87 7 Bioethanol from Starchy Biomass Part I Production of Starch Saccharifying Enzymes Subhash U Nair, Sumitra Ramachandran, and Ashok Pandey ABSTRACT There has been a substantial shift in global perception of the production of ethanol from plant-based biomass. These could be of starchy or cellulosic (or lignocellulosic) CONTENTS Abstract 87 7.1 Introduction 88 7.2 Enzymes Hydrolyzing α-1,4-Glucosidic Linkages 88 7.2.1 Endo-amylases (EC 3.2.1.1) 88 7.2.2 Exo-amylases (β-amylases EC 3.2.1.2) 88 7.3 Enzymes Hydrolyzing α-1,6-Glucosidic Linkages 88 7.3.1 Pullulanases (EC 3.2.1.41) 90 7.3.2 Isoamylases (EC 3.2.1.68) 90 7.4 Transglycosylation to Form α-1,4 and/or α-1,6-Glucosidic Linkages 90 7.4.1 Cyclodextrin Glucotransferase (EC 2.4.1.19; CGTases) 90 7.5 Hydrolysis of Both α-1,4- and α-1,6-Glucosidic Linkages 90 7.5.1 Glucoamylases (EC. 3.2.1.3) 90 7.6 Sources 91 7.7 Production 94 7.7.1 α- and β-Amylases 95 7.7.2 Glucoamylase 96 7.7.3 Pullulanase 96 7.8 Purication 96 References 102 © 2009 by Taylor & Francis Group, LLC 88 Handbook of Plant-Based Biofuels nature, which requires a step of hydrolysis to produce fermentable sugars which then can be fermented to ethanol. The hydrolysis of the starchy raw materials can be done by acids or enzymes, but due to several advantages enzymatic hydrolysis offers, it is the preferred choice for industrial applications. The enzymes involved in hydrolysis include α-amylase, β-amylase, glucoamylase, and pullulanase. These enzymes can be obtained from plant and microbial sources but industrial demand is met through the latter. This chapter presents a brief description of the sources, applications, and production of these enzymes. 7.1 INTRODUCTION The amylases (a term that refers to α-amylase and β-amylase here) can be generally dened as the enzymes that hydrolyze the O-glycosyl linkage of starch. α-Amylases are one of the most popular and important forms of the industrial amylases and have different reaction and product specicities, which include exo- and endo-specicity, preference for the hydrolysis, or the transglycosylation, α-(1,1), α-(1,4) or α-(1,6)- glycosidic bond specicity and glucan synthesizing activity. Depending on the type of cleavage caused on starch, the enzymes belonging to the amylase family are of the following types (see Figure 7.1; Kuriki and Umanaka 1999). 7.2 ENZYMES HYDROLYZING α-1,4-GLUCOSIDIC LINKAGES 7. 2.1 e n d o -a m y l a S e S (ec 3.2.1.1) This group of enzymes known as α-amylases cleave the α-1,4-glucosidic linkages. These enzymes generally do not cleave the α-1,6-glucosidic linkages. It is the most widespread enzyme among aerobes and anaerobic microbes. α-Amylases from dif- ferent sources have been puried and many have been crystallized. These enzymes are chiey required in the thinning of starch in the liquefaction process in the sugar, alcohol, and brewing industries. 7.2.2 ex o -a m y l a S e S (β-a m y l a S e S ec 3.2.1.2) The β-amylases generally occur in plants such as malt, sweet potato, soy bean, etc., and also as extracellular enzymes in some aerobic and anaerobic microorganisms. This enzyme degrades the amylose, amylopectin, and glycogen in an exo-fashion from the nonreducing ends, by hydrolyzing the alternate glucosidic linkages. It is incapable of bypassing the α-1,6 linkages and, hence, causes the incomplete hydro- lysis to form limit dextrins. 7.3 ENZYMES HYDROLYZING α-1,6-GLUCOSIDIC LINKAGES The debranching enzymes hydrolyze the α-1,6-glycosidic linkages in branched poly- mers. Two types of direct debranching enzymes, namely pullulanase and isoamylase are known. © 2009 by Taylor & Francis Group, LLC Production of Starch Saccharifying Enzymes 89 Maltotriose, Linear oligosaccharides Debranching enzymes Glucose CH 2 OH CH 2 OH O O O O H,OH α-amylase (endo) Linear oligosaccharides Maltose and beta limit dextrin Alpha limit dextrin, linear oligosaccharides, glucose and maltose Cyclodextrin glycosyl transferase Cyclodextrin β-amylase (exo) CH 2 OH CH 2 OH CH 2 OH CH 2 OH CH 2 OH CH 2 OH CH 2 OH O O O O O O O O O 4 1 4 1 4 1 O O O O CH 2 OH CH 2 OH CH 2 OH CH 2 OH CH 2 OH 4 4 4 1 4 1 1 1 O O O O O O O O O O CH 2 OH 6CH 2 CH 2 OH CH 2 OH CH 2 OH O O O O O O O O O CH 2 OH CH 2 OH O O CH 2 OH O O O CH 2 OH O CH 2 OH O O O 6CH 2 O CH 2 OH 6CH 2 CH 2 OH CH 2 OH CH 2 OH O O O O O O O O O H,OH O O O CH 2 OH O O CH 2 OH O O CH 2 OH O O 1 CH 2 OH O O 1 Isoamylase Pullulanase-1 Glucoamylase FIGURE 7.1 Schematic representation of different enzymes belonging to the amylase family. © 2009 by Taylor & Francis Group, LLC 90 Handbook of Plant-Based Biofuels 7. 3.1 Pu l l u l a n a S e S (ec 3.2.1.41) The enzymes that hydrolyze certain α-1,6-linkages are classied as pullulanases. These enzymes are capable of hydrolyzing the α-1,6-linkages in polysaccharides such as starch, amylopectin, and especially in pullulan to give the trisaccharide maltotriose. True pullulanases do not hydrolyze the α-1,4-linkages in pullulan. The pullulanases are different from isoamylase for their ability to degrade the branches of pullulan. 7. 3. 2 iS o a m y l a S e S (ec 3.2.1.68) Isoamylase generally occurs in higher plants (the enzyme from such sources is referred to as R-enzyme) but has also been found in yeast and bacteria. These enzymes have little or no effect on pullulan, but cleave all the α-1,6-linkages in amylopectin and glycogen. 7.4 TRANSGLYCOSYLATION TO FORM α-1,4 AND/OR α-1,6-GLUCOSIDIC LINKAGES 7.4.1 c y c l o d e x t r i n Gl u c o t r a n S f e r a S e (ec 2.4.1.19; cGta S e S ) This is a unique enzyme capable of converting starch and related substances to cyclodex- trin (CD). It is a multifunctional enzyme and catalyzes the conversion of the starch into CD by intramolecular transglycosylation (cyclization) and in the presence of acceptors it also catalyzes intermolecular transglycosylation (coupling and disproportionation). 7.5 HYDROLYSIS OF BOTH α-1,4- AND α-1,6-GLUCOSIDIC LINKAGES 7. 5.1 G l u c o a m y l a S e S (ec. 3.2.1.3) This class of enzymes, also referred to as amylogucosidases or saccharogenic amy- lase (1,4-α--glucan glucohydrolase), are capable of cleaving both α-1,6- and α-1,4- glucosidic linkages. These enzymes remove one glucose unit at a time from the nonreducing end of the large carbohydrate molecule. Although they are capable of hydrolyzing certain α-1,6-glucosidic linkages, they hydrolyze the α-1,4-glucosidic linkages much more rapidly. The microbial glucoamylase is important in the starch bioprocessing and brewing industry where the hydrolysis of starch is necessary. These enzymes can convert starch to glucose even in the absence of other enzymes. There are a few deviations from the above classication, since many enzymes are reported to show mixed types of the specicity. For example, the α-amylases weakly catalyze the α-1,4 transglycosylation; the CGTases feebly catalyze the α-1,4 hydrolysis in addition to the main reaction, the α-1,4 transglycosylation. There are a few α-amylases that catalyze the α-1,6 hydrolysis. Some pullulanases from ther- mophilic microorganisms have recently been reported to hydrolyze not only α-1,6- but also α-1,4-glucosidic linkages (Guzman-Maldonado and Paredas-Lopez 1995; Kuriki and Umanaka 1999). © 2009 by Taylor & Francis Group, LLC Production of Starch Saccharifying Enzymes 91 Most starch-converting enzymes belong to the GH-13 family. The α-amylase family or GH-13 family, is a large enzyme family that constitutes about 20 enzymes. This family can be further classied into clans, based on the three-dimensional structure of their catalytic components. Each of the clans may consist of two or more families with similar three-dimensional structure of their catalytic domain but with few sequence similarities, since protein structure is conserved to a large extent by the evolution as compared to the amino acid sequence. The α-amylase family (GH-13) belongs to the eighth clan among the fourteen clans described (Reddy, Nim- magadda, and Rao 2003). An enzyme should have the following characteristics to be included in this family: it should have specicity for α-1,4- or 1,6-glucosidic link- ages and be capable of hydrolyzing them to produce mono- or oligosaccharides, or capable of transglycosylations to form α-glucosidic linkages. They should have Asp, Glu, and Asp residues as the catalytic sites, corresponding to the Asp 206, Glu 230, and Asp 297 of Taka amylase A and should possess four highly conserved regions in their primary structures consisting of catalytic and important substrate-binding sites. α(ß/α) 8 ; the TIM barrel catalytic domain is also a required feature (Kuriki and Umanaka 1999, Henrissat 1991). 7.6 SOURCES The starch saccharifying enzymes play a dominant role in carbohydrate metabolism and are produced by many plants and animals. They are also produced by several microorganisms, which are the preferred sources for commercial production. These enzymes are either cell bound, intracellular, or extracellular. From a commercial point of view, extracellular enzymes are preferred and since the applications of these enzymes are usually at temperatures higher than 50°C, it is always advantageous to isolate enzymes that are thermostable. The major microbial sources of the thermo- stable α-amylases are Bacillus sp, especially B. subtilis, B. stearothermophilus, B. licheniformis, and B. amyloliquefaciens, and are widely used for the commercial production of the enzyme for various applications (Bertoldo and Antranikian 2001). However, for industrial applications, B. licheniformis (Thermamyl) and B. amyloliq- uefaciens are commonly employed. Equally important are the lamentous fungi, predominantly from the genus Aspergillus, which are widely used for the production of these enzymes, especially glucoamylase (Pandey et al. 2000a; Gupta et al. 2003; Sivaramakrishnan et al. 2006). However, in comparison to the ubiquitous presence of the α-amylases, β-amylases are produced by relatively fewer organisms. As regards glucoamylase, the bacterial sources do not provide sufcient glucoamylase for commercial needs. Currently, glucoamylase is produced mainly by the solid-state or submerged fermentation of many molds and bacteria, mainly from Aspergillus niger, A. oryzae, A. phoenicis, A. candidus, A. awamori, Rhizopus delemar, Mucor rouxianus, etc. The debranching enzymes R-enzymes or pullulanases are reported from several plant, animal, and microbial sources. Pullulanase was rst discovered in Klebsiella species. In the industry, the thermostable and acidophilic pullulanases are produced from Bacillus acidopullulyticus. The pullulanases from Thermococcus sp., T. litora- lis, Pyrococcus furiosus, and P. woesei have been described as type II as they cleave © 2009 by Taylor & Francis Group, LLC 92 Handbook of Plant-Based Biofuels both the α-1,4- and α-1,6-glucosidic bonds in starch and related polysaccharides. Neopullulanase, which hydrolyzes pullulan to panose (6-α--glucosylmaltose) by hydrolyzing its α-(1,4)-glucosidic linkages, are found in Bacillus, Micrococcus, Ther- moactinomycetes, and Listeria species. The isopullulanases (IPU) hydrolyze α-(1,4) glucosidic linkages of pullulan to produce isopanose (6-O-α-maltosyl-glucose). Few strains of fungi produce these types of pullulanases, for example, Aspergillus niger and Arthrobacter sp. The IPU does not attack the starch or dextran. Table 7.1 sum- marizes different bacterial and fungal sources of amylase, glucoamylase, and pul- lulanase (Doman-Pytka and Bardowski 2004). TABLE 7.1 Microbial Sources of Starch Saccharifying Enzymes α -Amylase β-Amylase Glucoamylase Pullulanase Aeromonas caviae Aspergillus terreus Acremonium zonatum Aerobacter aerogenes Acinetobacter Bacillus cereus Amylomyces rouxii Anaerobranca gottschalkii Alicyclobacillus acidocaldarius B. circulans Arxula adeninivorans Bacillus acidopullulyticus Alteromonas haloplanetis B. megatarium Aspergillus sp. Bacillus stearothermophilus Archaeobacterium pyrococcus woesei B. polymyxa A. awamori Bacillus cereus Aspergillus sp. Brettanomyces naardensis A. candidus Bacillus circulans A. awamori C. thermocellum A. foetidus Bacillus avocaldarius A. avus Clostridium thermosulfurogenes A. niger Bacillus macerans A. fumigatus Corynascus sepedonium A. oryzae Bacillus naganoensis A. kawachi Debaromyces sp. A. phoenicus Bacteroides thetaiotaomicron A. niger Emericella nidulans 45 A. saitri Clostridium thermohydrosulfuricum A. oryzae E. nidulans MNU 82 A. terreus Clostridium thermosaccharolyticum A. usanii Malbranchea sulfurea Bacillus rmus/lentus Desulfurococcus mucosus Bacillus sp. Pafa rhodozyma B. stearothermophillus Fervidobacterium pennavorans B. acidocoldarius Pichia anomala Candida famata Klebsiella aerogenes B. amyloliquefaciens P. holestii Cephalosporium charticola Klebsiella pneumoniae B. brevis Pseudomonas sp. Chalara paradoxa Klebsiella planticola B. circulans Rhizopus japonicus Clostridium sp. Klebsiella oxytica B. coagulans Saccharomyces sp. C. acetobutylicum Micrococcus sp. © 2009 by Taylor & Francis Group, LLC Production of Starch Saccharifying Enzymes 93 TABLE 7.1 Microbial Sources of Starch Saccharifying Enzymes α -Amylase β-Amylase Glucoamylase Pullulanase Bacillus avothermus Sacchoromyces cerevisiae Clostridium thermohydrosulfuricum Pyrococcus woesei B. globisporus Syncephalastrum racemosum RR96 C. thermosaccharolyticum Rhodothermus marinus B. licheniformis Streptomyces sp. C. thermosulfurogenes Streptococcus mitis B. megaterium Thermomyces lanuginosus Cladosporium resinae Streptococcus pneumoniae B. stearothermophilus Trichosporon beigelii Coniophora cerebella Streptococcus pyogenes B. subtilis Endomyces sp. Thermoactinomyces thalpophilus B. halmapalus Flavobacterium sp. Thermoanaerobacter ethanolicus Clostridium acetobutylicum Fusidium sp. Thermoanaerobacter nnii C. butricum Halobacter sodamense Thermoanaerobacter saccharolyticum C. thermohydrosulfuricum Humicola lanuginosa Thermoanaerobacter thermohydrosulfuricus C. thermosulfurogenes Lactobacillus brevis Thermoanaerobacter thermosulfurogenes Filobasidium capsuligenum Magnaporthe grisea Thermoanaerobium sp. Halobacterium halobium Mucor rouxianus Thermococcus hydrothermalis H. salinarium Neurospora crassa Thermotoga maritima Humicola insolens Penicillium italicum Thermus aquaticus H. lanuginosa P. oxalicum Thermus caldophilus H. stellata Piricularia oryzae Thermus thermophilus Lactobacillus brevis Rhizoctania solani Malbrachea pulchella var. sulfurea Rhizopus sp. Micrococcus luteus R. delemar M. varians R. javanicus Micromonospora vulgaris R. niveus Mucor pusillus R. oligospora Myceliophthora thermophila R. oryzae Myxococcus coralloides Sachharomyces diastaticus Nocardia asteroides Thermomyces lanuginosus © 2009 by Taylor & Francis Group, LLC 94 Handbook of Plant-Based Biofuels 7.7 PRODUCTION The starch saccharifying enzymes can be produced by submerged fermentation (SmF) and solid-state fermentation (SSF). For ease of handling and greater control of the physicochemical factors such as temperature and pH, submerged fermentation traditionally has been the preferred method of production. However, SSF has been considered superior in several aspects to SmF (Pandey 1992, 1994, 2003). It is cost effective due to the use of simple fermentation media comprised mainly of agro- industrial residues, uses little water, which consequently releases negligible or considerably less efuent, thus reducing pollution. The SSF processes are simple, provide easy aeration, use low volume equipment (lower cost), and are yet effec- tive by providing high product titers (concentrated products) (Pandey et al. 2004). Both natural and synthetic media are used for the production of amylolytic enzymes. Because the synthetic components are expensive, alternative cheaper sources such as agricultural by-products for the reduction of the cost of the medium are useful. In general, the production of these enzymes is affected by a variety of physicochemical factors, notably the composition and pH of the growth medium, inoculum size and age, temperature, aeration and agitation, and the carbon and nitrogen sources. TABLE 7.1 Microbial Sources of Starch Saccharifying Enzymes α -Amylase β-Amylase Glucoamylase Pullulanase Penicillium brunneum Trichoderma reesei Pseudomonsa stutzeri T. viride Pyrococcus woesei Rhizopus sp. Scytalidium sp. Talaromyces thermophilus Thermus sp. Thermoactinomyces sp. T. vulgaricus Thermococcus profundus Thermoascus aurantracus Thermomonospora viridis Thermonospora curvata T. vulgaris Thermomyces lanuginosus Thermotoga maritime Torula thermophila © 2009 by Taylor & Francis Group, LLC Production of Starch Saccharifying Enzymes 95 7.7.1 α- a n d β-am y l a S e S To counter the increasing commercial need for the amylases, continuous efforts are underway to reduce the cost of production of α-amylase. Employing SSF to uti- lize agricultural polymeric wastes is one of the most signicant ways of reducing the cost of amylase production. These wastes provide both support and nutrition to the microbes, and include wheat bran, spent brewing grain, maize bran, rice bran, rice husk, coconut oil cake, mustard oil cake, corn bran, amaranths grains, gram bran, palm oil cake, sunower meal, pearl millet bran, soy meal, etc. (Pandey et al. 2000a; Ramachandran et al. 2004a, 2004b;, Bogar et al. 2002; Francis et al. 2002, 2003; Satyanarayana et al. 2004; Sivaramakrishnan et al. 2006). Among these, wheat bran is generally considered the most suitable substrate. Filamentous fungi are generally reported to produce high titers of extracellular enzymes, and several strains of Aspergillus sp. and Rhizopus sp. are commonly used. A strain of Thermo- myces lanuginosus, a thermophilic fungus, was reported as an excellent producer of α-amylase (Jensen and Olsen 1992). The α-amylase from Pycnoporus sanguine by cultivation in SSF resulted in fourfold higher enzyme production than in SmF. Several yeast strains such as Saccharomycopsis capsularia and Cryptococcus sp. are also employed in α-amylase production using SSF (Satyanarayana et al. 2004; Sivaramakrishnan et al. 2006). The industrial submerged fermentation process is generally carried out in batch or in fed-batch mode. Simple and cheap media containing corn steep liquor and soy- bean meal or whey-based media offer benets of cost reduction and higher yields. The enzyme production is induced by the presence of some natural materials such as beet pulp, corn cob, rice husk, wheat bran, and wheat straw in the production medium and is generally affected by the pattern of growth of the microorganism and any morphological changes (in case of fungus). The cell growth and α-amylase pro- duction patterns are generally similar regardless of the limiting nutrient, suggesting that there exists stationary phase gene control of α-amylase production as opposed to a direct response to nutrient limitation. The dissolved oxygen tension is an impera- tive factor for α-amylase production and higher aeration rates improve the yields. Bacillus sp. is the most preferred and important source for the production for most amylolytic enzymes. Most applications of α-amylase require it to be thermotolerant, which is generally obtained from B. licheniformis; thermostable fungal α-amylase is obtained from A. niger and A. oryzae. As compared to α-amylase, not much is known about the production of β-amylase using microorganisms. Some microorganisms that produce β-amylase include B. polymyxa, B. cereus, B. megatarium, Streptomyces sp., Pseudomonas sp., and Rhizopus japonicus (Crueger and Crueger 1989; Pandey et al. 2000a). The technique of immobilization has also been employed for the production of the amylases (Pandey et al. 2000b). Various immobilization techniques can be uti- lized for α-amylase production, such as entrapment in gels using calcium alginate, kappa-carrageenan, agar and their combinations with polyethylene oxide, adsorption on cut disks of polymerized polyethylene oxide, and xation on formaldehyde acti- vated acrylonitrile-acrylamide membranes. © 2009 by Taylor & Francis Group, LLC 96 Handbook of Plant-Based Biofuels 7.7. 2 Gl u c o a m y l a S e Most of the commercial production of glucoamylase (GA) is carried out by sub- merged fermentation. The production is generally characterized by the simultaneous production of small quantities of other enzymes, referred to as associated activities. These include transglucosidase, which, however, is an undesirable phenomenon and should be controlled. The production of α-amylase in very small quantities is con- sidered desirable as it acts on the starch to catalyze the formation of saccharides of lower molecular weight, which are broken down to dextrose with relative ease by the glucoamylase. However, larger amounts of α-amylase may be detrimental to the production of dextrose as it produces saccharides, which may polymerize to unfer- mentable dextrose polymers by transglucosidase (Pandey 1995; Pandey et al. 2000a; Soccol et al. 2004; Sandhya and Pandey 2005). The fungal strains of Aspergillus and Rhizopus are the main sources of the glu- coamylase, although bacterial cultures such as Lactobacillus brevis and yeasts can also be used. The commercial production of GA is carried out by SmF as well as SSF using fungal strains and strains of A. awamori are frequently employed. Several agro-industrial residues, in combination and individually, imparted different pat- terns of GA induction in Aspergillus. In SSF, particle size of the substrate, moisture content, and water activity inuence GA production (Pandey and Radhakrishnan 1992, 1993; Selvakumar, Ashakumary, and Pandey 1998). The type of bioreactor used, such as asks, trays, rotary reactors, and columns (vertical and horizontal), inuence the production of glucoamylase. SSF often yields higher GA titers than SmF (Pandey et al. 1995). The technique of immobilization is also widely employed in the production of the GA. Strains of A. niger, Candida sp. and Endymycopsis sp. have been used for this purpose (Pandey et al. 2000b, Sandhya and Pandey 2005). 7.7.3 Pu l l u l a n a S e The pullulanases are either cell bound, intracellular, or extracellular. The enzyme can be produced by different microorganisms. The selection of an appropriate car- bon source is critical in pullulanase production, since the enzyme production is con- trolled by substrate induction and catabolite repression. Most saccharides, such as soluble starch, potato starch, amylopectin, potato dextrin, maltodextrin, and maltose, induce varying levels of pullulanase production. This could be attributed to the pres- ence of α-(1,6) linkages, present in complex polysaccharides, that can induce pro- duction of pullulanase. Soluble starch is a good source of carbon for the production of pullulanase, since it can induce the release of the enzyme in the medium (Nair 2006; Nair, Singhal, and Kamat 2006, 2007). Several other starches, dextrins or maltosaccharides, polypeptone, yeast extract, manganese also induce the production of pullulanase. 7.8 PURIFICATION Conventional techniques for the purication of amylases involve several steps, which include centrifugation of the culture to separate the solid media followed by selec- © 2009 by Taylor & Francis Group, LLC [...]... Mass (kDa) 34 57 75 67 70 49, 59 90 – 22, 39 42 112, 104 4, 61 75 Optimum pH 4.0–4.4 – 4.4–5.6 4.8 5.0 4 .7 4.5 4.5 5.5 4.5–5.5 4.4 5.0 4.5 Optimum Temperature (°C) 55–60 70 70 60 60 55 50 50 55 65 60 70 65 – 77 5.5 95 110 Nature of Protein 2.0 95 141, 95 82 5.0–5.4 5.0–6.2 60 4 0-5 0 5.1 – 62 nium sulfate precipitation, acid-clay treatment, and Sephadex G-100 gel filtration Two kinds of GA were obtained... Thermoanaerobacter finnii 80 7 Thermoactinomyces thalpophilus – – – – – – – – – 3.5–8 – 5–5.5 – – 80 95 – – – – 80 – 75 80 – 843 – 416 – – 113 1, 279 – – – 1,481 – 96,262 – 47, 041 – – 13 ,76 3 142,431 – – – 166,363 Production of Starch Saccharifying Enzymes 101 102 Handbook of Plant- Based Biofuels one-step purification of pullulanase and amylases from standard preparation of the enzyme Techniques like... Micrococcus sp 8 50 – – – – Pyrococcus woesei 6 100 – 100 404 46, 875 Rhodothermus marinus – 80 – 85 – – Streptococcus pneumoniae – – – – 75 9 86,5 17 Streptococcus pyogenes – – – – 1,165 128,898 Bacteroides thetaiotaomicron Handbook of Plant- Based Biofuels – 6.5 Bacillus flavocaldarius 70 © 2009 by Taylor & Francis Group, LLC 90 – – – 75 70 95 – 5 – – 6.5 5.5 – 6.5 – Thermobacteroides acetoethylicus Thermoanaerobacter... Taylor & Francis Group, LLC 98 Handbook of Plant- Based Biofuels maltotriose and maltotetraose from starch (Table 7. 2A) Amylases from B licheniformis were purified by enhanced two-phase separation in a PEG-Dextran system, followed by gel filtration and ion-exchange chromatography; B subtilis amylase gene was cloned into a plasmid and transferred to Escherichia coli The α-amylase so produced was purified... purification of α-amylase include autofocusing the enzyme produced by B subtilis, followed by gel filtration and ion-exchange chromatography; sequential steps of amylopectin affinity chromatography, DEAEion-exchange chromatography, and Sephacryl S-200 HR gel filtration for an A oryzae α-amylase; anion exchange (DEAE-cellulose) and affinity (α-cyclodextrinSepharose) chromatography for α-amylase from... Table 7. 2C Biochemical Characteristics of Pullulanases Optimum pH Optimum Temperature (°C) pH Stability Temperature Stability (°C) No of Amino Acids Aerobacter aerogenes 6 50 4.5–12 50 – – Anaerobranca gottschalkii 8 70   70 865 98, 973 Bacillus acidopullulyticus 5 65 4–8.5 55 – – Bacillus cereus 6 50 5–9 50 853 97, 502 Bacillus circulans 7 50 5–8 50 – – Organism Mol Weight (Da) – – – 475 53, 875 – – – – 75 ,000... weight calculated from the derived amino acid sequences of the B subtilis complete α-amylase ( 57, 700 Da) The α-amylase produced by the recombinant cells was purified by specific elution from the anti-peptide antibodies corresponding to the C-terminal region of target α-amylase, and then specifically eluted by the eluent containing low concentration of the antigen peptide used for immunization (Pandey... affinity binding on cross-linked starch, and DEAE-Biogel A chromatography for α-amylase from the yeast Lipomyces kononenkoae, etc (Pandey et al 2000a; Satyanarayana et al 2004) The purification of β-amylase is also done generally in the same way as that of α-amylase A β-amylase from Hendersonula toruloidea was separated by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, and gel... G -7 5 An extracellular β-amylase from a new isolate of B polymyxa was purified by adsorption on raw corn starch to 22.5-fold A thermostable β-amylase was purified with ammonium sulfate, DEAE-cellulose column chromatography, and gel filtration using Sephadex G-200 (Reddy, Swamy, and Seenayya 1998) The purification and characterization of the GA also follows generally the conventional methods Table 7. 2B... with a molecular weight of 75 kDa The GA from A niger contained four different forms and was purified to 32.4-fold with a final specific activity of 49.25 U/mg protein Two forms of the GA, homogeneous in nature, were purified from Monascus kaofiang nov sp F-I, which exhibited pH optima at 4.5 and 4 .7 A commercial preparation of the GA from A niger showed six different forms of GA, having apparently . -1 ,6-Glucosidic Linkages 90 7. 5.1 Glucoamylases (EC. 3.2.1.3) 90 7. 6 Sources 91 7. 7 Production 94 7. 7.1 - and β-Amylases 95 7. 7.2 Glucoamylase 96 7. 7.3 Pullulanase 96 7. 8 Purication 96 References. & Francis Group, LLC 96 Handbook of Plant- Based Biofuels 7. 7. 2 Gl u c o a m y l a S e Most of the commercial production of glucoamylase (GA) is carried out by sub- merged fermentation. The. perception of the production of ethanol from plant- based biomass. These could be of starchy or cellulosic (or lignocellulosic) CONTENTS Abstract 87 7.1 Introduction 88 7. 2 Enzymes Hydrolyzing -1 ,4-Glucosidic