ABSTRACT
This study was aimed at the production of glucoamylase which can be utilised for starch hydrolysis. A fourteen days experimental study was carried out to determine the day of highest glucoamylase activity. Day five and day twelve of the fourteen days experimental study had the highest glucoamylase activity. The specific activity for the crude enzyme was found to be 729.45 U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from guinea corn starch as the carbon source after five days of fermentation (GluAgGC5), and 1046.82 U/mg for glucoamylase isolated from Aspergillus niger in submerged fermentation using amylopectin fractionated from guinea corn starch as the carbon source after twelve days of fermentation (GluAgGC12).The crude enzyme was purified by ammonium sulphate precipitation and by gel filtration (using sephadex G 100 gel). Ammonium sulphate saturations of 70% and 20% were found suitable to precipitate proteins with highest glucoamylase activity. After ammonium sulphate precipitation, the specific activities of the enzyme were found to be 65.98 U/mg and 61.51 U/mg for GluAgGC5 and GluAgGC12, respectively. Similarly, after gel filtration, the specific activities of the enzyme were found to be 180.52 U/mg and 272.81 U/mg for GluAgGC5 and GluAgGC12, respectively. The optimum pH for GluAgGC5 were found to be 7.5,7.5 and 6.0 when using tiger nut starch, cassava starch and guinea corn starch as substrates, respectively, while the optimum pH for GluAgGC12 were found to be 5.0, 8.5 and 7.0 when using tiger nut starch, cassava starch and guinea corn starch as substrates, respectively. The enzyme activity in GluAgGC5 was enhanced by Ca2+,Co2+, Fe2+, Mn2+and Zn2+ but Pb2+ had inhibitory effect on the enzyme. Similarly, the enzyme activity of GluAgGC12 was enhanced by Ca2+, Zn2+, Co2+, Fe2+ and Mn2+ while Pb2+ had inhibitory effect on the enzyme. The optimum temperatures were found to be 50˚C and 45˚C for GluAgGC5 and GluAgGC12, respectively. The Michaelis Menten’s constant, Km and maximum velocity Vmax of GluAgGC5 obtained from the Lineweaver-Burk plot of initial velocity data at different substrate concentrations were found to be 770.75 mg/ml and 2500µmol/min using cassava starch as substrate, 158.55 mg/ml and 500 µmol/min using guinea corn starch as substrate and 46.23 mg/ml and 454.53µmol/min using tiger nut starch as substrate. Also, the Km and Vmax of GluAgGC12 were found to be 87.1 mg/ml and 384.61µmol/min using cassava starch as substrate, 29.51 mg/ml and 243.90 µmol/min using guinea corn starch as substrate and 2364 mg/ml and 2500µmol/min using tiger nut starches as substrate.
CHAPTER ONE
INTRODUCTION
Starch degrading enzymes are currently becoming and gaining more importance among the industrial enzymes because of the importance of starch, sugars and other products in modern biotechnological era (Omemu et al., 2008). Majority of these starch degrading enzymes are carbohydrases (that is, the amylases or starch converting enzymes), and they can be grouped into four types; the endoamylases, the exoamylases, the debranching enzymes and the transferases (Siew et al., 2012).
The endoamylases otherwise referred to as the endoacting enzymes are able to cleave α-1, 4 glucosidic bonds present in the inner part (endo-) of the amylose or amylopectin chain, the enzyme, α-amylase (EC3.2.1.1) is a well-known endoamylases (Van Der Maare et al., 2002 ). Similarly, the exoamylases cleave either or both the α-1, 4 and α-1, 6 bonds on the external glucose residues of amylose or amylopectin from the nonreducing end and thus produce only glucose (Bertoldo et al., 2002), glucoamylases (EC3.2.1.3) and α-glucosidases (EC 3.2.1.20) are very good examples of the exoamylases. The transferases are another group of starch- converting enzymes that cleave an α-1, 4 glucosidic bond of the donor molecule and transfer part of the donor to a glucosidic acceptor with the formation of a new glucosidic bond (Tharanathan and Mahadevamma, 2003). Enzymes such as amylomaltase (EC 2.4.1.25) and cyclodextrin glycosyltransferase (EC 2.4.1.19) form a new α-1, 4 glucosidic bond while branching enzyme (EC 2.4.1.18) forms a new α-1, 6 glucosidic bond. The debranching enzymes catalyse the hydrolysis of α-1, 6-glucosidic bonds in amylopectin and/or glycogen and related polymers. The affinity of debranching enzymes for the α-1, 6-bond distinguishes these enzymes from other amylases which have primary affinity for α-1, 4-glucosidic linkages (Siew et al., 2012). The enzyme pullulanase and isoamylase are well known examples of the debranching enzymes.
Carbohydrases, therefore, are those groups of enzymes which catalyses the breakdown of carbohydrates (e.g. starch, oligosaccharides as well as polysaccharides), into simple sugars. Examples of the carbohydrases include α – amylase, glucoamylase, etc. Alpha -amylase (E.C.3.2.1.1) hydrolyses α-l, 4- glycosidic bonds randomly in amylose, amylopectin and glycogen in an endo fashion. All α-amylases bypass α-1, 6-glycosidic bonds, but do not cleave
them. Hydrolysis of amylose by α -amylase causes its conversion into maltose and maltotriose, followed by a second stage in the reaction, the hydrolysis of maltotrioses. Glucoamylase (EC
3.2.1.3) is the exo-acting enzyme that hydrolyzes both 1,4-alpha- and 1,6-alpha-glucosidic linkages in amylose, amylopectin, glycogen as well as other related oligo and polysaccharides, yielding β-D-glucose as the end product. Hence, glucoamylases can serve as an industrially useful enzyme (Siddhartha et al., 2012).
Currently, amylases are of great importance in biotechnology with a wide spectrum of applications, such as in textile industry, cellulose, leather, detergents, liquor, bread, children cereals, ethanol production, and high fructose syrups production and in various strategies in the pharmaceutical and chemical industries such as the synthesis of optically pure drugs and agrochemicals (Mervat, 2012).
Figure 1: Schematic presentation of the starch degrading enzymes (endoamylases, exoamylases, debranching enzymes and the transferases). Black circles indicate reducing sugars. (Siew et al., 2012).
Glucoamylase belong to the amylase family, and the amylases are among the most important enzymes that are of great significance in present day biotechnology (Ritesh and Barkha, 2011). The amylase family has two major classes, namely: amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3). Glucoamylase is produced by a variety of fungi but the exclusive production of this enzyme in industries have been achieved mainly by Aspergillus niger, Aspergillus oryzae, Aspergillus awamori and Aspergillus terreus, probably because of their ubiquitous nature and non-fastidious nutritional requirements of these organisms (Siddhartha et al., 2013). In fungal cultures, glucoamylase rarely occurs without alpha amylase production. Other amylolytic enzymes such as alpha glucosidase are also likely to be concomitantly produced (Kshipra et al.,
2011).
1.1. Glucoamylase
Glucoamylase (1, 4-α- D-glucan glucohydrolases, EC 3.2.1.3) is an exo enzyme of great importance for saccharification of starchy materials and other related oligosaccharides and polysaccharides. This enzyme hydrolyzes 1,4-alpha-glycosidic linkages from the non-reducing end of starch as well as the 1,6-alpha-glucosidic linkages of starch and other related oligosaccharides and polysaccharides, yielding, β-D-glucose as the end product (Uma and Nasrin, 2013). Glucoamylase (E.C. 3.2.1.3) is an enzyme that cleaves glucosyl units from the non reducing end of amylose chain, glycogen and amylopectin linkages. This enzyme catalyses the hydrolysis of α-1,4 – linkages faster (Muhammad et al., 2012), and to a lesser extent, hydrolyzes α -1, 6 linkages, resulting in β -D- glucose as the end-product (Abdalwahab et al.,
2012).
Microbial enzymes are currently becoming increasingly important due to their technical and economic advantages (Damisa et al., 2013). In the production of glucoamylase from microbial sources, the organism needs essential elements such as nitrogen, carbon, phosphorus and sulphur for growth and subsequent amylase production. The concentrations of these elements have a profound effect on the yield of the enzyme.
Generally, amylases, (that is α- amylases, β-amylases and glucoamylases) can be produced either by submerged fermentation (SmF) or solid state fermentation (SSF) procedures.
However, the convectional amylase production is carried out by submerged fermentation (Radha et al., 2012). Glucoamylase production from microbial sources especially from Aspergillus niger is generally extracellular, and the enzyme can be recovered from culture filtrates (Sarojin et al., 2012). However, the extensive production of glucoamylase is obtained by using the fungus Aspergillus niger in enzyme production industries. This enzyme (glucoamylase) is generally regarded as safe (GRAS) by the Food and Drug Administration (FDA) (Siddhartha et al., 2013).
1.2 Aspergillus niger as a Microbial Source of Glucoamylase
Aspergillus is a large genus composed of more than a hundred and eighty accepted anamorphic species, with teleomorphs described in nine different genera (Pitt and Samson, 2000). The genus is subdivided in seven subgenera, which in turn are further divided into sections (Klich,
2002). Aspergillus mold species are found throughout the world and are the most common type of fungi in our environment (Suhaib et al., 2012). About sixteen species of these molds are dangerous to humans, causing diseases and infections. Aspergillus molds have a powdery texture. However, the colour of the mold’s surface differs from species to species and can be used to identify the type of Aspergillus.
Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. The black aspergilli are among the most common fungi causing food spoilage and bio- deterioration of other materials. They have also been extensively used for various biotechnological purposes, including production of enzymes and organic acids (Schuster et al.,
2002). Aspergillus niger is one of the most important microorganisms used in biotechnology. It is the most frequently reported species, and has often been included in biotechnological processes that are generally regarded as safe (GRAS) (Samson et al., 2007).
Since 1960s, Aspergillus niger has become a source of a variety of enzymes that are well established as technical aids in fruit processing, baking, and in the starch and food industries. This is because they are filamentous fungus growing aerobically on organic matter. In nature, they are found in soil and litter, in compost and on decaying plant material. They are able to grow in the wide temperature range of 6–47°C with a relatively high temperature optimum at
35–37°C. Aspergillus niger is able to grow over an extremely wide pH range: 1.4–9.8. These abilities and the profuse production of conidiospores, which are distributed via the air, secure the ubiquitous occurrence of the species, with a higher frequency in warm and humid places (Schuster et al., 2002).
1.2.1 Taxonomy of Aspergillus niger
Domain:Eukaryota Kingdom: Fungi Phylum: Ascomycota
Subphylum: Pezizomycotina Class: Eurotiomycetes Order: Eurotiales
Family: Trichocomaceae
Genus: Aspergillus
Species : Aspergillus niger
Source : (Samson et al., 2007)
1.2.2 Identification of Aspergillus Species
Generally, identification of the Aspergillus species is based on the morphological characteristics of the colony and microscopic examinations; although molecular methods continue to improve and become more rapidly available, microscopy and culture remains the commonly used and essential tools for identification of Aspergillus species (McClenny, 2005).
1.2.3 Morphological Identification of Aspergillus Cultures
Morphological features of Aspergillus cultures have been studied. The major and remarkable macroscopic features in species identification were the colony diameter, colour (conidia and reverse), exudates and colony texture. Microscopic characteristics for the identification were conidial heads, stipes, colour, length vesicles shape and seriation, metula covering, conidia size, shape and roughness also colony features including diameter after seven (7) days, colour of conidia, mycelia, exudates and reverse, colony texture and shape ( Diba et al., 2007).
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PRODUCTION OF Aspergillus Niger GLUCOAMYLASE USING GUINEA CORN STARCH AMYLOPECTIN AS THE ONLY CARBON SOURCE>
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