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). Bacillus sp. 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 a–amylase
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.
This material content is developed to serve as a GUIDE for students to conduct academic research
PRODUCTION AND OPTIMIZATION OF GLUCOAMYLASES FROM PLANTS AND ASPERGILLUSNIGER FOR STARCH HYDROLYSIS IN A BATCH BIOREACTOR
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