ABSTRACT
Three fungi isolates were obtained from soil collected from a laundry waste water disposal site. These organisms were identified morphologically as Aspergillus niger, Aspergillus nidulans and Trichoderma hazianum. Ten days pilot study on these organisms indicated that A. nidulans produced α-amylase of highest activity on day 6 and was therefore chosen for further studies. The internal transcribed spacer (ITS) region of the organism showed an amplicon size of approximately
500-600 bp from agarose gel electrophoresis. Optimum pH for α-amylase production was observed to be pH 5.0 with an activity of 140.706 µmolmin-1. Forty percent (40%) Ammonium sulphate saturation was found suitable to precipitate protein with maximum α-amylase activity. The enzyme was further purified through dialysis and gel filtration, giving purification fold of 5.99 and percentage yield of 19.62. The specific activity increased from 371. 43 to 2226.37 U/mg showing that the purification processes were ideal for the enzyme. The total protein decreased with the purification processes showing that low molecular weight proteins and other contaminants were removed with purification steps. The optimum pH and temperature of the purified α-amylase were
5.0 and 60°C, respectively. The Michaelis constant, KM (18.28mg/ml) and maximum velocity, Vmax
(144.927µmol/min) were obtained from the Lineweaver-Burk plot at different substrate concentrations. The study revealed that Co2+, Cu2+, Mg2+ and Fe2+ inhibited α-amylase activity whereas Ca2+ and Mn2+enhanced its activity. From the result of pH stability study, more than 50 % of the enzyme’s initial activity was retained at pH range of 4.0 to 9.0 after 60 min of incubation and was most stable at pH 5.0, with residual activity of 99 % after 60min and 90 % at 120 min. The thermal stability study revealed that the enzyme was most stable within 40 to 50°C with residual activity of 89 % at 40°C and 84% at 50°C after 120 min of incubation. At the optimum temperature, more than 50% of its initial activity was maintained after 90 min. The half-life and D-value decreased with temperature increase. The activation energy of denaturation Ea and Z- values were 66.793 Kjmol-1 and 31.25°C, respectively. At optimum temperature, the results of the thermodynamic parameters, ΔH0, ΔG0, and ΔS0 were 64.024, 53.242 and 0,039 Kjmol-1, respectively. The results of the study showed that Aspergillus nidulans can be exploited as a cheap and efficient source of α-amylase for many biotechnological industries that operate within these observed conditions.
CHAPTER ONE
INTRODUCTION
Owing to their specificity and catalytic efficiency, enzymes are today applied in many industries to obtain a valuable final product. This has stimulated interest in the exploration of known and potentially useful enzymes for biotechnological applications (Saranraj and Stella, 2013). Amylase was the very first enzyme to be produced industrially in the year 1833 and was used for the treatment of digestive disorders (Akansha and Varsha, 2013). Alpha Amylase (E.C.3.2.1.1) catalysis the random hydrolysis of internal α-1,4-glycosidic linkages in starch to low molecular weight products, such as glucose, maltose and maltotriose units (Paula and Pérola, 2010). It is an enzyme with diverse applications and constitutes a class of industrial enzymes of approximately 25 % of the world enzyme market (Paula and Pérola, 2010; Rajendra et al., 2016). Alpha Amylase (α-amylase) has found application in food industries, textile, paper, detergent, and pharmaceutical industries. However, with the advances in biotechnology, its application has expanded to many fields such as clinical, medicinal and analytical chemistry (Paula and Pérola, 2010; Saranraj and Stella, 2013).
Today, amylases are commercially available and this has caused an almost complete shift from chemical hydrolysis of starch in starch processing industries to eco-friendlier enzyme hydrolysis. Microorganisms in recent years have been a biotechnological source of α-amylase because of their many benefits over plant and animal sources. The use of microorganisms for the production of α- amylase is mainly because of economical bulk production capacity and the fact that microbes are easily engineered to obtain enzymes of desired characteristics (Saranraj and Stella, 2013). Among the microorganisms capable of secreting α-amylase, fungi are preferred because of their more acceptable GRAS (generally regarded as safe) status. In addition, the hyphal mode of growth, good tolerance to low water activity (����) and acidity which allows the avoidance of bacterial contamination make fungi most efficient for enzyme production (Jiby et al., 2016).
Solid state fermentation (SSF) method has in recent years gained attention for production of enzymes. Compared to submerged fermentation, Solid state fermentation proves to be more simple; requiring lower capital, simple fermentation media, absence of rigorous control of
fermentation parameters, uses less water and so produces lower wastewater (Saranraj and Stella, 2013; Priya and Mrunal, 2016). However, submerged fermentation (SmF) which was the very first method adopted for industrial enzyme production, is still very relevant for enzyme production because of better monitoring, ease of sterilization of the medium and purification process (Saranraj and Stella, 2013). Virtually all the large-scale enzyme producing facilities uses the proven technology of SmF (Jiby et al., 2016).
With constant rise in demand of α-amylase in different industries, there is need for producing one with better characteristics suitable for industrial applications. Therefore, the current study focuses on the production, purification and characterization of α-amylase from organism isolated from a laundry waste water disposal site.
1.1 Alpha amylases (α-amylase)
Alpha amylases (E.C.3.2.1.1) are extracellular enzyme group that hydrolysis the interior α-1,4- glycosidic linkages in starch. They belong to the enzyme class of hydrolases and they are a calcium metalloenzyme, completely unable to function in the absence of calcium (Ajita and Thirupathihalli, 2014). Hydrolysis of internal α-1,4-glycosidic linkages by α-amylase occurs randomly and generates maltotriose and maltose from amylose, or maltose, glucose and “limit dextrin” from amylopectin (Saranraj and Stella, 2013). α-Amylases being endo-amylases act along the chain length of its substrate and therefore, tends to be faster-acting than glucoamylase which is the exo-amylase (Jiby et al., 2016). Terminal glucose residues and α-1,6-linkages cannot be cleaved by α-amylase (Gupta et al., 2008; Ajita and Thirupathihalli, 2014). In human physiology, both salivary and pancreatic amylases function as digestive enzymes and operate at optimum pH of 6.7 – 7.0 (Tiwari et al., 2015).
1.2 Cassava starch as a substrate for α-amylase
Starch is a polymer of anhydrous glucose units which are typically accumulated in the unique and independent granules (Zia et al., 2017). It is the most abundant carbohydrate reserve in plants and is distributed in different parts such as leaves, flowers, fruits, seeds, stems and roots (Sandoval and Fernández, 2013). Structurally, as shown in Figure 1, starch consists of two polysaccharides; amylose (20 – 30 %) and amylopectin (70 – 80 %). Both consist of chains of α-(1,4)-linked D- glucose residues, which are interconnected through α-(1,6) glucosidic linkages, thus forming branches in the polymers (Eric, 2017). In biotechnological application, α-amylases display highest
specificity to starch more than any other type of polysaccharide (Saranraj and Stella, 2013). Cassava (Manihot esculenta) starch is predominantly found in the roots of the crop and is a highly suitable material for industrial use. This is because of its high carbohydrate content (80–90 % dry basis), consisting almost entirely of starch (Zia et al., 2017). Industrially, cassava starch plays an important role as raw material for bioethanol production among other applications. One of its advantages as an industrial choice is the possibility of being isolated in a pure form, having low contamination by non-starch components. Its functional properties include; low gelatinization temperature, non-cereal flavor, high viscosity, high water binding capacity, bland taste, translucent paste and a relatively good stability (Oluwatoyin et al., 2018).
Other enzymes as shown in Figure 2 participate in starch conversion. They include; β-amylase, γ- amylase, debranching enzymes and transferases. β-Amylases (EC 3.2.1.2) unlike α-amylases are exo-hydrolases acting from the non-reducing end of a polysaccharide chain (Ajita and Thirupathihalli, 2014; Ritu et al., 2017). β-amylase catalyzes the hydrolysis of the second α-1,4- glycosidic bond, cleaving off two glucose units (maltose) at a time. β-amylase cannot cleave at the branched point and therefore generates limit dextrin unit on hydrolysis of highly branched polysaccharide such as glycogen or amylopectin (Ajita and Thirupathihalli, 2014). The sweetness and flavor that accompany fruit ripening is as a result of starch breakdown to maltose by β-amylase (Ritu et al., 2017). γ-amylase (EC 3.2.1.3) in addition to cleaving the terminal α-1,4-glycosidic linkages at the non-reducing end of amylose and amylopectin, will also cleave α-1,6-glycosidic linkages yielding glucose (Ajita and Thirupathihalli, 2014). Unlike the other forms of amylase, γ- amylase is most efficient in acidic environments and has an optimum pH of 3 (Ajita and Thirupathihalli, 2014).
1.3 Sources of α-amylase
Plants, animals and microorganisms are all good sources of α-amylase, but the production from plants and animals are rather limited for several reasons (Asad et al., 2011). The concentrations of enzymes in plant materials are generally low and usually, starch processing industries require large quantities of enzymes (Asghar et al., 2007). α-Amylases from animal origin are usually limited to by-product of the meat industry and therefore its supply is also small (Hmidet et al., 2010). Microbial sources, mainly fungi and bacteria, are presently the preferred sources for its industrial production due to advantages such as cost effectiveness, consistency, less time and space required for production and ease of process modification and optimization (Swetha et al., 2006; Mohammadabadi and Chaji, 2012; Ahmadi, 2012; Saranraj and Stella, 2013). In plants, animals and microbes, α-amylases play an important role specifically for carbohydrate metabolism. There are many reports on its isolation, identification and characterization from these sources (Asghar et al., 2007; Bakri et al., 2009; Hmidet et al., 2010; Asad et al., 2011; Ahmad et al., 2013).
1.3.1 Plant α-amylases
There are three families of α-amylase genes that play a role in carbohydrate metabolism in plants and the relative involvement of α-amylases and other enzymes in starch degradation probably vary between plant species (Duncan et al., 2005).
The family one α-amylase is characterized by a secretory signal peptide. All the well-characterized cereal grain α-amylases fall within this family. This family of α-amylase is involved in the breakdown of extracellular starch in the endosperm of cereal grains and dicotyledonous seeds and probably in diseased tissue where cell death has occurred (Nakajima et al., 2004; Duncan et al.,
2005). The induction of family one α-amylase gene expression is promoted in response to the phytohormone gibberellic acid and repressed by abscisic acid (Arpana et al., 2012). Similarly, at the beginning of seed germination, there is active metabolism and a rise in respiration rate which causes rapid sugar exhaustion in embryo. This triggers the expression of α-amylase genes in general and degradation of starch in this tissue (Arpana et al., 2012). Since sugar reduces quantity of gibberellic acid, sugar starvation is suggested to be primary factor in initiating the synthesis of phytohormone gibberellic acid in the embryo which in turn brings about a remarkable up-shoot in sugar and α-amylase level. Therefore, the embryo’s nutritional requirement at early phase of its growth is met (Arpana et al., 2012).
The second family of α-amylase gene has no signal peptide and therefore is thought to be localized in the cytoplasm. They are thought to degrade a supposed cytosolic α-glucan, or a heteroglycan (Fettke et al., 2004). It is possible that family two α-amylase is involved in general stress responses. For example, cold acclimation in plants includes an increase in soluble sugars and hexosephosphates in the cytosol, and could lead to a transitory increase in cytosolic α-glucan, maintaining a high concentration of sugar within the cytosol, and limiting the need to transport sugar across plastid membranes (Duncan et al., 2005).
The third family is characterized by a large N-terminal domain, typically 400-500 amino acids in length, which contains a predicted chloroplast transit peptide (Duncan et al., 2005). Family three α-amylases may also be responsible for degrading plastid bound starch in starch storage tissues and in leaves in other plant species (Blennow et al., 2002).
1.3.2 Animal α-amylases
In animals, α-amylase occurs in pancreas, parotid, serum, urine and occasionally in smaller amounts in other tissues or tumors (Whitcomb and Lowe, 2007). Animal α-amylase consists of a single chain protein to which a carbohydrate is attached. Although, some species have an isoform with no carbohydrate attached (Ferey-Roux et al., 1998). Salivary amylase initiates carbohydrate digestion in the mouth and pancreatic amylase is the main enzyme for luminal digestion of carbohydrate in the small intestine. Human pancreatic α-amylase is a protein of about 57 kDa and 512 amino acids which includes a signal sequence (Whitcomb and Lowe, 2007). Human pancreatic juice amylase has no sugar groups and exists as two isoforms termed HPA I and HPA II (Ferey- Roux et al., 1998). Salivary amylase is coded for by the AMY1 gene and pancreatic amylase by AMY2; a third form present in some tumors is termed AMY2B (Ferey-Roux et al., 1998). The animal α-amylase contains three domains termed A, B, and C from the amino terminal with C being a globular domain of unknown function. The active site is located in a cleft between the A and B domains (Whitcomb and Lowe, 2007). Calcium and chloride ions bind to the A domain and may stabilize the active site. The active center contains 5 subsites which bind different glucose residues in the substrate (Ferey-Roux et al., 1998; Whitcomb and Lowe, 2007).
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PRODUCTION AND CHARACTERIZATION OF Α-AMYLASE FROM ASPERGILLUS NIDULANS UNDER SUBMERGED FERMENTATION SYSTEM
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