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Marine Microbial Amylases
CHAPTER EIGHT
Marine Microbial Amylases: Properties and Applications J. Suriya*, S. Bharathiraja†, M. Krishnan*, P. Manivasagan{, S.-K. Kim§,1 *School of Environmental Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India † CAS in Marine Biology, Annamalai University, Porto Novo, Tamil Nadu, India { Marine Bioprocess Research Center, Pukyong National University, Busan, Republic of Korea § Marine Bioprocess Research Center; Specialized Graduate School Science & Technology Convergence, Pukyong National University, Busan, Republic of Korea 1 Corresponding author: e-mail addresses: [email protected]; [email protected]
Contents 1. Introduction 2. Types of Amylase 2.1 α-Amylase 2.2 β-Amylase 2.3 Glucoamylase 3. Production Methods 4. Production of Amylases 4.1 Production of α-Amylase 4.2 Production of β-Amylase 4.3 Production of Glucoamylase 5. Purification of Amylases 6. Characterization of Amylases 7. Molecular Biology of Amylases 8. Determination of Enzyme Activity 8.1 Dinitrosalicylic Acid Method 8.2 Nelson–Somogyi Method 8.3 Determination of Activity Using Iodine 8.4 Dextrinizing Activity 8.5 Indian Pharmacopeia Method 8.6 Reduction in Viscosity of Starch Suspension 9. Industrial Applications Amylases 9.1 Enzymatic Hydrolysis of Starch for Fructose and Glucose Production 9.2 Bakery Industry 9.3 Other Industrial Applications 10. Conclusion Acknowledgment References
Advances in Food and Nutrition Research, Volume 79 ISSN 1043-4526 http://dx.doi.org/10.1016/bs.afnr.2016.07.001
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Abstract Amylases are crucial enzymes which hydrolyze internal glycosidic linkages in starch and produce as primary products dextrins and oligosaccharides. Amylases are classified into α-amylase, β-amylase, and glucoamylase based on their three-dimensional structures, reaction mechanisms, and amino acid sequences. Amylases have innumerable applications in clinical, medical, and analytical chemistries as well as in food, detergent, textile, brewing, and distilling industries. Amylases can be produced from plants, animals, and microbial sources. Due to the advantages in microbial production, it meets commercial needs. The pervasive nature, easy production, and wide range of applications make amylase an industrially pivotal enzyme. This chapter will focus on amylases found in marine microorganisms, their potential industrial applications, and how these enzymes can be improved to the required bioprocessing conditions.
1. INTRODUCTION More than 70% of the Earth surface is covered by sea, has unprecedented biological diversity, and comprises 95% of the biosphere (Qasim, 1999). Microbial diversity in marine environment is a promising agent for many novel industrial products (Berdy, 2005; Fenical & Jensen, 2006). The recent researches showed that the culturability of microorganisms in marine sediments (0.25%) and seawater (0.001–0.10%) is lower than soil microorganisms (0.30%) (Amann, Ludwig, & Schleifer, 1995). Henceforth, researchers switched over to marine environments for novel products from microorganisms. Microorganisms inhabitating in marine environment are always under stress conditions for space, nutrient, defence, offence, and salinity. Some marine eco regions are always changing its environmental conditions. Microorganisms living under this environment produce unique characteristic enzymes which are not produced by terrestrial organisms. These enzymes may have more than one independent function and these are considered as multifunctional enzymes. The main advantages of enzymes are the possibility to work at milder conditions, highly specific to substrate as well as eco-friendly (Krishna, 2011). Amylase is an extracellular enzyme which degrades starch by hydrolyzing glycosidic bonds at α-1,4 position (Ajayi & Fagade, 2007) and produces different polysaccharides such as glucose, maltose, and dextrin (Riaz, Haq, & Qadeer, 2003). Amylases have been used since ancient times (Gupta, Gigras, Mohapatra, Goswami, & Chauhan, 2003) for starch
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saccharification, beer production, digestive disorders treatment, and cheese production from milk (Drauz, Gr€ oger, & May, 2012). The enzymatic class of amylase accounts for 25% of world’s market (de Freitas, Escaramboni, Carvalho, de Lima, & de Oliva-Neto, 2014). Cost-effective medium is crucial to meet out the demands of amylase utilizing industries (Balkan & Figen, 2007). Utilization of agro-industrial wastes for the production of amylase enzyme is the cost-effective method and it also solves the problem of pollution (Amin, Bhatti, Zuber, Bhatti, & Asgher, 2015; Singh, Kapoor, & Kumar, 2012). The following sections elaborate on the types of amylases, their production, and their industrial applications.
2. TYPES OF AMYLASE 2.1 α-Amylase α-Amylase (EC 3.2.1.1) hydrolyze the internal α-1,4-glycosidic linkages in starch and produce glucose and maltose. α-Amylase depends on calcium for its catalysis and cannot cleave the terminal glucose residues and α-1,6-linkages. Starch is a polysaccharide that consists of 75–80% of amylopectin and 20–25% of amylase, and it is the natural substrate for the activity of α-amylase. Amylose is a linear chain molecule consisting of glucose units united by α-1,4-glycosidic linkage, whereas amylopectin has branched chains of glucose units. α-1,4-Glycosidic linkage joined the linear glucose units, while α-1,6-glycosidic bonds are present in branched chain which occurs at every 15–45 glucose units. The composition of glucose and maltose obtained after hydrolysis of starch is dependent on the hydrolysis conditions like the temperature and the enzyme origin. pH 7.0 is optimum for the activity α-amylase (Sundarram & Murthy, 2014).
2.2 β-Amylase β-Amylase (EC 3.2.1.2) produces maltose from starch by hydrolyzing the α-1,4-glucan linkages. Seeds of higher plants and sweet potatoes are the primary sources of β-amylase. β-Amylase is responsible for the sweetness of ripened fruit because it hydrolysis starch into maltose during ripening of fruits. pH 4.0–5.5 is the optimum for the hydrolytic activity of β-amylase. β-Amylase has been used for various research and industrial applications. It is utilized for structural studies of starch and glycogen molecules produced by different methods. It is used in the brewing and
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distilling industry. It is also applied for producing high-maltose syrups (Sivaramakrishnan, Gangadharan, Nampoothiri, Soccol, & Pandey, 2006).
2.3 Glucoamylase Glucoamylase (EC 3.2.1.3) breaks α (1-4)glycosidic bonds as well as α(1-6) glycosidic bonds in the nonreducing end of amylose and amylopectin, producing glucose. Its optimum pH is 3.0 and can be efficiently utilized in acidic environments (Sivaramakrishnan et al., 2006).
3. PRODUCTION METHODS Submerged fermentations (SmF) and solid-state fermentations (SSF) are the two methods widely employed for the production of amylases. The solid-state fermentation is a new method, whereas SmF is a traditional method for enzyme production from microorganisms which has been used for a longer period of time. In SmF, free-flowing liquid substrates like molasses and broths are used. The end products of the fermentation are liberated into the fermentation broth. Substrate utilization is very rapid in SmF; henceforth, substrate must be provided continuously for this fermentation process. This technique is well suited for the extraction of secondary metabolites from bacteria because it requires high moisture content for their growth (Couto & Sanroma´n, 2006). SmF has several advantages in which genetically modified organisms are grown well compared to SSF and media sterilization, purification, and recovery of the end products. Further, the control of process parameters such as pH, temperature, moisture, oxygen transfer, and aeration can be done easily (Kunamneni, Permaul, & Singh, 2005). Solid-state fermentation (SSF) is suitable for the less moisture content required microorganisms. In SSF, nutrient-rich waste materials such as bran, bagasse, and paper pulp can be used as substrate for the microorganisms and they are consumed very slowly and constantly. Hence, there is no need to supply the substrate for longer time (Kunamneni et al., 2005). Major advantages of SSF are easy to handle, recovery of higher concentration of products, and generation of lesser effluent (Couto & Sanroma´n, 2006). Therefore, SSF is considered as a promising method for commercial enzyme production. α-Amylase production by SmF and solid-state fermentation techniques has been examined for fungal species. The results showed that
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SSF was well suited for developing countries due to cost-effective production process (Kunamneni et al., 2005).
4. PRODUCTION OF AMYLASES Despite several microorganisms can produce amylases, it is very difficult to find a most suitable strain for this enzyme production. Some microorganisms produce more than one enzyme, for e.g., α-amylase and glucoamylase. For example, Aspergillus niger can produce as many as 19 enzymes, whereas α-amylase can be produced in reasonably good titers by as many as 28 strains (Pandey, 1992). pH, temperature, nutrient supplementation, aeration, and the control of contamination during fermentation are the very crucial parameters necessary for the growth of the microorganisms in fermentation for large-scale production.
4.1 Production of α-Amylase Major source for the α-amylase production is Bacillus species, which can either be produced by solid substrate fermentation (SSF) (Babu & Satyanarayana, 1995; Krishna & Chandrasekaran, 1996; Ramesh & Lonsane, 1987) or by SmF (Kellly, Bloton, & Fogarty, 1997; Omidji, Amund, Braimoh, & IIori, 1997). The SSF is considered as a most economic technique in enzyme production as well as for starch hydrolysis. Cheese whey, corn steep liquor, and soya bean meal are the cheap sources for the industrial production of α-amylase (Bajapai, Gera, & Bajapai, 1992; Omidji et al., 1997). Marine bacteria produce α-amylases with specific features such as pH and thermo stability, salt tolerance, and cold activity. For example, deep sea bacterium Nocardiopsis sp. 7326 (Zhang & Zeng, 2008) and marine Wangia sp. C52 (Liu et al., 2011) produced cold-adapted α-amylase, pH stable α-amylase was derived from marine bacterium Bacillus subtilis S8-1 (Kalpana & Pandian, 2014) and Bacillus sp. ALSHL3 (Vidilaseris et al., 2009), and Zunongwangia profunda (AmyZ2) (Wu, Qin, Cheng, & Liu, 2014) have potential to produce salt-tolerant α-amylase. Alkalophilic amylase produced by sponge-associated marine bacterium Halobacterium salinarum MMD047 had optimum activity at 40°C and pH 9.0 (Shanmughapriya et al., 2009). The impact of calcium ion and EDTA on α-amylase production was evaluated and results revealed that calcium ions stimulated the activity as well as it was very essential for the thermostability of the enzyme whereas EDTA decreased the activity of α-amylase
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(Chakraborty et al., 2011; Vieille & Zeikus, 2001). In contrast to this, calcium ions neither stimulate nor decrease the activity of α-amylase in Streptomyces strain A3 and indicated the calcium independency of this strain (Chakraborty, Raut, Khopade, Mahadik, & Kokare, 2012). It was also reported that the activity of calcium-dependent α-amylase is reduced in the presence of EDTA (Kiran & Chandra, 2008), whereas calciumindependent enzyme retain their activity even in the presence of EDTA (Chakraborty et al., 2012). Chakraborty et al. (2014) isolated a novel 45 kDa α-amylase from marine Nocardiopsis sp. and immobilized it in gellan gum. They found 74.76 1.32% to 87.64 1.52% entrapment efficiency of this immobilized enzyme. A novel 66 kDa α-amylase was isolated from the marine Streptomyces sp. by Chakraborty, Khopade, Kokare, Mahadik, and Chopade (2009). The specific activity was 113.64 U/mg protein and almost 50% of this enzyme activity was retained at 85°C and the activity was not inhibited by the presence of commercially available detergent and oxidizing agents. Surfactants, oxidant, and detergent stable 66 kDa α-amylase were isolated from marine haloalkaliphilic Saccharopolyspora sp. A9. The enzyme was active in a wide range of NaCl concentration with maximum activity at 11% NaCl (Chakraborty et al., 2011). Marine actinomycetes isolated from South Indian coastal region produced 6.48 U/mL of amylase (Selvam, Vishnupriya, & Subash Chandra Bose, 2011). While comparing with this result, Streptomyces sp. NIOT-VKKMA02 isolated from Andaman & Nicobar Islands produced 13.27 U/mL of amylase enzyme, which is twofold increases to that of previous report (Meena, Anbu Rajan, Vinithkumar, & Kirubagaran, 2013). Mohapatra, Banerjee, and Bapuji (1998) isolated α-amylase from Mucor sp. and found that the optimum activity at pH 5.0 and temperature 60°C. It has also been reported that the activity was not inhibited in the presence of 3% NaCl, 10 mM Ca2+, and 25 mM Mg2+, whereas EDTA strongly inhibited the activity. Starch is considered as the major substrate for yeast cells and their primary, secondary metabolites productions because it is very cheap and easily available raw material in the world. In recent years, amylase-producing yeasts has been utilized drastically because of its potential role in the ethanol and single-cell protein production from starch (Chi, Liu, Ji, & Meng, 2003; Gupta et al., 2003).
4.2 Production of β-Amylase Usually plants are the major source of β-amylases. Henceforth, not much work has been done on the microbial β-amylase. Despite some
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microorganisms have the potential to produce β-amylase like Pseudomonas sp., Bacillus cereus, Bacillus polymyxa, Bacillus megaterium, Streptomyces sp., and Rhizopus japonicus (Crueger & Crueger, 1989; Fogarty & Kellly., 1990). β-Amylase production from starch waste by B. megaterium B6 mutant UN12 was compared in the SmF and SSF. Arum and wheat bran gave the highest yields (Ray, Jana, & Nanada, 1997).
4.3 Production of Glucoamylase Glucoamylase production from A. niger in solid cultures has been widely reported (Pandey, 1992; Selvakumar, Ashakumary, & Pandey, 1998). Agroindustrial residues such as wheat bran, rice bran, rice husk, gram flour, wheat flour, corn flour, tea waste, and copra waste were used (Pandey, 1992; Pandey & Radhakrishnan, 1990, 1993; Selvakumar et al., 1998). Marine yeast Aureobasidium pullulans N13d (Li, Chi, Wang, & Ma, 2007) produces 58.5 U/mg protein within 56 h of fermentation. The results revealed that crude glucoamylase actively digests potato starch granules, but poorly hydrolyzes raw corn and sweet potato starch. During hydrolysis only glucose molecules are released from starch, indicating that glucoamylase can effectively break both α-1,4 and α-1,6 linkages of starch (Li, Chi, Duan, et al., 2007; Li, Chi, Wang, et al., 2007).
5. PURIFICATION OF AMYLASES High purity amylases have been only used in pharmaceutical and clinical sectors as well as for studying structure–function relationships and biochemical properties of the enzymes (Gupta et al., 2003). Henceforth, it is very crucial for developing economic process for enzyme purification to obtain pure enzymes with maximum specific activity. Purification of amylases has been carried out in the following steps. Supernatant is obtained by centrifugation then it is concentrated by ultra filtration. The crude amylase can be precipitated and concentrated by ammonium sulfate precipitation or organic solvents such as ethanol in the cold. The precipitated sample is subjected to dialysis against water or a buffer for further concentration (Shih & Labbe, 1995) and followed by the chromatographic techniques such as ion exchange, gel filtration, and affinity. The number of steps used for purification of enzymes will depend on the extent of purity (Gangadharan, Sivaramakrishnan, Nampoothiri, & Pandey, 2006; Kundu & Das, 1970; Moreira et al., 1999; Sivaramakrishnan, Gangadharan, Nampoothiri,
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Soccol, & Pandey, 2007). Combination of filtration and centrifugation is used for obtaining extracellular enzyme from the fermented mass. For intracellular enzymes, raw corn starch may be added followed by filtration and subsequent steps. Enzyme produced by Aspergillus falvus var. columnaris was precipitated, dialyzed, and then subjected to column chromatography for purification (Shih & Labbe, 1995). For purification of amylase from Preussia minim, the sample was precipitated by trichloroacetic acid/acetone and eluted through Sephadex G-200 gel filtration column, and the resulting fractions were pooled and applied to a DEAE–Sepharose ion-exchange column (Erdal & Taskin, 2010). Ammonium sulfate (80%) precipitation, TSK Toyopeal column chromatography, ultrafiltration, dialysis, and SP Sepharose column chromatography steps were used for the purification of α-amylase from mutant Bacillus (Ballschmiter, F€ utterer, & Liebl, 2006). For thermostable amylase purification, the cell extract obtained after centrifugation is subjected to high temperatures for denaturing thermolabile proteins. This step was followed by anion exchange chromatography for purification of α-amylase from Thermotoga maritima MSB8. Then SDSPAGE and size exclusion chromatography were used for checking the purity of the enzyme as well as for the determination of its molecular weight (Iefuji, Chino, Kato, & Iimura, 1996).
6. CHARACTERIZATION OF AMYLASES After purification, characterization is carried out for enzymes. SDS-PAGE is mostly used for characterization of purified enzyme along with molecular markers like BSA (67 kDa) and ovalbumin (43 kDa). Then the gel is stained with either Coomassie Brilliant Blue (Paquet, Croux, Goma, & Soucaille, 1991) or silver nitrate (Mukesh Kumar, Jayanthisiddhuraj, Monica Devi, Bala Kumaran, & Kalaichelvan, 2012) and visualized. Purity and homogeneity of the enzyme is also observed in the PAGE. Molecular mass of α-amylase produced by Bacillus MNJ23 and mutant B. subtilis was characterized by SDS-PAGE (12% and 10%, respectively) (Ballschmiter et al., 2006; Singhania, Patel, Soccol, & Pandey, 2009). SDSPAGE in a Phast Gel gradient (10–15%) was used for the characterization of enzyme produced by Clostridium acetobutylicum ATCC 824 (Mukesh Kumar et al., 2012). Size exclusion chromatography was also used for determining the molecular mass of the enzyme (Satoh, Uchimura, Kudo, & Komagata, 1997).
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Intracellular α-amylase of Streptococcus bovis 148 showed a single band on a SDS-PAGE gel which indicated the purification to homogeneity. The molecular mass was found to be 57,000 Da, near the 55,000 Da protein obtained with the gel filtration method, confirming that the intracellular α-amylase exists in a monomeric form (Melasniemi, 1987). Dong, Vieille, Savchenko, and Zeikus (1997) used Sephacryl S200 gel filtration chromatography for determining molecular weight of α-amylase produced from Pyrococcus furiosus. In addition to SDS-PAGE, isoelectric focusing is also used for enzyme characterization. The isoelectric point of the desired enzyme has been determined by using protein markers of broad range of pI values (Mukesh Kumar et al., 2012). α-Amylase produced by Clostridium thermohydrosulfuricum E 101-69 was ultrafiltered for concentration of crude enzyme sample and then it was subjected to isoelectric focusing with pI range of 3.5–5.0 (Nielsen & Borchert, 2000). pI 3–10 as standards was used for determining isoelectric point of C. acetobutylicum ATCC 824 α-amylase (Mukesh Kumar et al., 2012).
7. MOLECULAR BIOLOGY OF AMYLASES Recombinant DNA technology offers great features for cloning of amylase-producing strains, particularly α-amylase and glucoamylase. The main aim of gene cloning is the overexpression of thermostable enzymes, maximum productivity, and expression of the two enzymes simultaneously by the same strain. Much work has been carried out on the cloning of α-amylase genes either in Escherichia coli or in Saccharomyces cerevisiae. Like α-amylase, the number of glucoamylase-encoding genes also cloned in E. coli. Only very few reports are available on β-amylase expression in E. coli, usually from Bacillus sp. and only one report from Thermoanaerobacterium.
8. DETERMINATION OF ENZYME ACTIVITY Reducing sugars released as a result of starch hydrolysis by amylase enzyme is measured for determining the enzyme activity. Reading the absorbance of starch–iodine complex is another method for measuring enzyme activity. The following methods are usually used for enzyme assay.
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8.1 Dinitrosalicylic Acid Method In this method, enzyme solution is added to the aliquots of the substrate stock solution and incubated at 50°C for 10 min followed by the addition of dinitrosalicylic acid (DNS) method and the mixture is incubated in a boiling water bath for 5 min. After cooling to room temperature, the absorbance of the supernatant is measured at 540 nm. The activity is calculated by subtracting the A540 values of substrate and enzyme blanks from the A540 value of the analyzed sample (Feller, Le Bussy, & Gerday, 1998; Gusakov, Kondratyeva, & Sinitsyn, 2011; Kobayashi et al., 1992). DNS method was used for measuring alkalophilic α-amylase activity of Bacillus strain GM8901 (Gusakov et al., 2011).
8.2 Nelson–Somogyi Method In this method, enzyme solution and substrate stock solutions are heated at 50°C for 5 min separately. Then enzyme solution is added to the substrate stock solution and this mixture is incubated at 50°C for 10 min. After 10 min, Somogyi copper reagent is added to this mixture to terminate the reaction. This is followed by incubation of this mixture in a boiling water bath for 40 min and cooled to room temperature. Then Nelson arsenomolybdate reagent is added and incubated at room temperature for 10 min. Finally, water is added and absorbance of supernatant is read at 610 nm (Kobayashi et al., 1992). Haloalkaliphilic α-amylase of archaebacterium Natronococcus sp. Strain Ah-36 was subjected to NS method for determining enzyme activity (El-Safey & Ammar, 2004).
8.3 Determination of Activity Using Iodine This method is used for enzyme assay based on the principle that the formation of blue colored complex by the reaction of starch with iodine. On hydrolysis of starch by amylase enzyme this complex is changed into reddish brown color. Then the absorbance is read after the termination of enzyme substrate reaction. This gives a measure of the extent of hydrolysis of starch by α-amylase (Gupta et al., 2003).
8.4 Dextrinizing Activity In this enzyme assay, the crude enzyme is incubated at 92°C with soluble starch for 10 min. Then the reaction mixture is kept in ice to terminate the reaction. Iodine is added to this reaction mixture to form a colored
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complex with starch and then water is added to this complex to dilute the color to a measurable range that can be read at 600 nm (Haki & Rakshit, 2003).
8.5 Indian Pharmacopeia Method This method is used for determining the enzyme activity by measuring the amount of starch (grams) hydrolyzed by enzyme of a given volume. In this assay different concentration of enzyme solution is incubated with starch at 40°C for 1 h. After incubation, iodine is added to this mixture to produce colored complex (Gupta et al., 2003).
8.6 Reduction in Viscosity of Starch Suspension This method is mostly used in baking industry to check the quality of flour. Falling number (FN) method and Amylograph/Farinograph test are used for determining enzyme activity based on decreasing viscosity of starch suspension. In FN method, enzyme substrate preparations are measured at 100°C. Usually malted flour has a FN of around 400. Amylograph test is used to test the relationship between the enzyme activity and peak starch slurry viscosity. The lesser viscosity indicates the higher activity of the enzymes. Optimal Brabender units for bread baking flour ranges from 400 to 600 units (Gupta et al., 2003).
9. INDUSTRIAL APPLICATIONS AMYLASES In recent years interest in amylase production has been increased drastically due to its starch hydrolyzing properties and utilization of easily available and low cost raw material. It has many potential and widely used applications on the industrial front. In various industrial sectors, chemical methods used for the hydrolysis have been replaced by enzymes and it makes the process easier and eco-friendly.
9.1 Enzymatic Hydrolysis of Starch for Fructose and Glucose Production Fructose and glucose syrups are produced by the hydrolysis of starch by amylase (Van Der Maarel, Van Der Veen, Uitdehaag, Leemhuis, & Dijkhuizen, 2002). This process consists of three steps. Gelatinization is the first step followed by liquefaction and saccharification. In gelatinization starch granules are dissolved in water to produce a viscous starch suspension. Then
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amylase is added into the water to initiate hydrolysis. Starch is partially hydrolyzed by amylase and form short chain of dextrins resulting in reduction of the starch suspension. This process is called liquefaction. Release of glucose from starch by further hydrolysis by glucoamylase is called saccharification. α-1,4-Glycosidic linkages of nonreducing terminal ends are hydrolyzed by glucoamylase cleaves from the nonreducing terminal ends of starch, hence, it is called as an exoamylase. Pullulanase activity along with glucoamylase produces high-glucose syrup. Glucose isomerase converts the high-glucose syrup into high-fructose syrup by catalyzing the isomerization. The fructose syrup is usually used as a sweetener, especially in the beverage industry. Traditionally, acid hydrolysis is used for the production of glucose and fructose syrup. This process had many drawbacks. Due to the acidic nature of this process, it required corrosion-resistant equipment and high temperatures would inactivate the thermolabile enzymes. Henceforth, enzymatic hydrolysis is preferred for glucose and fructose syrup production. The α-amylase used in the liquefaction step can be derived from different microbial sources, for example, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, and P. furiosus are mostly used for α-amylase production. Hydrolysis of starch at a high temperature can be carried out with the help of thermostable amylases (Gupta et al., 2003; Kulp, Ponte, & D’Appolonia, 1981).
9.2 Bakery Industry For starch hydrolysis of dough, it is supplemented with α-amylase followed by yeast fermentation. Undesirable changes such as increase of crumb firmness, loss of crispness, decrease in moisture content, and loss of bread flavor occur during storage of baked bread. This together is called staling. α-Amylase acts as an antistaling agent to increase the shelf life of baked breads (Gupta et al., 2003; Kulp et al., 1981). However, a slight overdose of this enzyme produces gumminess of the bread (Chi et al., 2009). To prevent this, pullulanase is applied along with amylase for breakdown of gumminess responsible compounds (Kulp et al., 1981).
9.3 Other Industrial Applications Enzymes are employed in detergent industry due its eco-friendly nature and have the potential to work at mild conditions. The potential of α-amylase to work at low temperature and alkaline pH makes it extensively used in detergents. Calcium dependency and oxidant sensitivity are the major drawbacks
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of α-amylase. The oxidant resistance potential of amylase is increased by replacing the oxidant-sensitive amino acid met by leu in B. licheniformis amylase (Gupta et al., 2003). Starch is a preferred sizing agent due to its easy availability, low cost and can be easily removed from the fabric. α-Amylase is used for hydrolyzing starch into water-soluble components during desizing process where that can be removed by washing. The enzyme acts only on the starch molecules, it does not affect the fibers (Gupta et al., 2003). For protection of papers from stains in processing it is also coated with sizing agents. Starch is the most preferred sizing agent in paper industry. Starch is partially hydrolyzed by α-amylase and reduces the viscosity in a batch or a continuous process and makes it a suitable sizing agent for paper industry (Gupta et al., 2003). Mostly starch is used as a starting material for biofuel production due its low cost and easy availability. First step is liquefaction where starch is converted into viscous suspension. This suspension is subjected to saccharification for the production of fermentable sugars through hydrolysis by α-amylase. Finally, ethanol is produced from these fermentable sugars by yeast fermentation. S. cerevisiae and amylolytic yeast Saccharomyces fibuligera are fused for protoplasmic fusion to generate a new yeast strain to yield biofuel from starch to exclude saccharification process (Chi et al., 2009).
10. CONCLUSION The use of enzyme for various industrial needs has been increased recently due to their biodegradability and can be obtained from biological sources. By-products and waste products of other processes can be utilized for amylase production from microorganisms. The enzyme has many industrial applications which include production of fructose syrup, eco-friendly detergents, and baked products. It is also used as alternative for fossil fuels depletion by producing biofuel such as ethanol from starch. From these applications, it can be concluded that it is a promising agent to replace the conventional chemical process usually employed in the industries.
ACKNOWLEDGMENT Dr. J.S. is grateful to University Grants Commission—Dr. D.S. Kothari acknowledges the Postdoctoral Fellowship for their financial support.
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Li, H. F., Chi, Z. M., Wang, X. H., & Ma, C. L. (2007b). Amylase production by the marine yeast Aureobasidium pullulans N13d. Journal of Ocean University of China, 6(1), 61–66. Liu, J., Zhang, Z., Zhul, H., Dang, H., Lu, J., & Cui, Z. (2011). Isolation and characterization of α-amylase from marine Pseudomonas sp. K6-28-040. African Journal of Biotechnology, 10, 2733–2740. Meena, B., Anbu Rajan, L., Vinithkumar, N. V., & Kirubagaran, R. (2013). Novel marine actinobacteria from emerald Andaman & Nicobar Islands: A prospective source for industrial and pharmaceutical byproducts. BMC Microbiology, 13, 145. Melasniemi, H. (1987). Characterization of alpha-amylase and pullulanase activities of Clostridium thermohydrosulfuricum. Evidence for a novel thermostable amylase. Biochemical Journal, 246, 193–197. Mohapatra, B. R., Banerjee, U. C., & Bapuji, M. (1998). Characterization of a fungal amylase from Mucor sp. associated with the marine sponge Spirastrella sp. Journal of Biotechnology, 60, 113–117. Moreira, F. G., de Lima, F. A., Fazzano Pedrinho, S. R., Lenartovicz, V., Giatti, C., de Souza, M., et al. (1999). Production of amylases by Aspergillus tamari. Revista de Microbiologia, 30, 157–162. Mukesh Kumar, D. J., Jayanthisiddhuraj, A. B., Monica Devi, D., Bala Kumaran, M. D., & Kalaichelvan, P. T. (2012). Purification and characterization of α-amylase and β-galactosidase from Bacillus sp. MNJ23 produced in a concomitant medium. American-Eurasian Journal of Agriculture and Environmental Sciences, 12, 566–573. Nielsen, J. E., & Borchert, T. V. (2000). Protein engineering of bacterial alpha-amylases. Biochimica et Biophysica Acta, 1543, 253–274. Omidji, O., Amund, O. O., Braimoh, A. A., & IIori, M. O. (1997). Production of α-amylase in a corn steep liquor-soya bean meal medium by a strain of Bacillus stearothermophilus. Microbios, 90, 155–162. Pandey, A. (1992). Production of starch saccharifying enzyme in solid cultures. Strach/Starke, 39, 75–77. Pandey, A., & Radhakrishnan, S. (1990). Bioconversion of agricultural biomass into value added products: The solid-state fermentation approach. Proceedings of Kerala Academy of Sciences, 1, 69–79. Pandey, A., & Radhakrishnan, S. (1993). The production of glucoamylase by Aspergillus niger NCIM 1245. Process Biochemistry, 28, 305–309. Paquet, V., Croux, C., Goma, G., & Soucaille, P. (1991). Purification and characterization of the extracellular α-amylase from Clostridium acetobutylicum ATCC 824. Applied and Environmental Microbiology, 57, 212–218. Qasim, S. Z. (1999). The Indian Ocean: Images and realities (pp. 57–90). New Delhi: Oxford and IBH. Ramesh, M. V., & Lonsane, B. K. (1987). A novel bacterial thermostable alpha-amylase system produced under solid state fermentation. Biotechnology Letters, 9, 501–504. Ray, R. R., Jana, S. C., & Nanada, G. (1997). Production of β-amylase from starchy wastes by a hyperamylolytic mutant of Bacillus megaterium. Indian Journal of Experimental Biology, 35, 285–288. Riaz, N., Haq, I., & Qadeer, M. (2003). Characterization of α-amylase by Bacillus subtilis. International Journal of Agriculture and Biology, 5(3), 249–252. Satoh, E., Uchimura, T., Kudo, T., & Komagata, K. (1997). Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148. Applied and Environmental Microbiology, 63(12), 4941–4944. Selvakumar, P., Ashakumary, L., & Pandey, A. (1998). Biosynthesis of glucoamylase from Aspergillus niger by solid-state fermentation using tea waste as the basis of a solid state. Bioresource Technology, 65, 83–85.
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Selvam, K., Vishnupriya, B., & Subash Chandra Bose, V. (2011). Screening and quantification of marine actinomycetes producing industrial enzymes amylase, cellulase and lipase from South Cost of India. International Journal of Pharmaceutical Biology, 2, 1481–1487. Shanmughapriya, S., Seghal Kiran, G., Selvin, J., Gandhimathi, R., Bastin Baskar, T., Manilal, A., et al. (2009). Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047. Biotechnology and Bioprocess Engineering, 14, 67–75. Shih, N.-J., & Labbe, R. G. (1995). Purification and characterization of an extracellular a-amylase from Clostridium perfringens type A. Applied and Environmental Microbiology, 61, 1776–1779. Singh, R., Kapoor, V., & Kumar, V. (2012). Utilization of agro-industrial wastes for the simultaneous production of amylase and xylanase by thermophilic actinomycetes. Brazilian Journal of Microbiology, 43(4), 1545–1552. Singhania, R. R., Patel, A. K., Soccol, C. R., & Pandey, A. (2009). Recent advances in solidstate fermentation. Biochemical Engineering Journal, 44(1), 13–18. Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K. M., Soccol, C. R., & Pandey, A. (2006). α-Amylases from microbial sources—An overview on recent developments. Food Technology & Biotechnology, 44(2), 173–184. Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K. M., Soccol, C. R., & Pandey, A. (2007). Alpha amylase production by Aspergillus oryzae employing solid-state fermentation. Journal of Scientific and Industrial Research, 66(8), 621. Sundarram, A., & Krishna Murthy, T. P. (2014). α-Amylase production and applications: A review. Journal of Applied & Environmental Microbiology, 2(4), 166–175. Van Der Maarel, M. J., Van Der Veen, B., Uitdehaag, J., Leemhuis, H., & Dijkhuizen, L. (2002). Properties and applications of starch-converting enzymes of the α-amylase family. Journal of Biotechnology, 94(2), 137–155. Vidilaseris, K., Hidayat, K., Retnoningrum, D., Nurachman, Z., Noer, A., & Natalia, D. (2009). Biochemical characterization of a raw starch degrading a-amylase from the Indonesian marine bacterium Bacillus sp. ALSHL3. Biologia, 64, 1047–1052. Vieille, C., & Zeikus, G. J. (2001). Hyperthermophilic enzymes: Sources, uses, and molecular mechanisms for thermostability. Microbiology and Molecular Biology Reviews, 65, 1–43. Wu, G., Qin, Y., Cheng, Q., & Liu, Z. (2014). Characterization of a novel alkali-stable and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda. Journal of Molecular Catalysis B: Enzymatic, 110, 8–15. Zhang, J.-W., & Zeng, R.-Y. (2008). Purification and characterization of a cold-adapted alpha-amylase produced by Nocardiopsis sp. 7326 isolated from Prydz Bay, Antarctic. Marine Biotechnology, 10, 75–82.
Marine Microbial Amylases: Properties and Applications J. Suriya*, S. Bharathiraja†, M. Krishnan*, P. Manivasagan{, S.-K. Kim§,1 *School of Environmental Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India † CAS in Marine Biology, Annamalai University, Porto Novo, Tamil Nadu, India { Marine Bioprocess Research Center, Pukyong National University, Busan, Republic of Korea § Marine Bioprocess Research Center; Specialized Graduate School Science & Technology Convergence, Pukyong National University, Busan, Republic of Korea 1 Corresponding author: e-mail addresses: [email protected]; [email protected]
Contents 1. Introduction 2. Types of Amylase 2.1 α-Amylase 2.2 β-Amylase 2.3 Glucoamylase 3. Production Methods 4. Production of Amylases 4.1 Production of α-Amylase 4.2 Production of β-Amylase 4.3 Production of Glucoamylase 5. Purification of Amylases 6. Characterization of Amylases 7. Molecular Biology of Amylases 8. Determination of Enzyme Activity 8.1 Dinitrosalicylic Acid Method 8.2 Nelson–Somogyi Method 8.3 Determination of Activity Using Iodine 8.4 Dextrinizing Activity 8.5 Indian Pharmacopeia Method 8.6 Reduction in Viscosity of Starch Suspension 9. Industrial Applications Amylases 9.1 Enzymatic Hydrolysis of Starch for Fructose and Glucose Production 9.2 Bakery Industry 9.3 Other Industrial Applications 10. Conclusion Acknowledgment References
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Abstract Amylases are crucial enzymes which hydrolyze internal glycosidic linkages in starch and produce as primary products dextrins and oligosaccharides. Amylases are classified into α-amylase, β-amylase, and glucoamylase based on their three-dimensional structures, reaction mechanisms, and amino acid sequences. Amylases have innumerable applications in clinical, medical, and analytical chemistries as well as in food, detergent, textile, brewing, and distilling industries. Amylases can be produced from plants, animals, and microbial sources. Due to the advantages in microbial production, it meets commercial needs. The pervasive nature, easy production, and wide range of applications make amylase an industrially pivotal enzyme. This chapter will focus on amylases found in marine microorganisms, their potential industrial applications, and how these enzymes can be improved to the required bioprocessing conditions.
1. INTRODUCTION More than 70% of the Earth surface is covered by sea, has unprecedented biological diversity, and comprises 95% of the biosphere (Qasim, 1999). Microbial diversity in marine environment is a promising agent for many novel industrial products (Berdy, 2005; Fenical & Jensen, 2006). The recent researches showed that the culturability of microorganisms in marine sediments (0.25%) and seawater (0.001–0.10%) is lower than soil microorganisms (0.30%) (Amann, Ludwig, & Schleifer, 1995). Henceforth, researchers switched over to marine environments for novel products from microorganisms. Microorganisms inhabitating in marine environment are always under stress conditions for space, nutrient, defence, offence, and salinity. Some marine eco regions are always changing its environmental conditions. Microorganisms living under this environment produce unique characteristic enzymes which are not produced by terrestrial organisms. These enzymes may have more than one independent function and these are considered as multifunctional enzymes. The main advantages of enzymes are the possibility to work at milder conditions, highly specific to substrate as well as eco-friendly (Krishna, 2011). Amylase is an extracellular enzyme which degrades starch by hydrolyzing glycosidic bonds at α-1,4 position (Ajayi & Fagade, 2007) and produces different polysaccharides such as glucose, maltose, and dextrin (Riaz, Haq, & Qadeer, 2003). Amylases have been used since ancient times (Gupta, Gigras, Mohapatra, Goswami, & Chauhan, 2003) for starch
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saccharification, beer production, digestive disorders treatment, and cheese production from milk (Drauz, Gr€ oger, & May, 2012). The enzymatic class of amylase accounts for 25% of world’s market (de Freitas, Escaramboni, Carvalho, de Lima, & de Oliva-Neto, 2014). Cost-effective medium is crucial to meet out the demands of amylase utilizing industries (Balkan & Figen, 2007). Utilization of agro-industrial wastes for the production of amylase enzyme is the cost-effective method and it also solves the problem of pollution (Amin, Bhatti, Zuber, Bhatti, & Asgher, 2015; Singh, Kapoor, & Kumar, 2012). The following sections elaborate on the types of amylases, their production, and their industrial applications.
2. TYPES OF AMYLASE 2.1 α-Amylase α-Amylase (EC 3.2.1.1) hydrolyze the internal α-1,4-glycosidic linkages in starch and produce glucose and maltose. α-Amylase depends on calcium for its catalysis and cannot cleave the terminal glucose residues and α-1,6-linkages. Starch is a polysaccharide that consists of 75–80% of amylopectin and 20–25% of amylase, and it is the natural substrate for the activity of α-amylase. Amylose is a linear chain molecule consisting of glucose units united by α-1,4-glycosidic linkage, whereas amylopectin has branched chains of glucose units. α-1,4-Glycosidic linkage joined the linear glucose units, while α-1,6-glycosidic bonds are present in branched chain which occurs at every 15–45 glucose units. The composition of glucose and maltose obtained after hydrolysis of starch is dependent on the hydrolysis conditions like the temperature and the enzyme origin. pH 7.0 is optimum for the activity α-amylase (Sundarram & Murthy, 2014).
2.2 β-Amylase β-Amylase (EC 3.2.1.2) produces maltose from starch by hydrolyzing the α-1,4-glucan linkages. Seeds of higher plants and sweet potatoes are the primary sources of β-amylase. β-Amylase is responsible for the sweetness of ripened fruit because it hydrolysis starch into maltose during ripening of fruits. pH 4.0–5.5 is the optimum for the hydrolytic activity of β-amylase. β-Amylase has been used for various research and industrial applications. It is utilized for structural studies of starch and glycogen molecules produced by different methods. It is used in the brewing and
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distilling industry. It is also applied for producing high-maltose syrups (Sivaramakrishnan, Gangadharan, Nampoothiri, Soccol, & Pandey, 2006).
2.3 Glucoamylase Glucoamylase (EC 3.2.1.3) breaks α (1-4)glycosidic bonds as well as α(1-6) glycosidic bonds in the nonreducing end of amylose and amylopectin, producing glucose. Its optimum pH is 3.0 and can be efficiently utilized in acidic environments (Sivaramakrishnan et al., 2006).
3. PRODUCTION METHODS Submerged fermentations (SmF) and solid-state fermentations (SSF) are the two methods widely employed for the production of amylases. The solid-state fermentation is a new method, whereas SmF is a traditional method for enzyme production from microorganisms which has been used for a longer period of time. In SmF, free-flowing liquid substrates like molasses and broths are used. The end products of the fermentation are liberated into the fermentation broth. Substrate utilization is very rapid in SmF; henceforth, substrate must be provided continuously for this fermentation process. This technique is well suited for the extraction of secondary metabolites from bacteria because it requires high moisture content for their growth (Couto & Sanroma´n, 2006). SmF has several advantages in which genetically modified organisms are grown well compared to SSF and media sterilization, purification, and recovery of the end products. Further, the control of process parameters such as pH, temperature, moisture, oxygen transfer, and aeration can be done easily (Kunamneni, Permaul, & Singh, 2005). Solid-state fermentation (SSF) is suitable for the less moisture content required microorganisms. In SSF, nutrient-rich waste materials such as bran, bagasse, and paper pulp can be used as substrate for the microorganisms and they are consumed very slowly and constantly. Hence, there is no need to supply the substrate for longer time (Kunamneni et al., 2005). Major advantages of SSF are easy to handle, recovery of higher concentration of products, and generation of lesser effluent (Couto & Sanroma´n, 2006). Therefore, SSF is considered as a promising method for commercial enzyme production. α-Amylase production by SmF and solid-state fermentation techniques has been examined for fungal species. The results showed that
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SSF was well suited for developing countries due to cost-effective production process (Kunamneni et al., 2005).
4. PRODUCTION OF AMYLASES Despite several microorganisms can produce amylases, it is very difficult to find a most suitable strain for this enzyme production. Some microorganisms produce more than one enzyme, for e.g., α-amylase and glucoamylase. For example, Aspergillus niger can produce as many as 19 enzymes, whereas α-amylase can be produced in reasonably good titers by as many as 28 strains (Pandey, 1992). pH, temperature, nutrient supplementation, aeration, and the control of contamination during fermentation are the very crucial parameters necessary for the growth of the microorganisms in fermentation for large-scale production.
4.1 Production of α-Amylase Major source for the α-amylase production is Bacillus species, which can either be produced by solid substrate fermentation (SSF) (Babu & Satyanarayana, 1995; Krishna & Chandrasekaran, 1996; Ramesh & Lonsane, 1987) or by SmF (Kellly, Bloton, & Fogarty, 1997; Omidji, Amund, Braimoh, & IIori, 1997). The SSF is considered as a most economic technique in enzyme production as well as for starch hydrolysis. Cheese whey, corn steep liquor, and soya bean meal are the cheap sources for the industrial production of α-amylase (Bajapai, Gera, & Bajapai, 1992; Omidji et al., 1997). Marine bacteria produce α-amylases with specific features such as pH and thermo stability, salt tolerance, and cold activity. For example, deep sea bacterium Nocardiopsis sp. 7326 (Zhang & Zeng, 2008) and marine Wangia sp. C52 (Liu et al., 2011) produced cold-adapted α-amylase, pH stable α-amylase was derived from marine bacterium Bacillus subtilis S8-1 (Kalpana & Pandian, 2014) and Bacillus sp. ALSHL3 (Vidilaseris et al., 2009), and Zunongwangia profunda (AmyZ2) (Wu, Qin, Cheng, & Liu, 2014) have potential to produce salt-tolerant α-amylase. Alkalophilic amylase produced by sponge-associated marine bacterium Halobacterium salinarum MMD047 had optimum activity at 40°C and pH 9.0 (Shanmughapriya et al., 2009). The impact of calcium ion and EDTA on α-amylase production was evaluated and results revealed that calcium ions stimulated the activity as well as it was very essential for the thermostability of the enzyme whereas EDTA decreased the activity of α-amylase
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(Chakraborty et al., 2011; Vieille & Zeikus, 2001). In contrast to this, calcium ions neither stimulate nor decrease the activity of α-amylase in Streptomyces strain A3 and indicated the calcium independency of this strain (Chakraborty, Raut, Khopade, Mahadik, & Kokare, 2012). It was also reported that the activity of calcium-dependent α-amylase is reduced in the presence of EDTA (Kiran & Chandra, 2008), whereas calciumindependent enzyme retain their activity even in the presence of EDTA (Chakraborty et al., 2012). Chakraborty et al. (2014) isolated a novel 45 kDa α-amylase from marine Nocardiopsis sp. and immobilized it in gellan gum. They found 74.76 1.32% to 87.64 1.52% entrapment efficiency of this immobilized enzyme. A novel 66 kDa α-amylase was isolated from the marine Streptomyces sp. by Chakraborty, Khopade, Kokare, Mahadik, and Chopade (2009). The specific activity was 113.64 U/mg protein and almost 50% of this enzyme activity was retained at 85°C and the activity was not inhibited by the presence of commercially available detergent and oxidizing agents. Surfactants, oxidant, and detergent stable 66 kDa α-amylase were isolated from marine haloalkaliphilic Saccharopolyspora sp. A9. The enzyme was active in a wide range of NaCl concentration with maximum activity at 11% NaCl (Chakraborty et al., 2011). Marine actinomycetes isolated from South Indian coastal region produced 6.48 U/mL of amylase (Selvam, Vishnupriya, & Subash Chandra Bose, 2011). While comparing with this result, Streptomyces sp. NIOT-VKKMA02 isolated from Andaman & Nicobar Islands produced 13.27 U/mL of amylase enzyme, which is twofold increases to that of previous report (Meena, Anbu Rajan, Vinithkumar, & Kirubagaran, 2013). Mohapatra, Banerjee, and Bapuji (1998) isolated α-amylase from Mucor sp. and found that the optimum activity at pH 5.0 and temperature 60°C. It has also been reported that the activity was not inhibited in the presence of 3% NaCl, 10 mM Ca2+, and 25 mM Mg2+, whereas EDTA strongly inhibited the activity. Starch is considered as the major substrate for yeast cells and their primary, secondary metabolites productions because it is very cheap and easily available raw material in the world. In recent years, amylase-producing yeasts has been utilized drastically because of its potential role in the ethanol and single-cell protein production from starch (Chi, Liu, Ji, & Meng, 2003; Gupta et al., 2003).
4.2 Production of β-Amylase Usually plants are the major source of β-amylases. Henceforth, not much work has been done on the microbial β-amylase. Despite some
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microorganisms have the potential to produce β-amylase like Pseudomonas sp., Bacillus cereus, Bacillus polymyxa, Bacillus megaterium, Streptomyces sp., and Rhizopus japonicus (Crueger & Crueger, 1989; Fogarty & Kellly., 1990). β-Amylase production from starch waste by B. megaterium B6 mutant UN12 was compared in the SmF and SSF. Arum and wheat bran gave the highest yields (Ray, Jana, & Nanada, 1997).
4.3 Production of Glucoamylase Glucoamylase production from A. niger in solid cultures has been widely reported (Pandey, 1992; Selvakumar, Ashakumary, & Pandey, 1998). Agroindustrial residues such as wheat bran, rice bran, rice husk, gram flour, wheat flour, corn flour, tea waste, and copra waste were used (Pandey, 1992; Pandey & Radhakrishnan, 1990, 1993; Selvakumar et al., 1998). Marine yeast Aureobasidium pullulans N13d (Li, Chi, Wang, & Ma, 2007) produces 58.5 U/mg protein within 56 h of fermentation. The results revealed that crude glucoamylase actively digests potato starch granules, but poorly hydrolyzes raw corn and sweet potato starch. During hydrolysis only glucose molecules are released from starch, indicating that glucoamylase can effectively break both α-1,4 and α-1,6 linkages of starch (Li, Chi, Duan, et al., 2007; Li, Chi, Wang, et al., 2007).
5. PURIFICATION OF AMYLASES High purity amylases have been only used in pharmaceutical and clinical sectors as well as for studying structure–function relationships and biochemical properties of the enzymes (Gupta et al., 2003). Henceforth, it is very crucial for developing economic process for enzyme purification to obtain pure enzymes with maximum specific activity. Purification of amylases has been carried out in the following steps. Supernatant is obtained by centrifugation then it is concentrated by ultra filtration. The crude amylase can be precipitated and concentrated by ammonium sulfate precipitation or organic solvents such as ethanol in the cold. The precipitated sample is subjected to dialysis against water or a buffer for further concentration (Shih & Labbe, 1995) and followed by the chromatographic techniques such as ion exchange, gel filtration, and affinity. The number of steps used for purification of enzymes will depend on the extent of purity (Gangadharan, Sivaramakrishnan, Nampoothiri, & Pandey, 2006; Kundu & Das, 1970; Moreira et al., 1999; Sivaramakrishnan, Gangadharan, Nampoothiri,
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Soccol, & Pandey, 2007). Combination of filtration and centrifugation is used for obtaining extracellular enzyme from the fermented mass. For intracellular enzymes, raw corn starch may be added followed by filtration and subsequent steps. Enzyme produced by Aspergillus falvus var. columnaris was precipitated, dialyzed, and then subjected to column chromatography for purification (Shih & Labbe, 1995). For purification of amylase from Preussia minim, the sample was precipitated by trichloroacetic acid/acetone and eluted through Sephadex G-200 gel filtration column, and the resulting fractions were pooled and applied to a DEAE–Sepharose ion-exchange column (Erdal & Taskin, 2010). Ammonium sulfate (80%) precipitation, TSK Toyopeal column chromatography, ultrafiltration, dialysis, and SP Sepharose column chromatography steps were used for the purification of α-amylase from mutant Bacillus (Ballschmiter, F€ utterer, & Liebl, 2006). For thermostable amylase purification, the cell extract obtained after centrifugation is subjected to high temperatures for denaturing thermolabile proteins. This step was followed by anion exchange chromatography for purification of α-amylase from Thermotoga maritima MSB8. Then SDSPAGE and size exclusion chromatography were used for checking the purity of the enzyme as well as for the determination of its molecular weight (Iefuji, Chino, Kato, & Iimura, 1996).
6. CHARACTERIZATION OF AMYLASES After purification, characterization is carried out for enzymes. SDS-PAGE is mostly used for characterization of purified enzyme along with molecular markers like BSA (67 kDa) and ovalbumin (43 kDa). Then the gel is stained with either Coomassie Brilliant Blue (Paquet, Croux, Goma, & Soucaille, 1991) or silver nitrate (Mukesh Kumar, Jayanthisiddhuraj, Monica Devi, Bala Kumaran, & Kalaichelvan, 2012) and visualized. Purity and homogeneity of the enzyme is also observed in the PAGE. Molecular mass of α-amylase produced by Bacillus MNJ23 and mutant B. subtilis was characterized by SDS-PAGE (12% and 10%, respectively) (Ballschmiter et al., 2006; Singhania, Patel, Soccol, & Pandey, 2009). SDSPAGE in a Phast Gel gradient (10–15%) was used for the characterization of enzyme produced by Clostridium acetobutylicum ATCC 824 (Mukesh Kumar et al., 2012). Size exclusion chromatography was also used for determining the molecular mass of the enzyme (Satoh, Uchimura, Kudo, & Komagata, 1997).
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Intracellular α-amylase of Streptococcus bovis 148 showed a single band on a SDS-PAGE gel which indicated the purification to homogeneity. The molecular mass was found to be 57,000 Da, near the 55,000 Da protein obtained with the gel filtration method, confirming that the intracellular α-amylase exists in a monomeric form (Melasniemi, 1987). Dong, Vieille, Savchenko, and Zeikus (1997) used Sephacryl S200 gel filtration chromatography for determining molecular weight of α-amylase produced from Pyrococcus furiosus. In addition to SDS-PAGE, isoelectric focusing is also used for enzyme characterization. The isoelectric point of the desired enzyme has been determined by using protein markers of broad range of pI values (Mukesh Kumar et al., 2012). α-Amylase produced by Clostridium thermohydrosulfuricum E 101-69 was ultrafiltered for concentration of crude enzyme sample and then it was subjected to isoelectric focusing with pI range of 3.5–5.0 (Nielsen & Borchert, 2000). pI 3–10 as standards was used for determining isoelectric point of C. acetobutylicum ATCC 824 α-amylase (Mukesh Kumar et al., 2012).
7. MOLECULAR BIOLOGY OF AMYLASES Recombinant DNA technology offers great features for cloning of amylase-producing strains, particularly α-amylase and glucoamylase. The main aim of gene cloning is the overexpression of thermostable enzymes, maximum productivity, and expression of the two enzymes simultaneously by the same strain. Much work has been carried out on the cloning of α-amylase genes either in Escherichia coli or in Saccharomyces cerevisiae. Like α-amylase, the number of glucoamylase-encoding genes also cloned in E. coli. Only very few reports are available on β-amylase expression in E. coli, usually from Bacillus sp. and only one report from Thermoanaerobacterium.
8. DETERMINATION OF ENZYME ACTIVITY Reducing sugars released as a result of starch hydrolysis by amylase enzyme is measured for determining the enzyme activity. Reading the absorbance of starch–iodine complex is another method for measuring enzyme activity. The following methods are usually used for enzyme assay.
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8.1 Dinitrosalicylic Acid Method In this method, enzyme solution is added to the aliquots of the substrate stock solution and incubated at 50°C for 10 min followed by the addition of dinitrosalicylic acid (DNS) method and the mixture is incubated in a boiling water bath for 5 min. After cooling to room temperature, the absorbance of the supernatant is measured at 540 nm. The activity is calculated by subtracting the A540 values of substrate and enzyme blanks from the A540 value of the analyzed sample (Feller, Le Bussy, & Gerday, 1998; Gusakov, Kondratyeva, & Sinitsyn, 2011; Kobayashi et al., 1992). DNS method was used for measuring alkalophilic α-amylase activity of Bacillus strain GM8901 (Gusakov et al., 2011).
8.2 Nelson–Somogyi Method In this method, enzyme solution and substrate stock solutions are heated at 50°C for 5 min separately. Then enzyme solution is added to the substrate stock solution and this mixture is incubated at 50°C for 10 min. After 10 min, Somogyi copper reagent is added to this mixture to terminate the reaction. This is followed by incubation of this mixture in a boiling water bath for 40 min and cooled to room temperature. Then Nelson arsenomolybdate reagent is added and incubated at room temperature for 10 min. Finally, water is added and absorbance of supernatant is read at 610 nm (Kobayashi et al., 1992). Haloalkaliphilic α-amylase of archaebacterium Natronococcus sp. Strain Ah-36 was subjected to NS method for determining enzyme activity (El-Safey & Ammar, 2004).
8.3 Determination of Activity Using Iodine This method is used for enzyme assay based on the principle that the formation of blue colored complex by the reaction of starch with iodine. On hydrolysis of starch by amylase enzyme this complex is changed into reddish brown color. Then the absorbance is read after the termination of enzyme substrate reaction. This gives a measure of the extent of hydrolysis of starch by α-amylase (Gupta et al., 2003).
8.4 Dextrinizing Activity In this enzyme assay, the crude enzyme is incubated at 92°C with soluble starch for 10 min. Then the reaction mixture is kept in ice to terminate the reaction. Iodine is added to this reaction mixture to form a colored
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complex with starch and then water is added to this complex to dilute the color to a measurable range that can be read at 600 nm (Haki & Rakshit, 2003).
8.5 Indian Pharmacopeia Method This method is used for determining the enzyme activity by measuring the amount of starch (grams) hydrolyzed by enzyme of a given volume. In this assay different concentration of enzyme solution is incubated with starch at 40°C for 1 h. After incubation, iodine is added to this mixture to produce colored complex (Gupta et al., 2003).
8.6 Reduction in Viscosity of Starch Suspension This method is mostly used in baking industry to check the quality of flour. Falling number (FN) method and Amylograph/Farinograph test are used for determining enzyme activity based on decreasing viscosity of starch suspension. In FN method, enzyme substrate preparations are measured at 100°C. Usually malted flour has a FN of around 400. Amylograph test is used to test the relationship between the enzyme activity and peak starch slurry viscosity. The lesser viscosity indicates the higher activity of the enzymes. Optimal Brabender units for bread baking flour ranges from 400 to 600 units (Gupta et al., 2003).
9. INDUSTRIAL APPLICATIONS AMYLASES In recent years interest in amylase production has been increased drastically due to its starch hydrolyzing properties and utilization of easily available and low cost raw material. It has many potential and widely used applications on the industrial front. In various industrial sectors, chemical methods used for the hydrolysis have been replaced by enzymes and it makes the process easier and eco-friendly.
9.1 Enzymatic Hydrolysis of Starch for Fructose and Glucose Production Fructose and glucose syrups are produced by the hydrolysis of starch by amylase (Van Der Maarel, Van Der Veen, Uitdehaag, Leemhuis, & Dijkhuizen, 2002). This process consists of three steps. Gelatinization is the first step followed by liquefaction and saccharification. In gelatinization starch granules are dissolved in water to produce a viscous starch suspension. Then
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amylase is added into the water to initiate hydrolysis. Starch is partially hydrolyzed by amylase and form short chain of dextrins resulting in reduction of the starch suspension. This process is called liquefaction. Release of glucose from starch by further hydrolysis by glucoamylase is called saccharification. α-1,4-Glycosidic linkages of nonreducing terminal ends are hydrolyzed by glucoamylase cleaves from the nonreducing terminal ends of starch, hence, it is called as an exoamylase. Pullulanase activity along with glucoamylase produces high-glucose syrup. Glucose isomerase converts the high-glucose syrup into high-fructose syrup by catalyzing the isomerization. The fructose syrup is usually used as a sweetener, especially in the beverage industry. Traditionally, acid hydrolysis is used for the production of glucose and fructose syrup. This process had many drawbacks. Due to the acidic nature of this process, it required corrosion-resistant equipment and high temperatures would inactivate the thermolabile enzymes. Henceforth, enzymatic hydrolysis is preferred for glucose and fructose syrup production. The α-amylase used in the liquefaction step can be derived from different microbial sources, for example, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, and P. furiosus are mostly used for α-amylase production. Hydrolysis of starch at a high temperature can be carried out with the help of thermostable amylases (Gupta et al., 2003; Kulp, Ponte, & D’Appolonia, 1981).
9.2 Bakery Industry For starch hydrolysis of dough, it is supplemented with α-amylase followed by yeast fermentation. Undesirable changes such as increase of crumb firmness, loss of crispness, decrease in moisture content, and loss of bread flavor occur during storage of baked bread. This together is called staling. α-Amylase acts as an antistaling agent to increase the shelf life of baked breads (Gupta et al., 2003; Kulp et al., 1981). However, a slight overdose of this enzyme produces gumminess of the bread (Chi et al., 2009). To prevent this, pullulanase is applied along with amylase for breakdown of gumminess responsible compounds (Kulp et al., 1981).
9.3 Other Industrial Applications Enzymes are employed in detergent industry due its eco-friendly nature and have the potential to work at mild conditions. The potential of α-amylase to work at low temperature and alkaline pH makes it extensively used in detergents. Calcium dependency and oxidant sensitivity are the major drawbacks
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of α-amylase. The oxidant resistance potential of amylase is increased by replacing the oxidant-sensitive amino acid met by leu in B. licheniformis amylase (Gupta et al., 2003). Starch is a preferred sizing agent due to its easy availability, low cost and can be easily removed from the fabric. α-Amylase is used for hydrolyzing starch into water-soluble components during desizing process where that can be removed by washing. The enzyme acts only on the starch molecules, it does not affect the fibers (Gupta et al., 2003). For protection of papers from stains in processing it is also coated with sizing agents. Starch is the most preferred sizing agent in paper industry. Starch is partially hydrolyzed by α-amylase and reduces the viscosity in a batch or a continuous process and makes it a suitable sizing agent for paper industry (Gupta et al., 2003). Mostly starch is used as a starting material for biofuel production due its low cost and easy availability. First step is liquefaction where starch is converted into viscous suspension. This suspension is subjected to saccharification for the production of fermentable sugars through hydrolysis by α-amylase. Finally, ethanol is produced from these fermentable sugars by yeast fermentation. S. cerevisiae and amylolytic yeast Saccharomyces fibuligera are fused for protoplasmic fusion to generate a new yeast strain to yield biofuel from starch to exclude saccharification process (Chi et al., 2009).
10. CONCLUSION The use of enzyme for various industrial needs has been increased recently due to their biodegradability and can be obtained from biological sources. By-products and waste products of other processes can be utilized for amylase production from microorganisms. The enzyme has many industrial applications which include production of fructose syrup, eco-friendly detergents, and baked products. It is also used as alternative for fossil fuels depletion by producing biofuel such as ethanol from starch. From these applications, it can be concluded that it is a promising agent to replace the conventional chemical process usually employed in the industries.
ACKNOWLEDGMENT Dr. J.S. is grateful to University Grants Commission—Dr. D.S. Kothari acknowledges the Postdoctoral Fellowship for their financial support.
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