PRODUCTION OF Aspergillus Niger GLUCOAMYLASE USING GUINEA CORN STARCH AMYLOPECTIN AS THE ONLY CARBON SOURCE

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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