoamylases are limited by moderate thermostability, acidic pH requirement and slow catalytic activity (Kumar and Satyanarayana, 2009; Das et al., 2011).

Starch hydrolysis is a widely used process in various industries. The production of low molecular mass products from starch substrate underlies the sugar, brewing, spirits, textile and other forms of processing in industries. Starch-converting enzymes are also used in industrial processes, such as laundry, porcelain and detergents or as anti-staling agents in baking (Apar and Özbek, 2004). The use of microbial amylases (enzymatic hydrolysis)  has  almost  completely replaced  chemical  hydrolysis  of  starch  in  starch processing industries (Morita et al., 2008; Imran et al., 2012; Lin et al., 2013). This has created a need for the production of glucoamylase with a higher catalytic efficiency capable of completely hydrolyzing starch. In view of this, several studies have been carried out on the isolation of glucoamylase from various fungi using amylopectin from cassava, corn and potato, rice, wheat and pastry wastes as sole carbon sources (Pavezzi et al., 2008; Suganthi et al., 2011; Lakshmi et al., 2013; Lin et al., 2013). However, no research has reported isolation of glucoamylase using amylopectin from the underutilized

tiger nut starch as the only carbon source. This could be the key to unlocking the door to the availability of highly efficient glucoamylase for applications in industry, simultaneously increasing the economic relevance of the underutilized sedge.

1.1      Tiger Nut

Although an important food in ancient Egypt, with evidence lying in ancient tombs from predynastic times, Cyperus esculentus has become less popular in many parts of the world. Cyperus esculentus is an edible perennial sedge of the family Cyperacheae which is under-utilized but yet, has been reported to be highly beneficial to health. It produces rhizomes from base that are somewhat spherical (Ekeanyanwu et al., 2010). The plant is not really a nut but a tuber and has common names for which it is known by including; yellow nut grass (or nutsedge), earth almond, chufa (in Spanish), aya (in Hausa), aki hausa (in Igbo) and ofio (in Yoruba) (Bamishaiye et al., 2010; Ekeanyanwu and Ononogbu, 2010; Oladele, et al., 2010; Bamishaiye and Bamishaiye, 2011). Native to most of the Western hemisphere as well as southern Europe, Middle East, Africa and India, C. escuentus is a temperate sedge (or weed in some areas) that requires moist sandy soils for survival and has become naturalized in other parts of Europe and Asia. Tiger nut can be eaten raw, roasted, dried, baked or in the form of milk (Ekeanyanwu et al., 2010; Bamishaiye and Bamishaiye, 2011).

Figure 1: Tiger nut sedge and tubers

1.1.1   Nutritional Components of Tiger Nut

C. esculentus has been found to be rich in carbohydrates (33.8%), unsaturated oils (22.5%), proteins (8.37%), and fibre (23.3%) (Oladele et al., 2010). Tiger nut flour has also been found to contain minerals (sodium, calcium, phosphorus, iron, zinc and magnesium), vitamins (A, C, E and D) and amino acids (Ekeanyanwu and Ononogbu,

2010). The absence of cholesterol in C. esculentus has been the basis for its stated role in prevention and management of heart diseases. Tiger nut has also been thought to be effective in weight reduction, flatulence, diarrhea, thrombosis, colon cancer treatment with no report of allergy thus far (Bamishaiye et al., 2010). This unpopular wonder tuber has served as food for poultry, some aquatic animals, livestock and humans and can be consumed as a variety of delicacies relative to region or culture (Oladele et al., 2010). For instance, in Spain the tubers are consumed mostly in the form of natural tiger nut milk called horchata as well as used in the form of flour for cakes, in Morroco it is dried and ground and used as spice, in Nigeria the tubers are consumed in their natural form as a side collation and in the form of locally made milk drink called kunnu (Bamishaiye and Bamishaiye, 2011).

  Table 1: Nutritional content of tiger nut in mg/100g                                                   

  Components                                      Raw                                      Roasted                         

Crude protein (%)	8.07 ± 0.37		6.80 ± 0.89
Crude fat (%)	24.3 ± 0.58		26.3 ± 0.10
Crude fibre (%)	24.0 ± 1.58		23.3 ± 0.58
Ash (%)	1.80 ± 0.10		1.78 ± 0.10
Carbohydrates (%)	30.0		31.7
Moisture (%)	11.4 ± 0.12		11.2 ± 0.02
Fatty acids (%)	19.44		20.16
Energy (kg/100g)	1546.3		1586.9
Vitamins and Minerals (mg/100g)
A	0.21 ± 0.01		0.20 ± 0.01
C	7.30 ± 0.97		4.59 ± 0.09
D	0.42 ± 0.02		0.41 ± 0.01
E	0.74 ± 0.09		0.57 ± 0.10
Sodium (Na)	34.3 ± 1.53		34.1 ± 1.44

Calcium (Ca)	100.0 ± 2.65	99.9 ± 2.86
Iron (Fe)	4.12 ± 0.10	4.11 ± 0.26
Zinc (Zn)	3.98 ± 0.31	3.96 ± 0.50
Potassium (K)	486.0 ± 0.59	491.0 ± 48.9
Magnesium (Mg)	94.4 ± 1.28	96.0 ± 0.38
Copper (Cu)	0.92 ± 0.05	0.88 ± 0.15
Manganese (Mn)	0.20 ± 0.01	0.30 ± 0.02
Phosphorus (P)	219.0 ± 10.0	217.0 ± 12.1

(Ekeanyanwu and Ononogbu, 2010)

1.1.2   Phytochemicals of Tiger Nut Tuber

Phytochemicals are biologically active, naturally occurring chemical compounds produced by plants. They provide health benefits for humans beyond those attributed to macronutrients and micronutrients (Hasler and Blumberg, 1991). Phytochemicals protect plants from disease and damage (caused by environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack), contribute to the plant’s color, aroma and flavor (Gibson et al., 1998; Saxena et al., 2013).

Table 2: Bioactive phytochemicals in medicinal plants

Classification           Main groups of compounds                       Biological function

NSA                          Cellulose, hemicellulose, gums,                   Water holding capacity, delay

Non-starch polysaccharides Antibacterial and Antifungal

mucilages, pectins, lignins                            In nutrient absorption, binding toxins and bile acids

Terpenoids, alkaloids, phenolics                  Inhibitors of micro-organisms, reduce the risk of fungal infection

Antioxidants             Polyphenolic compounds, flavonoids, carotenoids,

Anticancer                 Carotenoids, polyphenols, curcumine, Flavonoids

Oxygen free radical quenching, inhibition of lipid peroxidation

Inhibitors of tumor, inhibits the development of lung cancer,anti- metastatic activity

Detoxifying

Agents

Reductive acids, tocopherols, phenols, indoles, aromatic iso-Thiocyanates, coumatrins, Flavones, carotenoids,

retinoids, cyanates, phytosterols

Inhibitors of procarcinogen, activation, inducers of drug, binding of carcinogens, inhibitors of tumourigenesis

Other                         Alkaloids, terpenoids, volatile, flavor compounds, biogenic amines

Neuropharmacological agents, anti- oxidant.

(Saxena et al., 2013)

Studies have shown that upon roasting of the tuber, the levels of some nutrients and anti-nutrients may be reduced, however consumption of raw tiger nut tuber does not produce any undesirable effect to the body (Uwakwe and Monago, 2009; Adejuyitan,

2011; Ukwuru et al., 2011).

Table 3: Phytochemical composition of tiger nut tubers

Phytochemical	Raw	Roasted
Alkaloids	+++	+
Glycosides   Cyanogenic glycosides   Resins   Flavonoids	ND   + +++ ND	ND ND +++   ND
Cardiac glycosides   Tannins + Sterols Saponins	ND ND +++   +	ND ND +++ ND

+++ = Present in very high concentration, ++ = Present in moderately high concentration, + = Present

in trace concentration, ND = Not detected

(Ekeanyanwu et al., 2010)

1.1.3   Health Benefits of Tiger Nut

Tiger nut can be consumed raw, roasted, baked or cooked (Adejuyitan, 2011). Cyperus  esculentus  was  reported  to   be   beneficial  in  preventing  atherosclerosis, thrombosis and activates blood circulation (Mohamed et al., 2005). It was also speculated to be responsible for preventing and treating urinary tract and bacterial infection, this may be attributed to the presence in very high concentration of alkaloids and resins as shown in table 3 (Ekeanyanwu et al., 2010; Saxena et al., 2013). Tiger nut is also thought to assist  in  reducing  the  risk  of several  forms  of  cancer  including  colon  cancer,  also inhibiting tumourigenesis, possessing anti-diabetic, weight-losing effect and anti-sickling properties (Uwakwe and Monago, 2009; Adejuyitan et al., 2009). Research has indicated

that Cyperus esculentus may play an important role in enhancing fertility (Agbai and Nwanegwo, 2013a; 2013b). The anti-diuretic, anti-inflammatory, anti-analgesic, anti- cancer, anti-viral, anti-malarial, anti-bacterial and anti-fungal activities of tiger nut may occur as a result of the presence of nutrients and anti-nutrients (alkaloids, tannins, resins, saponins, polyphenols, non starch polysaccharides and sterols) that  occur naturally in the plant (Wadood et al., 2013).

1.1.4   Industrial Applications of Tiger Nut

In the industry, the underutilized tiger nut  is becoming increasingly relevant. Tiger nut is gradually gaining popularity in the production of yogurt. This may be due to the  desired  flavor  imparted  by  the  nut  (El-Shenawy  et  al.,  2012).  Tiger  nut  milk, popularly known as ‘Horchata’ (in Spain) or ‘kunnu’ (in Nigeria) is gradually becoming a favorite drink as a result of the increasing knowledge of the nutritional value of the sedge (Bamishaiye and Bamishaiye, 2011). It is also used as a flavoring agent for ice cream and biscuits, also in the manufacture of nutritious products like bread, cakes (Ade- Omowaye et al., 2008; Ukwuru et al., 2011; Gambo and Da’u, 2014) and pudding (Abo- El-Fetoh et al., 2010). There is speculation in the potential application of tiger nut as a biomaterial for industrial processes such as fermentation (Adama et al., 2014), however, this area is yet to be explored.

1.1.5   Tiger Nut Starch

Starch exists as a major carbohydrate storage product in all plants containing chlorophyll. In the process known as photosynthesis, green plants extract energy from sunlight to form glucose using carbon dioxide and water (Nelson and Cox, 2008). Starch is a carbohydrate polymer made by the linking of glucose units which are oriented within granules in specific crystalline patterns. The major constituents of starch granules are amylopectin and amylose (Bhatti et al., 2007). Amylopectin (~75%) is a semi- crystalline highly branched polysaccharide with an α-1,4 backbone and 4-5% α-1,6 branch points enabling structuring of the starch granule while amylose (~25%) is amorphous in the native starch granule and is composed of essentially linear chains of α-1,4 linked glucose units (Eliasson, 2004; Horváthova et al., 2001). Amylose and amylopectin are products from  different  genes.  In  many  different  plants  (e.g.  Chlamydomonas reinhardtii)  a granule-bound starch synthase has been shown to be involved in the synthesis of amylase. Plants defective in this  enzyme (waxy  mutants) give  rise to  starch granules having majorly amylopectin (Eliasson, 2004). Starch obtained from tiger nut appears as a brilliant white crystalline non- hygroscopic powder with yields ranging from 14% to 31% depending on the sizes of the tubers used (Defelice, 2002; Adejuyitan et al., 2009). Tiger nut starch has been observed to have physicochemical properties comparable to maize starch and corn starch (Abo-El- Fetoh et al., 2010; Adama et al., 2014). Amylose digested by amylase, has been reported to occur less frequently in tiger nut starch (about 28%). Amylopectin however, which is digested by the enzyme glucoamylase, has been shown to occur more frequently in tiger nut starch (about 72%) (Adama et al., 2014).

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