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PRODUCTION AND OPTIMIZATION OF GLUCOAMYLASES  FROM  PLANTS AND ASPERGILLUSNIGER FOR STARCH HYDROLYSIS  IN A BATCH BIOREACTOR

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ABSTRACT

Glucoamylases  were  produced  from both  plants  and microorganisms  and were  optimized  for starch hydrolysis in batch bioreactor. Amylase activity was monitored  in germinating guinea com seeds for seven days.  Highest  amylase  activity  was observed  on days  3  and 7.  A study of the amylopectin content of millet, guinea com, cassava, com and tigernut starch showed that tiger nut had the  highest  amylopectin  content  while  cassava  starch had the  lowest.  Moist  amylopectin frommillet, guinea com, cassava, com and tiger nut starch were exposed  on the shelf to triger microbial growth. Luxurial growths were noticed on amylopectin  from guinea com, tigernut and cassava starch. Pure isolates were obtained by subculturing and identified as Aspergillus niger.  A 14 day  fermentation  study  to  determine  the  optimal  production  time  using  the  organism  and amylopectin  from  guinea  com,  tigernut  and  cassava  starch was  carried  out.  The fermentation studies showed a two peak profile for each amylopectin  used. The first on day 3  or 4,  while the second peak on day 11  and 12, respectively.  Large scale production  of glucoamylase was carried out on these days of highest enzyme production.  Glucoamylase  activities from both germinating guinea  corn  seeds  and Aspergillusniger were  enhanced  by calcium  (Ca),  zinc (Zn), cobolt (Co),  iron  (Fe) and manganese  (Mn)  ionbut  Lead  ion  (Pb)  completely  inactivated  the enzymes.  The Michaelismenten  constant  (K,~) and the maximum  velocity  (V,~a)obtained  from Lineweaver-Burk  plot of initial velocity  data at different  substrate  concentrations  showed high affinity   of  the   glucomylases   for   their   substrates.   The   optimal  pH   and   temperature   of glucoamylases  from both germinating seeds and Aspergillusniger were in the range of 4.5-8.5 and 45-60  C, respectively.The  glucoamylases  were  screened for a and p  glucosidase  activities  and glucoamylase  obtained  on day 7 from germinating  guinea  com  seeds  (GluGERGC7)  and that obtained on days  11  and 12  from Aspergillusniger grown in broth containing  amylopectin  from cassava  and  tiger  nut  starch  (GluAgCSVl  1)  and  (GluAgTN12),  respectively  were  found  to exhibit high  a glucosidase  activity.  The rate of substrate utilization  or the efficiency  of batch bioreactor at the predetermined  optimal conditions was predicted using Ksand V,obtained from a  modified  form  of Michaelis-Menten’s  equation  and  were  found  within  the  range  of 40• 460mg/ml and 0.811-50 mol/min, respectively using cassava, guinea com and tiger nut starch as substrates.  The results  suggest that  the  glucoamylases  obtained  from both  germinating  guinea com seeds and Aspergillusniger possess the qualities  of biotechnological applications  in which the optimal conditions could be predicted.

CHAPTER ONE

INTRODUCTION

The huge demand  for starch in industries  for the production  of high glucose  syrups and ethanol has led to stiff competition with dietary starch. There is the need to discover other sources  of starch  for  industrial   purposes   in  other  to  spare  dietary   starch.  This  is complicated  by the problems  of completely  hydrolysing  starch  due to the difficulty  of obtaining the appropriate enzymes to hydrolyse its multiple branching especially thea-  1, 6 glucosidic bonds.

The study of plant and microbial  glucoamylases  is important from both basic and applied perspectives. Amylase  is a major  enzyme in the industry (Souzaand Magalhaes,  2010). It hydrolyses  the  starch molecules  into  glucose  units  (Raimi  et al.,  2012).  Cereal  grains synthesize    multiple    forms    of  a-amylase    during   germination    to   supply   soluble carbohydrates  for the developing  seedling.  Heterogeneity  of starch- degrading enzymes in germinating  seeds  enhances  the  conversion  of insoluble  granules  to  soluble  starch and dextrins (Donn et al., 1991).  These multiple  forms of amylase suggest that each isoform may  have  a  particular  metabolic  function.  The  individual  forms  act  cooperatively  to degrade  starch during  germination.  The exclusive  production  of amylases  has also been reported  in Aspergillus  niger, A.  oryzae, Aflavus  and A.  terreus(Zambare,  2010; Koc and Metin, 2010;Puriet al., 2013; Lawal et al., 2014).

The  most   well  known   amylolytic   enzymes   are  a-amylase   (EC  3.2.1.1),   [-amylase

(EC3.2.1.2)  and  glucoamylase   (EC  3.2.1.3)  (Sivaramakrishnan   et  al.,  2006;   Janecek,

2009).  a-Amylase  digests  starch  by  randomly  breaking  the  glycosidic  bonds  between glucose molecules.  The product is therefore a mixture of maltose and dextrins.  P-amylase digests  starch by cleaving  every second bond  starting from one end, producing  maltose. Glucoamylase   (a-1,  4-glucan-glucohydrolases,  EC  3.2.1.3)  is  of great  importance  for saccharification  of starchy  materials  and  other  related  oligosaccharides.  Glucoamylase consecutively hydrolyzes a-1,4-glycosidic  bonds from the non-reducing  ends of starch and a-1,6 glucsidic linkages in polysaccharides  yielding glucose as the end-product,  which in tum serves as a feedstock for biological fermentations  (Saueret al., 2000; Haq et al.,  2003; El-Gendy, 2012).

Tiger  nut  (Cyperus  esculentus)which  is underutilized has  the  credentials  to  become  the principal  feedstock  for Nigerian’s  energy fuel-alcohol  and glucose  syrup market.This  will go  a long  way  in helping  Nigeria  improve  her  economy  and  in achieving  one  of her Millennium  Development Goals (MDG’ s) which involves climate sustainability.

1.1 Starch

Starch  is a storage  form  of glucose  in plants.  It contains  two types of glucose  polymer: amylase  and amylopectin  (Nelson and Cox,  2005).  Amylase  consists  of long,  unbranched chains of D-glucose  residues  connected  by a-1, 4 linkages.  Such chains vary in molecular weight  from a few thousand  to more than a million.  Amylopectin  on the other hand has a high molecular  weight  (up to  100 million)  but  highly  branched.  The glycosidic  linkages joining  successive  glucose  residues  in amylopectin  chains  are a-1, 4 in the straight  chain

and a-1, 6 linkages at the branched points (Figure  1).

Nonreducing end

0                                0                                0                                0

H     OH                         H     OH                         H     OH

(a) amylose

Reducing end

Branch

(a1-+6) branch point

Amylose

—} Reducing ends

Nonreducing ends

Main chain

(b)                                                                                                            (c)

Figure  l:Amylose and amylopectin  components  of starch.  (a) A short segment of amylase,  a linear polymer  of D-glucoseresidues in (a-1, 4)) linkage.  A single chain can contain  several thousand  glucose  residues.  (b) An  (a-1, 6) branch  point  of amylopectin.  (c) A cluster  of amylase and amylopectin  (Nelson and Cox, 2008).

Amylose fraction is about 25-30% of the starch molecules found in com and has a molecular weight of about 250,000.  The percentage  of amylose in the starch is genetically determined. Genetic   modifications   producing   high-amylose   (50-70%)   com   starch  are  also   found. Amylopectin  comprises  about  70-75%  of the  starch  found  in the  com  kernel  and  has  a molecular weight of about 50-500 million.

1.1.1 Industrial hydrolysis of starch

Starch hydrolysis  is a widely employed process in many industries in the production  of low molecular  mass products.  Such industries  include,  sugar, brewing,  spirits, textile  and some food industries.  Starch hydrolysis  involves two methods:  acidic and enzymatic method (Kilic and Ozbek,  2004; Ma  et al.2006;  Kolusheva  and Marinova,  2007; Zamora  et al.,  2010; Betiku,  2010;  Dincbas  and  Demirkan,  2010;  Echegi  et  al.,  2013).  The  older  and  more traditional  method  is  acidic  hydrolysis   which  requires  highly  acidic  medium   (pH  1-2) obtained  through  mineral  acids  at high temperatures  (150-230C) and high pressure.  As a result of the thermal  processing,  acidic hydrolysis  produces  unnecessary  byproducts  which contaminate  the end product  hydrolysate. The enzymatic  hydrolysis  of starch is carried out under low temperatures, normal pressure and pH of around 6-8.

Enzyme  hydrolysis  of starch is always  carried  out using more of a-amylase  from different sources   than   [-amylase   (Kolusheva   and  Marinova,   2007).   Alpha-amylase   attacks  the polysaccharide   molecules   in  the  inner  part  of the  chain  to  destroy  the  spiral  of the polysaccharide   chain  and  thus  the  characteristic   blue  color  with  iodine  disappears.  This quickely  reduces  the  viscosity  of the  starch  solutions  given rice  to  dextrines.  Continuous action of the amylase activity produces maltose, in which one of the molecules of glucose has a free glucoside  group  and hence reducing  properties  of reducing  sugar.  Liquefaction  and saccharification  are the two major  processes  of starch hydrolysis.  The breakdown  of large particles drastically reduces the viscosity of gelatinized starch solution, resulting in a process called liquefaction.  The final stages of depolymerization  are mainly the formation of mono-, di-, and tri-saccharides  in a process known assaccharification.In enzyme hydrolysis of potato starch,  com, wheat and rice,  immobilized  and free a-amylases  have been reported to be used (Dincbas  and  Demirkan,  2010).  This  method  is carried  out  by  mixing  the  enzyme  with buffered  solution containing  2-10%  (w/v) of the starches  and incubated  at 37C  for 10 min and the enzyme activities determined  by the starch-iodine  method.  The susceptibility  of the starch  granules  are  also  greatly  affected  by their  amylose  content,  starch polymorphism, structure of amylopectin,  and the presence  of amylose-lipid  complex in the starch granules. Amylose negatively correlates with the susceptibility of starch to amylase hydrolysis because it intertwines with amylopectin,  and holds the integrity of the starch granules.  Amylopectin has larger proportions  of short branch-chains, which result in more open space (weak points) in the granule for amylase to penetrate  and hydrolyze  the starch.  Guinea  com,  com,  millet and cassava starches have low enzyme digestibility due to their high amylose content (Jane, 2006).

1.1.2 Problems of industrial starch hydrolysis

Due to thermal processing,  acid hydrolysis of starch produces unnecessary byproducts which contaminate the end product hydrolysate (Kilic and Ozbek, 2004; Ma et al., 2006; Kolusheva and  Marinova,  2007;  Zamora  et  al.,  2010;  Betiku,  2010;  Dincbas  and  Demirkan,  2010; Echegi et al., 2013) like glucose and maltose.  This old method of hydrolysis requires:the use of  corrosion   resistant   materials,gives   rise  to  high  colour   and  salt  ash  content   (after neutralization),  needs more energy for heating andis relatively difficult to control. As a result of these problems,  enzymatic hydrolysis becomes a better method for starch hydrolysis since the above mentioned  constraints  are not peculiar.  Although  enzyme  hydrolysis  of starch is carried out under milder conditions characterized  by high reaction rate,  starch hydrolysis  is faced with the problem  of achieving  completehydrolysis  of starch due to the difficulty  of obtaining  the  appropriate  enzyme  to hydrolyse  the multiple  branching  of a-1,6  glucosidic bonds.

1.2Aspergillus niger

Aspergillus  niger is a member of the genus Aspergillus  which includes a set of fungi that are generally  considered  asexual,  although  perfect  forms  (forms that reproduce  sexually) have been found.  Aspergilli  are ubiquitous  in nature.  They are geographically  widely distributed, and have been observed in a broad range of habitats because they can colonize a wide variety of substrates. A.  niger is commonly  found  as a saprophyte  growing on dead leaves,  stored grain, compost piles, and other decaying vegetation. The spores are everywhere, and are often associated  with  organic  materials  and  soil.  The  history  of safe  use  for A.  niger  comes primarily  from its use in the food industry for the production  of many enzymes  such as a• amylase, amyloglucosidase, cellulases,  lactase, invertase, pectinases, and acid proteases.  In addition,  the annual production  of citric acid by fermentation  is now approximately  350,000 tons,  using either A.  niger yeast as the producing  organisms.  Citric acid fermentation  using A.niger  is carried  out  commercially  in both  surface  culture  and  in  submerged  processes (Kubicek and Rohr,  1986). A. niger has some uses as the organism  itself, in addition to its products  of fermentation.  For  example,  due  to  its ease  of visualization  and  resistance  to several anti-fungal agents, A.  niger is used to test the efficacy of preservative  treatments.   In addition, A.  niger has been  shown to be exquisitely  sensitive to micronutrient  deficiencies prompting the use ofA.  niger strains for soil testing (Abdalwahab et al., 2012).

1.3Amylases

Amylases are enzymes that break down starch (Gouda and Elbahloul,  2008;Vidyalakshimi  et al.,  2009).  They  are obtained  from plants,  animal  and microorganisms  (MacGregor  et al.,

1988; Sivaramakrishnanet  al., 2006; Gouda and Elbahloul, 2008;Vidyalakshimi  et al., 2009; Parmar  and Pandya, 2012;Debet  al., 2013). Amylases  are of great  significance  in biotechnological  studies (Prassana, 2005; Souza and Magalhaes, 2010; Rahmaniet al., 2011; Parmar   and  Pandya,   2012;   Mukesh-Kumaret   al.,   2012;   Mobini-Dehkordi   and   Javan,

2012).Amylases are produced by plants,  animals and microbes, where they play a major role

in carbohydrate  metabolism.  Amylases from plant and microbial sources have been used for centuries  as food  additives.  Amylases  from barley have been  applied  in brewing  industry. Fungal  amylases  have  been  widely  used  for the  preparation  of oriental  foods.  Microbial amylases  are used  for  industrial  production  due  to  their  cost  effectiveness,  consistency, lesscumbersome  and ease of process  modification  and optimization (Burhan  et al., 2003). Bacillusp. has been widely used for thermostable  a-amylase  production  to meet industrial needs.   B.   subtilis,   B.   stearothermophilus,   B.   licheniformisB.   amyloliquefaciens   and filamentous  fungi are good producers  of amylases and have been widely used for large scale production of amylases for various applications.  Moulds are known to be major producers  of extracellular  amylases  and are widely  employed  for the industrial  production  of amylases. Aspergillihave been widely employed for the production of amylases (Sivaramakrishan  et al.,

2006). Production of enzymes by solid-state fermentation  (SSF) using these moulds has been reported to be a cost-effective  production technique (Sivaramakrishan et al., 2006).

1.3.lAmylase assay

Alpha  amylase  cleaves  internal  a-1,  4-  glycosidic  linkages  in starch  to  produce  glucose, maltose, or dextrins, while glucoamylase  cuts the a-1, 4- and a -1, 6-glycosidic  linkages to release glucose from the non-reducing  ends of starch.  There are mainly two types of assays that are used to determine the activity of a-amylase  and glucoamylase  (Xiao et al.,  2005).

One  is based  on  measunng  the  amount  of reducing  sugars  by  the  dinitrosalicylic   acid, whereas the other is based on the decreased  staining value of blue  starch-iodine  complexes (Fuwa,  1994).  The second method  is based  on color development  that results  from iodine binding to starch polymers. However, the starch-iodine assays were reported to vary based on iodine concentrations  ranging from 0.25mM 3 mM and with the wavelength used to measure color  development  varying  from  550nm to 700 nm (Thomaset  al.,  1980,  Gonzalez  et al., 2002).

Dinitrosalicyclic  acid method is carried out by taking an appropriate amount of the enzyme in

1 % soluble starch solution. The released glucose is measured with 3, 5-dinitrosalicyclic  acid (DNSA) reagent using glucose as a standard.  Glucoamylase  activity unit (U) is expressed as the amount  of enzyme releasing one µmol of glucose per minute per ml. Xiao et al.  (2005) compared  microplate  starch-iodine  assay  and the DNS reducing  sugar assay using  sets of enzyme  samples  prepared  with  an Aspergillus   oryzae  a-amylase  (Sigma  A-6211)  and  an Aspergillus  niger glucoamylase  (Sigma A-1602), respectively.  It was demonstrated  that both assays were highly reproducible. More so, both methods generated equivalent values for the number of enzyme units present in the set of glucoamylase  samples.Even though, the amount of a-amylase  activity in the samples,  as determined  with the two assays,  was very different, the amount of starch consumed in mg/min as measured by the iodine method was equal to the amount of glucose produced  in mg/min as measured by the DNS assay.It was reported  that the units  of a-amylase  activity  in samples  measured  with the  iodine  assay  (mg of starch equivalents  consumed/min)  was five times higher than the units of activity  (mg of glucose equivalents  produced/min)  measured  with the DNS method. Apparently, equivalent  units of glucoamylase  but not alpha-amylase  activity were obtained using the iodine and DNS assays for the following reasons. Glucoamylases  degrade starch by removing glucose units from the non-reducing  ends,  thereby  reducing  the  mass  of starch available  for  iodine  binding  and producing  an equivalent  mass  of glucose.  In contrast,  endo-acting  a-amylases  reduce  the concentration of starch polymers that are able to bind iodine.

1.3.2 Classification of amylases

Amylases  are classified based on their differences  in their primary and tertiary structures as well as in their catalytic machineries  and reaction mechanisms (Janecek, 2009). Amylases act by hydrolyzing  bonds between adjacent  glucose units,  yielding products characteristic of the particular  enzyme  involved.  A large variety  of enzymes  are able to act  on starch  (Aiyer,

2005). These enzymes can be divided basically into four groups: endoamylases, exoamylases, debranching   enzymes   and   transferases   (Sivaramakrishnanet    al.,   2006).   Endoamylases cleavethe a-1, 4 glycosidic bonds resulting in a-anomeric  products.  Exoamylases  cleave the a-1, 4 or a-1, 6 glycosidic bonds of the external glucose residues resulting in a or [ anomeric products.  Debranching  enzymes  hydrolyze  the a-1,  6 bond  exclusively  leaving  long linear polysaccharides.  Transferases  cleave  a-1,  4  glycosidic  bond  of the  donor  molecule  and transfer part of the donor to a glycosidic acceptor forming a new glycosidic bond.  The above classification is based on their mode ofreaction (Parmar and Pandya, 2012).

1.3.2.1 aamylase

Alpha-amylase   (EC  3 .2.1.1)  hydrolyses  starch  by breaking  the  glycosidic  bonds  between glucose molecules in a random fashion giving rise to a mixture of maltose and dextrin.  Some a-amylases  are metolloenzymes, which require  calcium  ions for their activity  and stability (Sivaramakrishnanet  al., 2006).  a-amylases  are grouped into families which can roughly be divided  into two  groups;  starch hydrolyzing  enzymes  and  starch modifying or transglycosylating enzymes.  The highly  specific  catalytic  groups  in the three-dimensional structure of a-amylase  showed the capability of binding to substrate there by promoting  the breakage  of the glycoside bonds.a-amylase from human has been reported  to be a calcium• containing enzyme composed of 512 amino acids in a single chain with a molecular weight of 57.6 kDa.   There are four conserved sequence regions found in strands  93, 94,  95 and~   of the  catalytic  ([/a)s barrelled  domainofa-amylase  structure.  These  regions  were  used  in defining  of a-amylase  family  (Reddy  et  al.,  2003;  Sivaramakrishnan,  2006;  Souza  and Magalhaes,  2010).  a-amylasewas  reported  to  contain  three  domains  A,  B  and  C.  The A domain  which  is the  largest  presents  a typical  barrel  shaped  ([/a)ssuper structure.  The B domain  is  inserted  between  the  A  and  C  domains  and  is  attached  to  the  A  domain  by disulphide bond. The C domain has a P-sheet structure linked to the A domain by polypeptide chain and is an independent domain with unknown function.



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