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, K_m and maximum velocity V_max 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 K_m   and  V_max   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).

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).

• 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).

 

 

 

 

Figure 2: Photographs of colonies of Aspergillus species in czapek yeast agar (CYA) and Malt extract agar (MEA) at 25 ⁰C, after seven days, showed morphological difference. A. aculeatus (A-B); A. carbonarius (C-D); A. foetidus (E-F); A. sp. UFLA DCA 01 (G-H); A. japonicus (I-J); A. niger (K-L); A. niger Aggregate (M-N), A. tubingensis (O-P)   (Silva et al., 2011).

 

 

Table 1: Microscopic Characteristics Used for Identification of Aspergillus Isolates

Fungus                                                        Microscopic Features
Aspergillus                           Stripe                            Vesicle       Metula                           Conidia Species              Size            Colour        Surface      Serration   Covering      Shape         Surface
A.  flavus	400-800	Pale Brown	Quietly spherical	Biseriate	3/4	Glubose ellipsoid	Smooth finely roughened.
A.  niger	400 -3000	Slightly brown	Smooth walled	Biseriate large size	Entirely	Glubose	Very rough, irregular
A. fumigatus	200-400	Greyish near apex	Smooth walled	Uniseriate pyriform	Upper 2/3	Glubose small in columns	Smooth or spinose
A. nidulans	70-150	Brown in age	Smooth walled	Biseriate Spatulate	Upper 1/2	Spherical	Smooth Slightly rough
A. tereus	100-250	Uncoloured	Smooth walled	Biseriate spherical	Upper ½ to 3/4	Glubose	Smooth walled
A. parasiticus	250-500	Colourless	Finely roughened	Uniseriate spherical	1/2	Glubose	Distinctly rough
A. oryzae	500-2500	Uncoloured	Rough	Uniseriate	½ or more	Glubose	Smooth
A. tamari	600-1500	Uncoloured	Rough walled	Biseriate Spatulate	Entirely	Spherical	Smooth
A. ochraceus	300-1700	Yellowish pale brown	Coarsely rough	Biseriate globose	Entirely	Spherical small	Smooth finely rough
A. sojae	300-900	Uncoloured	Rough	Uniseriate		Spherical Rough walled	Rough walled
A. niveus	100-500	Uncoloured	Smooth	Biseriate	Upper 2/3	Glubose	Smooth walled

 

Source: (Diba et al., 2007)

 

 

 

 

 

Table 2: Macroscopic Characteristics Used for Identification of Aspergillus Isolates

Macroscopic Features
Fungi                            On CYA                                                                 On MEA
Colour of           Reverse                                 Colour of        Reverse Conidium           Colour        Diameter(mm)  Conidium        Colour           Diameter(mm)
A. niger	Green	White to cream, yellow	28	Green	White to cream yellow	33
A. flavus	White to cream yellow	Brown	19	Green	Brown	23
A. fumigatus	Yellow orange	Pale Brown	44	Green	Yellow	50
A. japonicus	Green	White  to cream	31	Green	Brown	39
A. terreus	Green	Yellow	20	White to cream	White to cream	22

(CYA) Czapek yeast agar, (MEA) Malt extract agar.

Source: (Suhaib et al., 2012)

1.2.4 Uses of Aspergillus niger

Aspergillus niger shows an amazing nutritional flexibility and metabolic capacity, and produces high levels of secreted primary and secondary metabolites. This organism, Aspergillus niger, offers valuable advantages for enzyme secretion, such as facilitated proteolytic processing and protein folding as well as posttranslational modifications (Lubertozzi and Keasling, 2009). This has made this microorganism a potentially attractive host for the biotechnological industry, especially the genus Aspergillus, which is frequently applied in enzyme production due to the GRAS status. (Samson et al., 2007).

Due to enormous development of genetic engineering and efficient expression systems, Aspergillus species have also achieved increased attention as host for industrial production of homologues and heterologous proteins. Aspergillus niger is cultured for the industrial production of many substances. Many useful enzymes are produced using industrial fermentation of Aspergillus niger. For example, Aspergillus niger glucoamylase formed is used in the production of high fructose corn syrup, pectinases produced are used in cider and wine clarification, alpha-galactosidase, an enzyme produced are used to break down certain complex sugars (Schuster et al., 2002).

1.2.5 Safety Aspects of Aspergillus niger

Aspergillus niger is generally regarded as a safe organism. In rare cases when persons are exposed to intense spore dust, hyper-sensitivity reactions have been observed.

They are generally regarded as a non-pathogenic fungus widely distributed in nature. Humans are exposed to its spores every day without disease becoming apparent. Only in few cases has Aspergillus niger been able to colonise the human body as an opportunistic invader and in almost all these cases the patients have a history of severe illness or immunosuppressive treatment (Schuster et al., 2002).

 

1.3 Guinea Corn

Guinea corn (Sorghum bicolor or Sorghum vulgare) is a cereal crop commonly known as grain sorghum. It belongs to the family: Poaceae (or Gramineae), general class: sorghum (Chukwu et al., 2011). The guinea corn (Sorghum grain) is the fifth most important cereal in the world after wheat, rice, maize and barley, it varies in colour, shape, and size. The colour of the guinea corn grain varies from white to dark brown depending on the phenolic pigments present. This cereal seed is quite abundant in starch, amylose and amylopectin. Its starch or carbohydrate content is 72.12% for the dark brown variety and 73.98% for the white variety; similarly its amylose content is 35.00% for the dark brown species and 21.67% for the white species (Chukwu et al., 2011). In addition, guinea corn has 10.80 % and 10.00 % protein content for the dark brown variety and white variety respectively. Sorghum grain (guinea corn) however, does not contain gluten and cannot be used for leavened products unless mixed with wheat. Chemical composition of guinea corn shows that it contains carbohydrate, proteins, lipid, ash, fibre, moisture, oil.

1.3.1 Taxonomy of Guinea Corn

For the purpose of classification, guinea corn is classified as follows;

Kingdom:                    Plantae
Subkingdom:               Tracheobionta
Superdivision:             Spermatophyta
Division:                      Magnoliophyta
Class:                           Liliopsida
Subclass:                     Commelinidae
Order:                          Cyperales
Family:            Poaceae (Grass)
Genus:             Sorghum

Species:          Sorghum bicolor/ Sorghum vulgar

Source: (Akaninwor et al., 2007)

Table 3: Chemical Composition of Guinea Corn

Components	Content (%) (Brown)	Content (%) (White)
Oil	5.03±0.06	3.03±0.13
Crude protein	10.80±0.31	10.00±0.43
Crude fibre	2.33±0.12	1.97±0.03
Ash	1.87±0.01	1.97±0.02
Nitrogen free extract	71.66±0.12	73.97±0.32
Carbohydrate	72.12±0.14	73.98±0.17
Energy value	374.07±0.64	363.10±0.58
Amylose	35.00±0.04	21.67±0.31
Calcium	0.14±0.02	0.27±0.12
Potassium	0.19±0.01	0.21±0.02
Phosphorus	0.16±0.03	0.12±0.01

 

Source: (Chukwu et al., 2011)

Table 4: Essential Amino Acid Composition (mg/g) of Sorghum

Grain	Isoleucine	Leucine	Lysine	Methi- onine	Cystine	Phenylalanine	Tyrosine	Threonine	Tryptophan	Valine
Sorghum	245	832	126	87	94	306	167	189	63	313

Source: (Chukwu et al., 2011)

 

1.3.2 Amylopectin from Guinea Corn as a Carbon Source for Glucoamylase Production

A number of carbohydrase (amylases and glucoamylase) production by Aspergillus niger has been found to be of maximum yield when starch components (amylose and amylopectin) from seeds and tubers were used as substrates (Damisa and Otitolaiye, 2013). This therefore suggests that starch from seed such as guinea corn can be used as a substrate to produce products of an economic value as in glucoamylase. Starch is the major storage form of carbohydrate in plants. It is often stored in the seeds of plants, the roots or the leaves. Starch consists of two components: amylopectin, a branched chain polymer of glucose, and amylose, a straight chain polymer of glucose. Inotherwords, starch is a large polysaccharide, of considerable industrial importance that is mainly composed of amylose and amylopectin (Mahsa et al., 2003). These two components form a semi crystalline structure in the starch granules, which consist of crystalline lamellae (ordered, tightly packed of parallel glucan chains) and amorphous lamellae (less ordered regions) (Lawal et al., 2004). Starches of different origins have different degrees of crystallinity (range about 15-45 %). Amylose is the linear component or portion of starch. It is a polymer of glycopyranosyl monomers linked to each other by α (1, 4) linkages (Eric, 2013).

Amylopectin has the same backbone as amylose, but it is branched by α (1, 6) linkages (Shibanuma et al., 1994). They are polysaccharide and highly branched polymer of starch found in plants. It is one of the two components of starch, the other being amylose. In amylopectin, the glucose units are linked in a linear way with α (1, 4) glycosidic bonds. Branching takes place at the α (1,6) bonds occurring every 24 to 30 glucose units, resulting in a soluble molecule that can be quickly degraded as it has many end points for enzymes to attach onto. In contrast, amylose contains very few α (1, 6) bonds or linkages or even none at all. This, makes the amylose chain or linkages (that is, the α -1, 4- bonds) to be hydrolyzed more slowly, but however have higher density and are insoluble (Abdalwahab et al., 2012). A similar molecule to amylopectin is glycogen. It has the same composition and structure to amylopectin, but with more extensive branching that occurs at every 8 to 12 glucose units (Prasanna, 2005).

 

 

 

 

 

 

Figure 3: Structure of amylopectin and amylose (Eric, 2013).

The starch of guinea corn normally contains 70–80% amylopectin, although some species contain 100% amylopectin and others up to 62% amylose (Balole and Legwaila, 2006).

 

 

1.3.3 Other Substrates Commonly Used For Glucoamylase Production

Other alternative substrates that can be used as carbon sources for glucoamylase production include: dextrin, fructose, glucose, lactose, maltose and others. These sources are however expensive for commercial production of this enzyme (that is, glucoamylase). Various agricultural by products like wheat bran, rice husk, sugarcane bagasse, potato residue, wheat bran, rice bran, green gram bran, black gram bran, maize bran and others can be abundantly utilized (Siddhartha et al., 2012).

1.4 Properties of Glucoamylase

Glucoamylases are able to hydrolyse 88.5-100% of soluble starch, an advantage which would be beneficial in the starch processing industry. Similarly, the pH and temperature optima of glucoamylases isolated from fungi especially Aspergillus spp. are generally of  the range of  3.7-7.4 and 46-60 º C respectively (Nahar et al., 2008). However, depending on the source of the glucoamylase, they are a few exceptions. This enzyme (Glucoamylase) can be derived from a wide variety of plants, animals and microorganisms such as, bacteria, filamentous fungi and yeast.

1.5 Sources of Glucoamylase

Glucoamylase can be derived from a wide variety of plants, animals and microorganisms such as, bacteria, filamentous fungi (mold, yeast). Microbial sources of this enzyme (Glucoamylase) have advantages over other sources. A few of these advantages are, because of the short growth period of the microorganism, the enzymes from microbial sources can generally meet industrial demands because, of their short life span (Ritesh and Barkha, 2011). Moreover, the enzymes of microbial origin can be isolated easily and their characteristics can be manipulated by genetic engineering and biotechnology techniques (Muhammad et al., 2011). This technique of enzyme biotechnology is extensively used in enzymes production. Furthermore, another advantage associated especially with microbial glucoamylase particularly from Aspergillus niger is the control over bacterial contamination due to its capacity to tolerate a high degree of acidity (Radha et al., 2012). Glucoamylase enzyme used commercially originates from strains of either Aspergillus niger or Rhizopus sp. where they are used for the conversion of malto- oligosaccharide into glucose. However, the extensive utilization of glucoamylase is obtained by using the fungus Aspergillus niger in enzyme production industries. This enzyme (glucoamylase) is generally regarded as safe by the Food and Drug Administration (FDA) (Siddhartha et al., 2013).

1.6 Forms of Glucoamylase

Since the discovery of two forms of glucoamylase from black koji mold in the 1950’s, many reports have appeared on the multiplicity of glucoamylase. The various forms of glucoamylases are thought to be the result of several mechanisms which include mRNA modifications, limited proteolysis, variation in carbohydrate content or the presence of several structural genes. The glucoamylases are glycoprotein in nature and differ in their content and nature of carbohydrate from different sources. The carbohydrate moiety plays an important role in stabilising the native conformation of the enzyme and is not involved in activity and antigenecity (Shenoy et al., 1985).

Aspergillus niger produces two forms (isoenzymes) of glucoamylase that are separable by electrophoresis or by chromatography on DEAE-cellulose and are designated: G1 (Glucoamylase 1) and G2 (Glucoamylase 2) (Jorgen et al., 2013). The molecular weight of glucoamylase 1 is 99,000 Da, and that of glucoamylase 2 is 112,000 Da. Both forms of the glucoamylase, also described as glycoenzymes, contain covalently linked carbohydrate (containing d-mannose, d-glucose, and d-galactose residues). The carbohydrate-protein linkage in these glycoenzymes is primarily glycosidic to the hydroxyl group of l-serine and l-threonine residues. Glycosylamine linkages to l-asparagine and l-glutamine may also be present. Glucoamylase 1 and 2 possess identical amino acid compositions and, presumably, identical amino acid sequences. However, the two glycoenzymes differ in carbohydrate content, glucoamylase 2 containing nearly twice as many carbohydrate residues per molecule as glucoamylase 1. Consequently, it is suggested that the two forms of glucoamylase are isoglycoenzymes. The difference in electrophoretic and chromatographic properties of the isoglycoenzymes is probably due to a difference in the number of amide groups or glycosylaminically linked carbohydrate units in the polypeptide chains (Pazur et al., 1971).

 

 

 

1.7 Types of Glucoamylases

From the microbial sources of glucoamylase, two main types of glucoamylases are known: fungal glucoamylases and bacterial glucoamylases.

Glucoamylases have been identified from few bacterial species such as aerobic strains of B. stearothermophilus, Flavobacterium sp., Halobacterium sodomense and Arthrobacter globiformis. Anaerobic strains include C. thermohydrosuffuricum, Clostridium sp., Clostridium acetobutylicum, Clostridium thermosaccharolyticum, and the microaerophile, Lactobacillus amylovorus. Most of the glucoamylases produced by these bacteria except Lactobacillus amylovorus are thermostable and are thus potential enzymes for application in the saccharification of starch for glucose syrup production, where temperatures greater than 60 º C are employed. The pH optimum for activity was in the range of 5.5- 6.5 with a temperature optimum of 55 º C (Siddhartha et al., 2012).

1.8. Mechanism of Action of Glucoamylase

Mechanism of action of glucoamylase shows that it acts by a multichain mechanism, in which the enzyme acts randomly on all the substrate molecules. The widely accepted mechanism of hydrolysis involves proton transfer to the glycosidic oxygen of the scissile bond from a general acid catalyst, formation of an oxocarbonium ion, and a nucleophilic attack of water assisted by a general base catalyst (Jorgen et al., 2000).

The catalytic domain of glucoamylase is made up of the general acid and base functions of Glu 179 and Glu 400 respectively.

 

 

 

 

 

 

 

 

 

Figure 4: Mechanism of action of glucoamylase (Siddhartha et al., 2012).

 

1.8.1 Mechanism of Hydrolysis by Glucoamylase

In the mechanism of hydrolysis by glucoamylase, the D-glycosidic bond oxygen is protonated by hydrogen ions from an amino group or imidazole group of the enzyme active site. The electron deficient center at carbon 1 of the glycosidic bond attracts electrons from a donor group such as a hydroxyl group, either from water or a serine group in the active site. The resulting structure is the formation of an oxocarbonium ion intermediate. The final step involves the addition of a hydroxyl ion (or a water molecule) to the oxocarbonium ion intermediate, with the neutral D-glucosyl fragment functioning as the leaving group. The hydroxyl ion (OH) group is added in the β configuration; hence, β-D-glucose is formed as the product of hydrolysis. Glucoamylase catalysis involves the inversion of the anomeric configuration (Siddhartha et al., 2012).

 

Figure 5: The catalytic mechanism of glucoamylase illustrating the action of the catalytic base E400 (top) and acid E179 (bottom) in the water-assisted hydrolysis of substrate involving inversion of the configuration of the anomeric carbon. (Jorgen et al., 2000).

1.9 Structure of Glucoamylases

The structures of glucoamylases show that, majority of glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region (Jorgen et al., 2000). The catalytic domain or the active site folds as a twisted (α/α)₆  barrel forming a central funnel-shaped active site, while the starch-binding domain folds as an antiparallel β-barrel and has two binding sites for starch (Jorgen et al., 2013).

The catalytic domain (CD) of glucoamylase from Aspergillus niger contains 13 α -helixes of which 12 form an (α/α)₆ barrel. In this fold, six outer and six inner α-helixes surround the funnel-shaped active site, constituted by the six highly conserved α to α segments, which connect the N-terminal of the inner with the C-terminal of the outer helixes. The catalytic site includes the general acid and base functions of Glu179 and Glu400 situated at the bottom of a pocket. The catalytic domains (CDs) of glucoamylase from Aspergillus niger, Aspergillus awamorivar X100, and S. fibuligera share a very similar fold. The S. fibuligera glucoamylase contains 14 α-helices, 12 of which makes up the (α/α) 6-motif in an organisation identical to that of Aspergillus niger and Aspergillus awamorivar X10 catalytic domains (Jorgen et al., 2000).

Figure 6: Stereoview of the catalytic domain (amino acid range 1- 471) of Aspergillus niger and Aspergillus awamorivar X100 glucoamylase (rings A, B, C, D are indicated). The C- and N-termini are indicated together with the side chains of the two catalytic residues E179 and E400 (Jorgen et al., 2000).

Similarly, the starch binding domain (SBD) consists of eight β-strands organised in two β-sheets forming a twisted β-barrel structure. The structure of starch binding domain (SBD) from Aspergillus niger glucoamylase has been determined by NMR, showing existence of two starch binding domains. The starch binding domain (SBD) binds onto starch as an individual entity and disrupts the compact structure of the starch granule, facilitating the hydrolysis by the catalytic domain (Southall et al., 1999). The starch binding domain (SBD) has two functions: it binds to the starch, and also disrupts the surface, thereby enhancing the amylolytic rate.

 

 

Figure 7: Stereoview of the starch binding domain (SBD) from Aspergillus niger glucoamylase, with the C- and N-terminal indicated (Jorgen et al., 2000).

The linker region, this is the serine and threonine rich O-glycosylated region of the structure of Aspergillus niger glucoamylase (amino acid range 440-508). It contains a very highly O-glycosylated C-terminal segment of about 30 amino acid that connects with the starch binding domain. This particular part of the linker has been attributed roles in stability, secretion, and digestion of raw starch (Goto et al., 1999).

Figure 8: The linker region, the catalytic part and starch binding domain of glucoamylase (Bjarne et al., 1993).

 

1.10 Amino Acid Sequence of Glucoamylase

The amino acid composition of glucoamylase produced from Aspergillus species is expressed from the glucoamylase genes from the Aspergillus species. The glucoamylase gene from Aspergillus species have been cloned and the nucleotide sequences of the genes revealing little variation in the amino acid sequence of glucoamylase produced from Aspergillus species (Jorgen et al.,2013). The amino acid sequence of glucoamylase from Aspergillus niger showed that it consist of 640 amino acid residues while that of glucoamylase from Aspergillus awamori showed that it consist of 616 amino acid residues. However, glucoamylase from   Aspergillus niger has 94 % sequence identity to glucoamylase from Aspergillus awamori (Jorgen et al., 2013), and it is highly homologous in its nucleotide sequence as well as the amino acid sequence, to glucoamylase from Rhizopus spices.

From the amino acid sequence of glucoamylase, it shows that, the tryptophan residues and carboxyl groups are important in the catalytic activity of Aspergillus glucoamylase (Yoshikazu et al., 1986). Comparison on the amino acid sequences of three glucoamylases (from Aspergillus sp., Rhizopus, and S. diastaticus.) showed they were homologous. These comparisons explain the structure-function relationships of the glucoamylase (Yoshikazu et al., 1986), as it showed 30 to 44% homology, including conservative substitutions. In some areas, only two of the three glucoamylases are homologous. Also in the amino acid sequence of glucoamylase, there are four regions which have higher homology, suggesting that they are probably functionally constrained and essential to glucoamylase. Some of the tryptophan residues and carboxyl groups which are significant in catalytic activities by chemical modification are strictly conserved and their surrounding regions are well conserved (Clarke and Svensson, 1984).

 

 

Figure 9: Sequence comparison of the three glucoamylases (Yoshikazu et al., 1986).

Conserved amino acid residues and conserved substitution are emphasized by boxes and dotted boxes, respectively. Underlines show the four highly conserved regions. Arrows indicate conserved tryptophan, aspartic acid, and glutamic acid residues. Numbers in parentheses show the residue numbers from the initial codon.

 

 

 

 

Figure 10: Predicted secondary structure for glucoamylase. The downward arrow indicates the processing site of the signal peptide and the upward one indicates the probable proteolysis site (Yoshikazu et al., 1986).

 

 

1.11 Immobilisation of Glucoamylase

Enzymes have widely been used commercially due to their uses as biocatalyst which can work specifically and efficiently. However, there are some limitations or weaknesses of enzyme uses in industries, such as the instability of the enzymes, the availability and the limited use of the enzyme, which causes their use in industrial sectors to be limited (Yandri et al., 2012). In order to solve and diminish these weaknesses, immobilization techniques and processes are developed or employed.

Enzymes, such as glucoamylase, can be immobilised to a large number of different carriers by entrapment, adsorption, ionic binding, cross linking /covalent binding (Varavinit et al., 2001).  Due to the obvious advantages of processes using immobilized enzymes and also in order to make industrial glucose production a continuous process, several of the immobilization techniques such as adsorption, covalent/cross-linking, ionic binding, entrapment and making use of membrane like structures have been exploited for the immobilization of glucoamylase on various supports (Shenoy et al., 1985). Currently, glucose production in the world is by enzymatic methods. Industries make use of bacterial α-amylase and fungal glucoamylase for the conversion of starch to glucose, which is a well established commercial process. Various studies on the immobilization of glucoamylase on different supports have been extensively carried out. Of these, covalent/cross-linking and adsorption techniques have to be extensively studied.

For glucoamylase, covalent binding is very effective in retaining the enzyme, and can achieve high activity after immobilisation, if the amino acid residues that are covalently bound to the support material are not involved in the active site or substrate binding site. A number of materials can be used as insoluble carriers for immobilised enzymes. A few examples of materials used to achieve the immobilisation by covalent binding of glucoamylase include: binding with cellulose, activated by cyanogen halide, e.g. cyanogen bromide (Porath et al., 1967), binding with the use of triazine (Kay and Cook 1967), binding using bagasse (that is, a natural cellulosic material) after activation of the bagasse with periodic acid (Varavinit et al., 2001), binding onto a chemically synthesized polyaniline polymer, after the activation of the polymer with glutaraldehyde (Silva et al., 2005). That is, immobilization of Aspergillus niger glucoamylase onto a polyaniline polymer material.

 

1.12 Glucoamylase Immobilization with Other Enzymes

Due to the beneficial effects, the immobilization of enzymes on the same particle/carrier or on different particles/carriers used in separate or mixed systems has been exploited. Glucoamylase can be co-immobilized with other enzymes and studied as a model system (Gestrelius, 1972). In most of the cases, glucoamylase can be co-immobilized with α-amylase or with a mixture of α-amylase and glucose isomerase. A few examples of such studies are summarized in the table below; as such studies have been carried out to make use of such systems in the starch industries particularly for the continuous conversion of starch to glucose or high fructose syrup (Shenoy et al., 1985).

Table 5: Glucoamylase Immobilisation with other enzymes

ENZYMES IMMOBILISED	METHOD OF IMMOBILISATION	SUPPORT / CARRIERS USED	USE / APPLICATIONS
Glucoamylase  + Glucose oxidase	Covalent  (Co- immobilised)	CNBr  Activated, sepharose -4B	Model
Glucoamylase,  α- amylase and / or pullulanase	Covalent ( Separately immobilised)	DEAE-Cellulose, AE- cellulose (Activated)	Partial Hydrolysis of starch to glucose
Glucoamylase  +  Glucoisomerase	Covalent   (Co- immobilised)	Porous glass particle	High fructose syrup from partially hydrolysed starch
Glucoamylase + α- amylase + Glucose isomerase	Covalent   (Co- immobilised)	CNBr  Activated, sepharose -6MB	Single step production of starch to high fructose syrup
Glucoamylase+ α- amylase	Covalent   (Co- immobilised)	Polyarcylamide gel	Hydrolysis of starch
Glucoamylase+ yeast cells	Covalent   (Co- immobilised)	Living yeast cell	Beer Wort Fermentation

 

Source: (Shenoy et al., 1985).

Due to some limitations of enzymes (such as the instability of the enzymes, the availability and the limited use) in industries (particularly the starch industries), immobilisation is  very essential and attempts has been made for the “single step” conversion of starch to high fructose syrup using an immobilized multienzyme system of α-amylase, glucoamylase and glucose isomerase.

1.13 Inhibitors of Glucoamylase

Glucoamylase, just like most enzymes suffer from the effect of inhibitors. A very potent inhibitor of glucoamylase is acarbose, a starch blocker that inhibits glucoamylase. It is especially a competitive reversible inhibitor (Sharifi et al., 2008), that binds glucoamylase at its starch binding domain or subsites (Jorgen et al., 2013). Acarbose is a pseudotetrasaccharide, composed of an acarviosine moiety with a maltose at the reducing terminus. The pseudotetrasaccharide acarbose binds with high affinity to glucoamylase (Jorgen et al., 2000).

 

Figure11: Structure of Acarbose and its mode of action (Sharifi et al., 2008).

Other inhibitors of glucoamylase are:

1. Pseudodisaccharide acarviosine – which comprises of the first two units of acarbose at the non-reducing end, but binds with a much lower affinity.
2. D-gluco-Dihydroacarbose prepared by hydrogenation of the valeinamine ring in acarbose, shows a similar binding as acarbose.
3. L-dihydroacarbose – formed or obtained in the preparation of the D-glucoisomer of reduced acarbose and this inhibitor with an inverted chair conformation of the hydrogenated valeinamine ring shows an even weaker binding to glucoamylase.
4. L-deoxynojirimycin.

1.14 Activators of Glucoamylase

Enzymes, many of which require metal ions for their maximal catalytic activity, are termed as holoenzymes. Metals are responsible for the right orientation of the active site of holoenzymes (Bhatti et al., 2005). The interaction between enzyme protein and metal ions and their effect on structure can be stabilized by metal binding during which metal ions are coordinated usually by lone pair donation from oxygen or nitrogen atoms.

Glucoamylase can be activated by metals such as copper, calcium, manganese and others. In the study of the purified enzyme fraction and analysis for the effect of copper ions on glucoamylase from Aspergillus niger, the activation energy for the native glucoamylase was 94.46 KJ/mol and the copper modified glucoamylase was 119.98 KJ/mol implying that the copper modified glucoamylase was much stable when compared to that of native enzyme (Rangabhashiyam et al., 2011). The enzyme, glucoamylase from Aspergillus phoenicis was activated mainly by manganese (176%), and calcium (130%) ions (Benassi et al., 2014).

1.15 Applications of Glucoamylase

Glucoamylases are of enormous significance, particularly in the food industry, where they are useful in the saccharification of starch for glucose syrup, starch hydrolysis, high fructose corn syrup (HFCS) production, methane and alcohol production (Muhammad et al., 2011). They are mainly utilized in the production of glucose syrup, high fructose corn syrup, and in whole grain and starch hydrolysis for alcohol production (Jennylynd and Byong, 1997). Glucoamylase (EC 3.2.1.3) hydrolyzes starch (as well as other related oligosaccharides and polysaccharides) from its non reducing ends by cleaving the α-1, 4 and α-1, 6 glycosidic bonds consecutively (Uma and Nasrin, 2013).

In starch hydrolysis, glucoamylase is used to accomplish the hydrolysis of starch to glucose units, starch is hydrolysed by a two stage process: liquefaction then saccharification, using a mixture of amylolytic enzymes. The first step called liquefaction, involves treatment with amylase at    85 -100 ºC. The dextrin is soluble and so can be passed through a bed of immobilized glucoamylase which accomplishes further hydrolysis to glucose units. High glucose syrups contain 96-98% D-glucose. For high fructose corn syrup (HFCS) production, glucoamylase can be used for the production of fructose syrup from liquefied starch   (Abdalwahab et al., 2012).

In alcohol production, ethanol has been shown to be produced industrially from glucoamylase when starchy materials are used as substrates. Ethanol has been shown to be produced from very high gravity mashes of dry milled corn (35% w/w total dry matter) under simultaneous saccharification and fermentation conditions (Rasmus et al., 2005). The effects of glucoamylase dosage, pre-saccharification, ethanol yield and volumetric and specific productivity were determined. It was shown that higher glucoamylase doses and/or pre-saccharification accelerated the simultaneous saccharification and fermentation process and increased the final ethanol concentration.

Typically, the fermentations are carried out as simultaneous saccharification and fermentation (SSF), where glucoamylase and yeast (Saccharomyces cereviseae) are added simultaneously, prior to the simultaneous saccharification and fermentation, the starch source is milled and mixed with water, yielding a viscous slurry, which is liquefied by heat treatment (jet cooking) and α-amylase, there after the simultaneous saccharification and fermentation begins (Ingledew, 1993). The coarse particles generated after distillation and centrifugation are normally dried and can be referred to as dried distiller’s grain (with solubles), (DDG/DDGS). These ruminants are often rich in protein, fibre and vitamins.

Source: (Rasmus et al., 2005).

Figure 13: Schematic flow diagram for the ethanol production from dry milled corn. Pre-saccharification is only applied in some plants. Distiller’s dried grains with soluble (DDGS)

1.16 Aim and Objectives of the Study

 1.16.1 Aim of the Study

This study is aimed at production of glucoamylase from Aspergillus niger in a submerged fermentation system, using amylopectin fractionated from guinea corn starch as the sole carbon source.

1.16.2 Specific Objectives of the Study

This work was designed to achieve the following specific objectives:

1. To extract glucoamylase from Aspergillus niger.
2. To determine the protein content of the enzyme.
3. To assay for the activity of the enzyme using 3, 5- dinitrosalicyclic acid (DNSA).
4. To partially purify the enzyme by ammonium sulphate precipitation and gel chromatography (size exclusion chromatography).
5. To characterize the partially purified enzyme using the effect of pH, effect of temperature, effect of substrate concentration and the effect of ion concentration
6. To determine the kinetic parameters such as the K_m and Vmax values of the enzyme.

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