Process Biochemistry 48 (2013) 201211

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Review

Microbial acid-stable -amylases: Characteristics, genetic engineering and applications

Archana Sharma, T. Satyanarayana
Department of Microbiology, University of Delhi, South Campus, Benito Juarez Road, New Delhi 110021, India

a r t i c l e

i n f o

a b s t r a c t
Among the wide variety of amylolytic enzymes synthesized by microorganisms, -amylases are the most widely used biocatalysts in starch saccharication, baking industries and textile desizing. These enzymes randomly cleave the -1,4-glycosidic linkages in starch, generating maltose and malto-oligosaccharides. The commercially available -amylases have certain limitations, such as limited activity at low pH and Ca2+ -dependence, and therefore, the search for novel acid-stable and thermostable amylases from extremophilic microorganisms and the engineering of the already available enzymes have been the major areas of research in this eld over the years. Several attempts have been made to nd suitable microbial sources of acid-stable and thermostable -amylases. Acid-stable -amylases have been reported in fungi, bacteria and archaea. -Amylases that are active at elevated temperatures have been reported in bacteria as well as in archaea. -Amylases that possess both characteristics, to the extent required for their various applications are very scarce. The developments that have been made in molecular biology, directed evolution and structural conformation studies of -amylases for improving their properties to suit various industrial applications are discussed in this review. 2013 Elsevier Ltd. All rights reserved.

Article history: Received 17 October 2012 Received in revised form 24 December 2012 Accepted 26 December 2012 Available online 3 January 2013 Keywords: Starch saccharication -Amylase Acid-stable Site directed mutagenesis Directed evolution

Contents 1. 2. 3. 4. 5. 6. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Starch hydrolyzing enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acid-stable -amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Production of -amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purication and kinetics of acid-stable -amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of -amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Substrate specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Optimal temperature and pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Metal ion and stability of -amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -Amylase gene cloning and expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural conformation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein engineering of -amylase by site directed mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. Active site residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Thermostability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Determinants of the pH activity prole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alteration of other properties of -amylases by directed evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Correlation between the molecular dynamics of active site residues and the pH activity prole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Computational modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of -amylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 202 202 202 203 204 204 204 205 205 205 205 205 206 206 206 207 207 208 208 208 208 208 208

7. 8. 9.

10. 11. 12. 13. 14.

Corresponding author. Tel.: +91 11 24112008, fax: +91 11 24115270. E-mail address: tsnarayana@gmail.com (T. Satyanarayana). 1359-5113/$ see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.12.018

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1. Introduction Starch is the second major polysaccharide food reserve in nature after cellulose. Plants are unique in synthesizing this -glucan, which serves as an important source of nutrition for other living organisms. Starch is the most easily available source of carbon and energy on the Earth and is synthesized by plants in the presence of sunlight and water through photosynthesis. Starch is synthesized in plastids, which are present in leaves, and accumulates as insoluble granules in higher and lower plants. It is synthesized as semi-crystalline granules with different polymorphic types and degrees of crystallinity. The granules vary in size from 2 to 100 m and exhibit round, oval and irregular shapes. Starch is a major component of most staple foods and is used in many food and non-food industries. It is mainly utilized in the textile, paper, pharmaceutical, beverage and alcohol industries. In terms of its structural organization, starch is mainly composed of two high molecular weight compounds, amylose and amylopectin, and both of these contain -d-glucose as the sole monomer. Amylose is a linear, water-insoluble polymer of glucose subunits that are joined by -1,4-bonds (99%), and it possesses a molecular weight of 1 105 to 1 106 . In contrast, amylopectin is a branched, water-soluble polysaccharide with short -1,4-linked (95%) linear chains of 1060 glucose units and -1,6-linked (5%) side chains with 1545 glucose units that form the volume of the starch molecule [1,2]. The levels of amylose and amylopectin vary among starches, but representative percentages of amylose and amylopectin are 2528% and 7275%, respectively. Starch is mainly consumed after processing for domestic or industrial purposes. Hydrolyzed starch has applications in the food, beverage, pharmaceutical, textile and detergent industries. Until the 19th century, starch saccharication was achieved by acid hydrolysis using dilute HCl because the understanding of the potential advantages of biological catalysts was limited. Enzymatic starch processing has advantages over chemical starch hydrolysis, as the latter has drawbacks including a requirement for high temperature, low pH and corrosion-resistant vessels, low glucose yields, and the formation of unwanted color and bitter tasting compounds [3]. Today, starch saccharication is totally enzyme-based. The world market for industrial enzymes is estimated to be around 3 billion US dollars [4], and it is estimated to be even greater from the products obtained from these enzymes. The total enzyme market was 5.1 billion US dollars in 2009 [5]. Acid-stable extracellular enzymes are required because they are utilized in the degradation of polymeric or oligomeric carbon sources, which occurs at a pH ranges from 3.2 to 4.5 [6]. The promising property of enzymes from thermoacidophiles is activity at low pH and high temperature, and therefore, these enzymes can be used in starch, textile and fruit juice industries. The demand for enzymes from extremophiles may increase in the future because of their activity under harsh industrial conditions. A large number of bacteria, fungi and yeasts produce extracellular enzymes that degrade starch in different environmental niches. Variety of polysaccharide hydrolyzing enzymes that are suitable for various industrial applications have emerged in the last few decades, leading to the screening of these enzymes for novel properties. In this review, we review the developments in the genetic engineering of acidic microbial -amylases to improve their characteristics to suit varied applications.

[10] and GH 15 (glucoamylases) [11]. The enzymes differ in their amino acid sequences, reaction mechanisms, catalytic activities and structural characteristics. Based on their mode of action, the enzymes are divided into two categories: endoamylases (-amylases, pullulanases, isoamylase) and exoamylases (-amylase, glucoamylase). -Amylases are extracellular enzymes that catalyze the hydrolysis of -1,4-glycosidic linkages in starch liberating linear and branched oligosaccharides of varying chain lengths as well as glucose; the end products have an -conformation at C1 [12]. These are categorized based on their end product as saccharifying and liquefying -amylases. Saccharifying -amylases produce free sugars but reduce the viscosity of starch pastes slowly. On the contrary, liquefying -amylases rapidly reduce the viscosity of starch pastes without producing free sugars as compared to saccharifying amylases. The saccharifying -amylases are further classied as maltose-forming (Bacillus acidicola) [13], maltotetraose-forming (Pseudomonas sp. IMD 353) [14], maltopentaose-forming (Bacillus cereus NY-14) [15] and maltohexaose-forming (Bacillus stearothermophilus US100) [16] -amylases based on the end products formed. Liquefying -amylases are reported from Sclerotinia sclerotiorum [17,18] and B. stearothermophilus [19] and saccharifying -amylases are known from Streptococcus bovis JB 1 [20] and Scytalidium thermophilum [21]. Based on their optimal pH for activity, -amylases are also classied as acidic, neutral or alkaline. The optimal pH of amylases ranges from 2 to 12. Most -amylases are active in the neutral range [22]. The -amylases from Bacillus subtilis AX20, Bacillus licheniformis, Micromonospora melanospora and Geobacillus thermoleovorans display their highest activities at pH of 6.0, 6.5, 7 and 8, respectively [2325]. Acidic, neutral and alkaline amylases are suited to different industrial applications. The search for -amylases with the desired kinetic properties for diverse applications is encouraged to improve the industrial process in terms of economics and feasibility [26]. 3. Acid-stable -amylases The demand for -amylases that produce high levels of maltose is increasing, as they have diverse commercial applications [27]. The -amylases currently used in starch processing are active at 95 C and pH 6.8 and are stabilized by Ca2+ ; therefore, the industrial processes with these enzymes cannot be performed at low pH (3.24.5), the pH of native starch [28]. In order to be compatible with the optimal pH of the enzyme used for liquefaction, the pH of the starch slurry is raised from its native pH 3.24.5 to 5.86.2, and furthermore, Ca2+ is added to enhance the activity and/or stability of enzyme. The next saccharication step requires another pH adjustment to pH 4.24.5. Both of these steps (pH adjustment and salts removal) should be omitted, as they are time consuming and increase the cost of the products [12]. The need is, therefore, to identify extremozymes from extremophiles that are naturally endowed with the properties required for specialized industrial applications [29] (Fig. 1). -Amylases are widely distributed in plants, animals and microorganisms. Among amylases derived from various sources, microbial enzymes are known to fulll industrial demands. 4. Production of -amylases The production of -amylase in submerged and solid state fermentations has been studied extensively. The growth and enzyme production of various microorganisms are affected by a number of physical and chemical parameters, such as carbon, nitrogen and phosphate sources, metal ions, temperature, pH, agitation, aeration, inoculum age and size. Most -amylases are inducible enzymes.

2. Starch hydrolyzing enzymes Amylolytic enzymes (-glucanases) hydrolyze the glycosidic linkages in various -glucans. They belong to 3 families of glycoside hydrolases (GHs) [7]: GH 13 (-amylases) [8,9], GH 14 (-amylases)

A. Sharma, T. Satyanarayana / Process Biochemistry 48 (2013) 201211

203

Fig. 1. Conventional and ideal starch saccharication processes.

Their expression is induced in the presence of starch, maltose and other carbon sources, including lactose, trehalose and -methyld-glycoside. Maltose induces -amylase in Aspergillus spp., but in Aspergillus oryzae (NRC 401013) and A. oryzae (DSM 63303), both maltose and starch act as inducers [30,31]. Catabolite repression has been reported to involve glucose and other sugars. Carbon sources, such as glucose and maltose have been used in the production of -amylase, but the use of starch is ubiquitous [3234]. Industrially important enzymes have traditionally been produced in submerged fermentation, but recently, these enzymes have been produced by solid state fermentation. The combination of low molecular weight dextran with Tween-80 increases -amylase production by 2.7-fold [35]. Soybean meal, casamino acids [36], corn steep liquor [37] and meat extract [32] have all been employed for the economical production of -amylase. The level of phosphate in the medium signicantly affects growth and enzyme production [34,38]. Various metal ions, such as Ca2+ , Fe2+ , Mg2+ and K+ are added to the medium for -amylase production [32,33]. The presence of Co2+ in the production medium supports 13-fold higher biomass, but reduces the enzyme yield [39].

Traditionally, the one-variable-at-a-time approach has been used to optimize the production of -amylase [40], but it is time consuming and does not permit an understanding of the interactions among the process parameters [41]. Statistical approaches have proven to be very useful in optimizing medium components and cultural variables for maximizing enzyme titer in B. acidicola [34], Bacillus sp. KR 8104 [38] and Aspergillus awamori [42]. 5. Purication and kinetics of acid-stable -amylases A number of conventional and rapid, modern strategies used for the purication of (-amylases are listed in Table 1. The methods often used for the concentration and purication of acid-stable amylases are ammonium sulfate precipitation and ion exchange chromatography. The commercial use of -amylases does not require the enzyme to be in a puried form, but their application in the pharmaceutical and clinical sectors demand high purity. The puried enzyme is also required in studies of structurefunction relationships and biochemical properties. Some puried microbial acid-stable (-amylases and their characteristics are listed in Table 2.

Table 1 Strategies employed for purifying acid-stable amylases. Source Alicyclobacillus sp. A4 Purication strategy Ultraltration with 6 kDa (Motianmo, Tianjin, China) HiTrap SP XL column (Ion exchange) (Amersham Pharmacia, Uppsala, Sweden) Organic solvent fractionation Sephadex G-100, CM-Sephadex DEAE-Cellulose DE52 (pH 5.3) Afnity chromatography with alginic acid-CELBEDS (pH 5.0) Ammonium sulfate precipitation DEAE-Sepharose, phenyl sepharose 60% (NH4 SO4 ), DEAE Sepharose (pH 5.3) Sephadex G-75 Ammonium sulfate precipitation DEAE Sepharose fast ow Sephadex G-75 Afnity chromatography, Sepharose 6B (pH 5.5) Fold purication/% recovery 21.8 References [129]

Bacillus circulans GRS 313 Bacillus licheniformis NCIB 6346 Bacillus sp. B3 Bacillus sp. KR-8104 Bacillus sp. WN11 Bacillus sp. YX-1 Lactobacillus plantarum : data not available.

2.54 33/66 Amy I 65/13 Amy II 40.7/9.5 34

[52] [130] [131] [32] [132] [31] [133]

204 Table 2 Characteristics of acidstable microbial -amylases. Source Bacteria Alicyclobacillus sp. A4 A. acidocaldarius Bacillus acidocaldarius B. caldolyticus Bacillus circulans GRS 313 Bacillus licheniformis NH1 Bacillus stearothermophilus B. stearothermophilus US 100 Bacillus sp. Bacillus sp. KR8104 Bacillus sp. WN 11

A. Sharma, T. Satyanarayana / Process Biochemistry 48 (2013) 201211

Molecular mass (kDa) 64 160 66 70 48 58 53 59 Amy 1 76 Amy 2 53 56 53 52 76 135 48 52 54 68 50 68 41.5

pH/stability

Temperature/stability

pI

Km , Vmax , kcat

Reference

4.2 3.0 3.5 5.5 4.9 49 4.65.1 5.6 4.5 4.06.0 5.5/5.59.0 (1 h) 5.0 5.6/4.58.0 57 57 4.55.0/5.07.0 5.5 5.6 4.0 4.85.0 5.5 5.5 5.5 5.25/5.08.0 5.0

75/75 (>95% for 1 h) 75 70 70/70 (60 min) 48 90 5570 80 70 7580 7580/80 (4 h) 4050 82/9095 6570 80 70 55 115 50 50/40 (60 min) 40/60 (15 min) 50 40/60 (15 min) 50/55 (10 min) 45/35 (60 min)

4.82 3.5 4.0

0.16 mg ml1 (Km ) 11.66 mg ml1 (Km ) 68.97 U (Vmax ) 2.6 mg ml1 (Km ) 909 U mg1 (Vmax ) 0.8 g l1 (Km ) 622 s1 (Kcat ) 0.13% (Km ) 1.0 mg ml1 (Km ) 0.19 mg ml1 (Km ) 0.19 mg ml1 (Km ) 1.14 mg ml1 (Km ) [amylopectin] 2.5 mg ml1 (Km ) [starch] 7.65 mg ml1 (Km ) [amylose] 0.8 g ml1 (Km ), 622 s1 (kcat ) 0.056 mg ml1 (Km )

[129] [134] [135] [136] [52] [62] [19] [137] [51] [32] [132]

Bacillus sp. YX1 Bacillus sp. US 100 Bacillus subtilis KCC 103 Geobacillus sp. LH8 L. kononenkoae CBS 5608 Lactobacillus manihotivorans Pyrococcus furiosus Fungi Aspergillus oryzae M13 A. awamori ATCC 22342 A. chevalieri NSPRI A. hennebergi A. chevalieri NSPRI A. avus A. foetidus ATCC 10254

[33] [138] [139] [59] [140] [47] [141] [142,143] [144]

[145,146] [147] [148]

A. hennebergi Blochweitz A. oryzae A. usamii A. usamii Fusarium vasinfectum Lipomyces kononenkoae Paecilomyces sp. Thermomyces lanuginosus Trichoderma viride T. viride Yeast Cryptococcus avus Cryptococcus sp. S-2 : data not available.

50 54 54 76 69 42 84.5 66

5.5 5.0/6.08.0 4.0 3.05.0 4.45.0 5.0 4.0 5.6 5.5 5.5 6.0

50/40 (15 min) 40/55 (10 min) 65 6070 4550/50 (30 min) 70 45 65/50 (>7 h) 60 5.05.5/4.07.0 60 50 37

<3.5 (10 min) 4.2

[145] [147] [72] [142] [149] [140] [150] [151] [147] [114] [152] [153]

Several characteristics of acid-stable -amylases differ from those of neutral -amylases (Table 3).

6. Characterization of -amylases 6.1. Substrate specicity The substrate specicity of -amylases vary among microorganisms. -Amylases exhibits their highest specicity toward starch compared to other substrates, including amylose, amylopectin, cyclodextrin, glycogen and maltotriose [12].

The thermostability of these enzymes is an important characteristic and determines the primary structure of the protein. Optimal temperatures ranging between 45 and 115 C have been observed for -amylases (Table 2). A number of acid-stable -amylases have been puried and characterized from different microorganisms. It has been observed that acid-stable -amylases contain 30% less acidic and basic amino acids compared to neutral -amylases, and
Table 3 Distinguishable properties between acid-stable and neutral -amylases. Properties pH range Temperature range ( C) Molecular weight (kDa) pI Acid-stablity Thermostability ( C) Release of CNP from CNP--G3 Cleavage of G5 Number of subsite Acid-stable -amylases 3.06.0 40115 41160 3.44.8 3.55.5 6080 Suppressed by KSCN GGG() + GG 5 Neutral -amylases 6.58.0 3790 12.570 5.07.1 Unstable Unstable Stimulated by KSCN GG + GGG(/) 79

6.2. Optimal temperature and pH -Amylases display activity over a broad pH range from 2.0 to 12.0 [43]. The optimal pH of most of the -amylases falls in the acidic and neutral range [22]. The acid-stable -amylases from microorganisms are listed in Table 2.

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this feature prevents the electrostatic repulsion of charged groups at acidic pHs and contributes to the acid stability of the proteins [44]. As the nature of acid-stable -amylases depends on their isoelectric points, a pH lower than the isoelectric point signies that their basic amino acids carry a large number of positive charges, resulting in the expansion of the protein structure, which consequently affects the activity of the catalytic center [45]. Charged amino acids make up only 18% of the A. niger -amylase, suggesting the acid resistance of this enzyme [46]. Most of the known acidstable -amylases lack thermostability at elevated temperatures, which is a major constraint for their application in the starch industry. Research is in progress to isolate extremophilic microorganisms that produce enzymes with this desired property. 6.3. Molecular weight The molecular weights of microbial -amylases range from 12.5 to 160 kDa (Table 2). The acidic -amylase from A. niger has a lower molecular weight than that of acidic -amylase from other molds. Most acid-stable -amylases are high molecular weight enzymes as reported in B. stearothermophilus US 100 [16], Lactobacillus manihotivorans [47], G. thermoleovorans [48], Bacillus sp. KR8104 [38] and B. acidicola [49]. 6.4. Inhibitors Heavy metal ions, sulfhydryl group reagents, Nbromosuccinimide (NBS), p-hydroxy mercuribenzoic acid, iodoacetate, EDTA and EGTA are known to inhibit -amylases [50]. Many -amylases are also inhibited by the Hg2+ ion [51] indicating the presence of a carboxyl group in the enzyme molecules [52]. Furthermore, Hg2+ is known to oxidize indole rings and to interact with the aromatic rings present in tryptophan residues [53]. The inhibition of enzyme activity by NBS demonstrates the catalytic role of tryptophan [49]. Dithiothreitol and -mercaptoethanol are reducing agents, which suggest the role of SH groups in the catalytic activity of these enzymes. There are reports where DTT has stimulated and inhibited the activities of -amylases [54]. DTT has no effect on maltogenic -amylase from Bacillus sp. WPD616, indicating that SH groups are not involved in its catalytic activity or that this enzyme has no free, accessible SH groups [55]. The inhibition of -amylase by Woodwards reagent K (WRK) signies the involvement of acidic amino acids in the active site of the enzyme [56,57]. 6.5. Metal ion and stability of -amylase Various cations, substrates and other stabilizers inuence the thermostability of these enzymes [43]. -Amylases are metal activated enzymes that have a high afnity for the Ca2+ ion. The Ca2+ ion alters the activity and thermal stability of most -amylases, and it is known that their thermal stability is usually enhanced in the presence of the Ca2+ ion [58,59]. The number of bound Ca2+ ions varies from 1 to 10. Usually one Ca2+ ion is sufcient to stabilize the enzyme. However, the crystalline TAKA amylase A contains ten Ca2+ ions but only one is tightly bound [60]. Dialysis against EDTA can remove Ca2+ and the Ca2+ ion free enzyme can be reactivated with the addition of calcium ions. Although -amylase is known to be Ca2+ -dependent, there are reports of Ca2+ -independent acidstable -amylases that do not require Ca2+ for stability and activity [32,48,51]. 7. -Amylase gene cloning and expression -Amylase was one of the rst proteins adopted for molecular biological studies because of the existence of an easy screening

assay, the availability of amylase negative strains, the extensive knowledge of its genetics, protein production and fermentation technologies for the -amylase of B. subtilis. Sajedi et al. [61] reported the cloning of the -amylase gene (1328 bp) from Bacillus sp. KR-8104, which encodes 440 amino acids without 20 amino acids at its N- and C- termini. The -amylase gene of B. acidicola with an N- and C- terminal truncation has been recently cloned into the pET28a(+) vector and expressed in E. coli [49]. The 62 kDa recombinant -amylase is optimally active at pH 4.0 and 60 C. The gene encoding the 460 aa extracellular -amylase from Pyrococcus furiosus was also cloned in E. coli. The P. furiosus -amylase is a liquefying enzyme with a specic activity of 3900 U mg1 at 98 C. It is optimally active at pH 5.56.0 and 100 C with a T1/2 of 13 h at 98 C and does not require Ca2+ for its activity [58]. Another -amylase gene, Amy N, from B. licheniformis NH1 was also cloned, sequenced and expressed in E. coli using the pDEST17 expression system. This recombinant -amylase shows high thermostability at 85 C (60 min) compared to the native B. licheniformis NH1 -amylase (8 min) [62]. The gene encoding the acid-stable -amylase from Aspergillus niger was cloned in the pPIC9K vector and expressed in Pichia pastoris with a very high production of 2838 U ml1 . The 58 kDa recombinant -amylase is optimally active at pH 4.0 and 70 C [46]. A 1920 bp gene encoding 640 amino acids of an acid-stable -amylase was cloned from Aspergillus kawachii IF04308.

8. Structural conformation studies Circular dichroism (CD) spectroscopy and X-ray crystallography are extensively used techniques for acquiring information about protein structure and conformation. -Amylases have 3 domains. A central (/)8 TIM-barrel (named after triosephosphate isomerase, a protein fold consisting of eight -helices and eight parallel strands in an alternating pattern of -helices and -strands in a single domain), known as domain A forms the core of the molecule and contains three active site residues D231 , E261 and D328 [B. licheniformis -amylase (BLA) numbering], while domains B and C are situated at opposite sides of this TIM-barrel. The amino acid residues in the active site are strictly conserved, but a few positional changes particularly in the catalytic residues were observed when the B. stearothermophilus -amylase (BStA) was superimposed with BLA. This nding indicates the exible nature of the catalytic residues, which plays an important role in catalytic reactions. Domain B is a projection between the third strand and the third helix of the TIM barrel and forms an irregular -like structure that is possibly responsible for the differences in substrate specicity and stability between -amylases. The C-terminal portion of the sequence is present in domain C and contains a Greek key motif (a frequent beta sheet motif found in protein structures and is formed from four adjacent antiparallel beta strands). C-terminal truncation has been observed in some glycosyl hydrolases such as the -amylases from B. subtilis, Pseudomonas stutzeri and -amylase 1 in malt [63,64] while articial truncation has been performed on various amylolytic enzymes from B. subtilis, Bacillus sp. and B. stearothermophilus [6567] to study the function of the C-terminal region of -amylase. The C-terminus has been shown to be involved in the translocation of the enzyme across the outer membrane of E. coli, as reported in Alteromonas haloplanctis [68] and Bacillus sp. KR8104 [69], the binding of the enzyme to raw starch [70] and its thermal stability [66,67,70]. In contrast, Salimi et al. [71] suggested that C-terminal truncation does not affect the thermal stability, optimal pH or end products of starch hydrolysis. Some amylases contain a carbohydrate-binding module (CBM) for the hydrolysis of insoluble starch. A CBM is a noncatalytic ancillary domain of 40200 amino acids with a discrete fold that aids in the binding

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of enzyme to insoluble polysaccharide surfaces and thus improves hydrolysis [72,73]. CBM with afnity for starch are known as starch binding domains (SBDs). Approximately 10% of amylolytic enzymes possess a separate domain for binding to raw starch. Florencio et al. [74], Morlon-Guyot et al. [75] and Nakamura et al. [76] reported the presence of SBDs in three -amylases from Lactobacilli. The genes encoding the -amylases have been sequenced [77], and amino acid sequence analysis of these enzymes showed more than 96% identity. Santiago et al. [78] reported the involvement of ve-tandemmodule SBD, which act not only as distinct modules but also as a part of the whole amylase. Three steps are involved in the catalytic mechanism for retaining glycosyl hydrolases [79,80]. First, the glycosidic oxygen is protonated by the proton donor (E261 ) followed by a nucleophilic attack on the C1 of the sugar residue in subsite-1 by D231 . Once the aglycon group of the substrate leaves, a water molecule is activated presumably by the deprotonated E261 . This water molecule hydrolyzes the covalent bond between the nucleophilic oxygen and the Cl of the sugar residue in subsite-1, thereby completing the catalytic cycle [81]. The 3D structure and amino acid sequence of acidic -amylase from A. niger was described by a group at the company Novo [82]; this enzyme displayed a 3D structure that is similar to that of TAKA amylase A (TAA). Several applications involving -amylases are performed at pH that differs from the optimal pH of -amylases and therefore, there is a compelling need to change the pH performance prole of -amylases and their related enzymes. Binding and light scattering experiments using 8-anilino-1-naphthalenesulfonic acid (ANS) revealed that at an acidic pH, B. amyloliquefaciens -amylase (BAA) unfolds in such a way that its hydrophobic surface is exposed to a greater extent than the native form at a neutral pH. In addition, quenching of the intrinsic tryptophan residues in the protein molecules with acrylamide indicates that at pH 3.0, the protein is in a partially unfolded conformation with more tryptophan residues exposed to the solvent compared to the native conformation at a neutral pH. Ca2+ is required for the refolding of the molten globule state to the native form. All known -amylases contain a calcium ion, which is situated at the interface between domains A and B [82], and is required for the activity and/or stability of the enzyme. The function of the conserved Ca2+ has been suggested to be structural [83,84], as it is too distant from the active site to participate in catalysis. One or more Ca2+ ions are present in many structures, the rst calcium ion (Ca I) is strictly conserved in all distantly related -amylases and it connects domain A to domain B and helps to stabilize the active site structure and controls the formation of the extended substrate binding site. The second calcium ion (Ca II) is situated close to Ca I and in the presence of sodium ions, a CaNaCa arrangement was observed in BLA [83], BAA [85] and BStA [86]. By comparing the metal-containing and metal-free crystal structures of BLA, it was observed that the loss of metal ions causes numerous conformational changes around the metal triad and the active site, which contains 21 residues. In addition to Ca2+ , chloride ions have been shown to enhance the catalytic efciency of the enzyme. The deduced amino acid sequence of the Ca2+ -independent -amylase from Bacillus sp. KR-8104 (KRA) revealed maximum sequence homology to the BAA [85% identity and 90% similarity] and BLA [81% identity and 88% similarity] -amylases. The 3D structure of KRA shows one amino acid substitution in comparison with BLA and BAA in the region engaged in calcium binding, while at the interface of the A and B domains and around the metal triad and active site, many amino acid differences between BLA and KRA exist. The presence of chloride ions in the active sites is predominant in mammalian -amylases [84,87].

9. Protein engineering of -amylase by site directed mutagenesis Information from 3D structures, chemical modications and other studies aids in understanding the functions of various enzyme domains and offers ways to tailor the key amino acids for catalytic activity, and to improve stability under stressed conditions. Site directed mutagenesis is a technique that alters the properties of an enzyme based on its structural information. Rational protein engineering and site-directed mutagenesis studies on -amylases have been carried out to tailor glycosyl hydrolases in terms of increasing their thermostability [8890], altering their pH activity prole [81,9194], and altering their product specicity [9,95]. 9.1. Active site residues The three catalytic residues [(D231 , E261 and D328 ) in BLA] are fully conserved residues among all -amylase family enzymes in both their amino acid sequences and 3D structures [96]. Hasegawa et al. [97] studied the possible role and mechanism of the catalytic residues D193 , E219 and D294 of the P. stutzeri maltotetraose forming -amylase. D193 acts as base catalyst (nucleophile) whose side chain oxygen atom lies close to the C-1 atom of glucose-4 (Glc4), which is involved in the formation of the intermediate in the hydrolysis reaction. Residue D294 aids in tightly binding the substrate to form a twisted and distorted glucose ring at the 1 position (Glc4). The hydrogen bond between the side chain atom of E219 and the O-1 atom of Glc4 suggests the possible role of E219 as an acid catalyst (proton donor). This residue is protonated at the optimal pH for catalysis [98] (Fig. 2). The active site residues of the -amylase of B. subtilis N7 (D176 , E208 and D269 ) were mutated to their amide forms to evaluate the role of these active site residues in catalysis. Mutations resulted in a 15,000-fold decrease in the specic activity of the enzyme, with no effect on ability of the mutants to bind the substrate [99]. Vihinen et al. [100] showed that a specic mutation in the active site (D331 E) leads to an almost complete inactivation of the -amylase from B. stearothermophilus. 9.2. Thermostability Residues involved in salt-bridges, calcium binding or potential deamidation processes were reported to affect the thermostability of the protein and were subjected to site-directed mutagenesis, based on informational suppression. The mutations responsible for altering the thermostability are mainly present in domain B and its interface with domain A, where the triadic metal site (CaNaCa) and substrate binding site are located [90]. The triadic metal site is highly sensitive to any modication that can change the network of electrostatic interactions that entrap the metal ions. Priyadharshini et al. [101] also reported that the region around the calcium binding site of BLA is sensitive to any modication. The amino acid residues encoding the calcium-binding site (N104 , D161 , D183 , D200 and D430 ), which extends from residues 104 to 200 in the loop regions of domain B and D430 in domain C, were changed to D104 and N161 , N183 , N200 and N430 by site-directed mutagenesis and the resultant mutant amylase showed improved specic activity at pH 5.0 and 70 C and decreased activity at 30 C. However, the stability of the amylase was unaffected by the mutations N104 D and D430 N. Another mutation, D161 N, greatly decreased the thermostability of amylase. In another study, site-directed mutagenesis was used to enhance the thermal stability and calcium independence of a mesophilic -amylase from Bacillus megaterium WHO. Mutations (A53 S and H58 I) in the calcium-binding site affected the -amylase. The A53 S mutant showed that the enhanced calcium binding is most likely responsible for the increased stability of the enzyme.

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Fig. 2. Scheme of a possible mechanism for retaining glycosyl hydrolases and the roles of the catalytic residues in the -amylase family of enzymes (residue numbering of G4-amylase). (Adapted from Hasegawa et al. [97]).

In contrast, a calcium-independent mutant (H58 I) possessed high thermostability. Mutation of H238 D in the B. stearothermophilus amylase, which is involved in Ca2+ and substrate binding, resulted in decreased specic enzyme activity and thermal stability. However, Ca2+ had a greater effect on the catalytic efciency, kcat /Km , and half-life (at 60 C) of the A53 S mutant [102]. Structure-based mutagenesis of BLA revealed the signicance of seven residues (D121 , N126 , D164 , N192 , D200 , D204 and A269 ), which were sensitive to any amino acid substitutions. Replacement of three asparagine residues (N172 , N188 and N190 ) signicantly enhanced the thermostability of the mutant. The substitution of phenylalanine to asparagine at position 190, led to a 6 fold increase in the half-life of enzyme at 80 C [90]. Similarly, the mutations of H133 I and A209 V were the most favorable substitutions for enhancing the thermostability of -amylase [88]. Salt bridge residues D121 R127 also contributed to increasing the thermostability of BLA compared to other homologous bacterial -amylases. In a protein, hydrophobic residues are unfavorable at solventexposed sites, and amino acid substitutions on the surface have less of an impact on protein thermostability. Interestingly, hyperthermostable variants of BLA were obtained by incorporating hydrophobic residues at the surface [103]. There are several reasons why the protein is stabilized at high temperatures including increased hydrophobic packing (generally in cavities and surface indentations), aromaticaromatic interactions on the surface of the protein, stabilization of the intrinsic metal binding site and sheets, an extended network of salt bridges and hydrogen bonds, and the replacement of the residues responsible for irreversible chemical alterations of the protein structure. 9.3. Determinants of the pH activity prole A string of substitutions have been made in BLA to stabilize the protein and improve its performance at acidic pHs [8890,93,94,103105]. Site-directed mutagenesis and saturated mutagenesis have been employed to tailoring the optimal pH of a number of enzymes, including the -amylase from B. licheniformis [106] and soybean -amylase [107], but the catalytic rate of these enzymes was affected. Liu et al. [104] described the relationships between acid resistance and the structural features of different mutants of BLA with changes at two crucial residues, L134 and S320 (Fig. 3). The combined effect of the double mutant L134 R/S320 A indicated that the amino acids inserted at each site contribute independently to the overall stability of the protein, as is generally seen for stabilizing mutations in protein structures [108111]. Changes in the electrostatic eld due to charged groups play a signicant role in determining the stability of BLA in strongly

acidic environments. However, Neilson et al. [105] reported a signicant change in the pH activity prole of -amylase of Bacillus Ba2 between neutralneutral mutations and neutral-charged mutations. It was concluded that the dynamic aspects of the active site other than electrostatics are most likely responsible for the pH activity proles of -amylases. Mutations around the catalytic nucleophile in KRA that decreased its pKa value are as follows: D188 R, D246 K, D266 N, N280 K, and Q291 H (increased positive or decreased negative charges) and A52 S, V154 N, I212 T, Y265 N, T297 S, and A379 S (decreased hydrophobic effects) which may be responsible for the shift in its pH activity prole [61]. 10. Alteration of other properties of -amylases by directed evolution Naturally occurring enzymes are wonderful biocatalysts with abundant potential applications in industry and medicine. To be compatible with the specic requirements for an application, the catalytic properties of the enzyme must be tailored. Directed evolution mimics Darwinian evolution and has emerged as a powerful tool for engineering enzymes with new or improved functions. This technique can be used to modify various enzyme properties, such as activity, selectivity, substrate specicity, stability and solubility

Fig. 3. The position of the point mutation in the wild type -amylase gene from B. licheniformis CICC 10181. Domain A, blue; domain B, green; domain C, gray. The active site residues D231 , E261 , D328 are shown in red. The position of the point mutation is shown in yellow. (Adapted from Liu et al. [104]).

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[112]. Various strategies are used for directed evolution, including error-prone PCR, DNA shufing, staggered extension, random priming recombination, heteroduplex recombination, random chimera genesis on transient templates, recombinant extension on truncated templates, incremental truncation for the creation of hybrid enzymes, degenerate oligonucleotide gene shufing, random drift mutagenesis, sequence saturation mutagenesis and nucleotide excision and exchange technology [113]. The genetargeted mutants of extremely thermoacidophilic archaea are a major challenge, but some progress has been made in this area. The mutant of Sulfolobus solfataricus 98/2 termed S. solfataricus PBL 2025, lacks approximately 50 genes including lacS [114]. The inability of this mutant to grow on lactose based minimal media provides a selectable marker [115]. In addition, a decient mutant of S. solfataricus was used to study the function and regulation of -amylase [116]. At present, the starch industry has become the largest market for enzymes after the detergent industry. The properties of starch and -glucan acting enzymes have been altered by directed evolution because the naturally occurring enzymes from hyperthermophilic bacteria and archaea are unt for industrial applications or have restricted shelf lives. Richardson et al. [117] found two robust chimeric -amylases with superior properties suited for industrial applications using DNA shufing by high throughput screening. Directed evolution coupled with a high throughput robotic screen was employed to broaden the industrial use of the maltogenic -amylase (Novamyl) from Bacillus sp. Error prone PCR (epPCR) and DNA shufing with high throughput robotic screening was employed to improve the performance of this enzyme in baking. The promising variants showed a 10 C increase in their thermal stability at pH 4.5 [118].

13. Applications of -amylases Presently, amylases have the major world market share for enzymes [123]. Many amylase preparations are available from various enzyme manufacturers for specic applications, such as starch saccharication, baking, bioethanol production, textile desizing, in laundry and dish-washing detergents [22,124127]. Bacterial amylases are generally preferred over fungal amylases due to the characteristic advantages that the former offers [25,128]. Acidstable -amylases are preferred, as their application minimizes contamination risk. The acid-stable and Ca2+ -independent -amylases are preferred over the currently used enzymes in starch processing because the latter are active at 95 C and pH 6.8 and stabilized by Ca2+ . The process cannot be performed at low pH (3.24.5), the pH of the native starch [28]. The stress is, therefore, on extremozymes from extremophiles that are naturally endowed with the properties needed for specic industrial applications [29]. To prevent the staling of bread and other baked goods and to improve their texture and shelf-life, the dough is supplemented with -amylase. The bacterial maltogenic and acidic -amylases with intermediate thermostability have been reported to act as antistaling agents, thereby reducing the crumb rmness during storage and improving the volume, texture, avor and shelf-life of bread by shortening the amylopectin chain length due to the formation of malto-oligosaccharides (DP 2-12) [124]. 14. Conclusions -Amylase is one of the most important enzymes employed in the starch processing industry for the production of starch hydrolysates. The pH of native starch ranges between 3.2 and 4.5, and therefore, thermostable acidic and Ca2+ -independent amylases are better suited for conversion of starch to various sugar syrups. Acidic -amylases are produced by bacteria, archaea and fungi. -Amylases are also used in the removal of starch from beer, fruit juices, textiles and porcelain. Maltogenic amylase is used as an antistaling agent to prevent the retrogradation of starch in bakery products. Food and starch-based industries are the major markets for their application of -amylases, but they are now gaining importance in biopharmaceutical applications, and their demand of these enzymes is expected to rise in the future. Acknowledgements Authors gratefully acknowledge nancial assistance from the Ministry of Environment and Forests (MoEF) and Council of Scientic and Industrial Research (CSIR), Government of India, New Delhi while writing this review. References

11. Correlation between the molecular dynamics of active site residues and the pH activity prole The conformational dynamics of protein molecules are programmed into their structures and are a signicant element of their function. Alteration of the pKa values of catalytic residues affects the pH-prole of the enzyme. There are a number of microenvironmental determinants of the pKa of a titratable group in proteins such as electrostatic effects [104], hydrophobic effects (desolvation effects) [61], hydrogen binding, helix dipole interactions, as well as the dynamic aspects of the active site [105], which can be changed to inuence the pKa. BLA catalyzes the enzyme substrate reaction at a low pH by protonating the nucleophile (D231 ) and at a high pH by deprotonating hydrogen donor (E261 ); the correlation between the activity of the enzyme and the pH is determined by the pKa values of these two active site groups [119].

12. Computational modeling The availability of basic information on a protein aids in improving the properties of the protein by computational modeling. A number of computer programs have been employed to model amylase including MODELLER 9 VB [120], Frodo, Hydra (Polygen Waltham, Mass.), Insight (Biosym Technologies, San Diego, Calif.), Gromos (Biomos b.v., Groningen, The Netherlands). For validation of the model, PROCHECK [http://nihserver.mbi.ucla.edu/SAVES/ Info.php], WHAATIF [https://swift.cmbi.ru.nl/servers/html/index. html], ProSA [http://prosa.services.came.sbg.ac.at] and RAMPAGE [http://mordred.bioc.cam.ac.uk/] have been employed and SwissPdbViewer ver3.7 [121] and MOLMOL [122] programs have been used to analyze and compare 3D structures.

[1] Buleon A, Colonna P, Planchot V, Ball S. Starch granules: structure and biosynthesis. Int J Biol Macromol 1988;23:85112. [2] Tester RF, Karkalas J, Qi X. Starch structure and digestibility, enzymesubstrate relationship. Worlds Poult Sci J 2004;60:18695. [3] Jansen B, Olsen J. Amylases and their industrial potential. In: Johri BN, Satyanarayana T, Olsen J, editors. Thermophilic moulds in Biotechnology. Netherlands: Kluwer Academic Publishers; 1999. p. 11537. [4] Pandey A, Ramachandran S. General introduction. In: Pandey A, Webb C, Soccol CR, Larroche C, editors. Enzyme technology. New Delhi: Asiatech Publishers; 2005. p. 110. [5] Sanchez S, Demain AL. Enzymes and bioconversions of industrial, pharmaceutical and biotechnological signicance. Org Process Res Dev 2010;15:22430. [6] Futterer O, Angelov A, Liesegang H, Gottschalk G, Schleper C, Schepers B, Dock C, Ghollasi M, Khajeh K, Naderi-Manesh H, Ghasemi A. Engineering of a Bacillus -amylase with improved thermostability and calcium independency. Appl Biochem Biotechnol 2010;162:44459. [7] Henrissat B. A classication of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 1991;280:30916.

A. Sharma, T. Satyanarayana / Process Biochemistry 48 (2013) 201211 [8] MacGregor EA, Janecek S, Svensson B. Relationship of sequence and structure to specicity in the a-amylase family of enzymes. Biochim Biophys Acta 2001;1546:120. [9] Kuriki T, Takata H, Yanase M, Ohdan K, Fujii K, Terada Y, Takaha T, Hondoh H, Matsuura Y, Imanaka T. The concept of the a-amylase family: a rational tool for interconverting glucanohydrolases/glucanotransferases, and their specicities. J Appl Glycosci 2006;53:15561. [10] Pujadas G, Ramirez FM, Valero R, Palau J. Evolution of b-amylase: patterns of variation and conservation in subfamily sequences in relation to parsimony mechanisms. Proteins 1996;25:45672. [11] Coutinho PM, Reilly PJ. Glucoamylase structural, functional, and evolutionary relationships. Proteins 1997;29:33447. [12] Antranikian G. Microbial degradation of starch. In: Winkelmann G, editor. Microbial degradation of natural products. Weinheim, Germany: VCH; 1992. p. 2756. [13] Sharma A, Satyanarayana T. High maltose-forming, Ca2+ -independent and acidstable -amylase from a novel acidophilic bacterium Bacillus acidicola TSAS1. Biotechnol Lett 2010;32:15037. [14] Fogarty WM, Bourke AC, Kelly CT, Doyle EM. A constitutive maltotetraoseproducing amylase from Pseudomonas sp. IMD 353. Appl Microbiol Biotechnol 1994;42:198203. [15] Yoshigi N, Chikano T, Kamimura M. Characterisation of maltopentaose producing bacterium and its cultural conditions. Agric Biol Chem 1985;49:237984. [16] Ali MB, Mhiri S, Mezghani M, Bejar S. Purication and sequence analysis of the a typical maltohexaose-forming a-amylase of the B. stearothermophilus US100. Enz Microb Technol 2001;28:53742. [17] Brena BM, Pazos C, Franco-Fraguas L, Batista-Viera F. Chromatographic methods for amylases. J Chromatogr B Biomed Appl 1996;684:21737. [18] Imen BA, Urdaci MC, Ali MB, Denayrolles M, Chaignepain S, Limam F, Bejar S, Marzouki MN. Structural investigation and homology modeling studies of native and truncated forms of -amylases from Sclerotinia sclerotiorum. J Microbiol Biotechnol 2009;19(11):130618. [19] Manning BG, Campbell LL. Thermostable -amylase of Bacillus stearothermophilus. J Biol Chem 1961;236:29525. [20] Freer SN. Purication and characterization of the extracellular -amylase from Streptococcus bovis JB1. App Environ Microbiol 1993;59:1398402. [21] Aquino ACMM, Jorge JA, Terenzi HF, Polizeli MLTM. Studies on a thermostable -amylase from the thermophilic fungus Scytalidium thermophilum. Appl Microbiol Biotechnol 2003;61:3238. [22] Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. Advances in microbial amylases. Biotechnol Appl Biochem 2000;31:13552. [23] Naja MF, Deobagkar DN, Deobagkar DD. Purication and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20. Protein Expr Purif 2005;41(2):34954. [24] Robyt J, Ackerman RJ. Isolation, purication, and characterization of a maltotetraose-producing amylase from Pseudomonas stutzeri. Arch Biochem Biophys 1971;145:10514. [25] Malhotra R, Noorwez SM, Satyanarayana T. Production and partial characterization of thermostable and calciumindependent -amylase of an extreme thermophile Bacillus thermooleovorans NP 54. Lett Appl Microbiol 2000;31:37884. [26] Martin ML, Hoseney RC. A mechanism of bread rming. II: role of starch hydrolyzing enzymes. Cereal Chem 1991;68:5037. [27] Fogarty WM, Collins BS, Doyle EM, Kelly CT. The high maltose-forming -amylase of Saccharomonospora viridis: mechanisms of action. J Indust Microbiol 1993;11:199204. [28] Shivaramakrishnan S, Gangadharan D, Nampoothiri KM, Soccol CR, Pandey A. -Amylases from microbial sources an overview on recent developments. Food Technol Biotechnol 2006;44:17384. [29] Satyanarayana T, Rao JLUM, Ezhilvannan M. -Amylases. In: Pandey A, Webb C, Soccol CA, Larroche C, editors. Enzyme technology. New Delhi: Asiatech Publishers; 2005. p. 189220. [30] Eratt JA, Douglas PE, Moranelli F, Seligy VL. The induction of -amylase by starch in Aspergillus oryzae: evidence for controlled mRNA expression. Can J Biochem Cell Biol 1984;62:67890. [31] Canganella F, Andrae CM, Antranikian C. Characterization of amylolytic and pullulytic activities from thermophilic archaea and from a new Fervidobacterium species. Appl Microbiol Biotechnol 1994;42:23945. [32] Sajedi RH, Naderi-Mahesh H, Khajeh K, Ahmadvand R, Ranjbar BA, Asoodeh A, Moradian F. A calcium independent -amylase that is active and stable at low pH from the Bacillus sp. KR-8104. Enz Microb Technol 2005;36: 66671. [33] Liu XD, Xu Y. A novel raw starch digesting -amylase from a newly isolated Bacillus sp. YX-1: purication and characterization. Biores Technol 2008;99:431520. [34] Sharma A, Satyanarayana T. Optimization of medium components and cultural variables for enhanced production of acidic high maltose-forming and Ca2+ -independent -amylase by Bacillus acidicola. J Biosci Bioeng 2011;111:5503. [35] Arnesen S, Eriksen SH, Olsen J, Jensen B. Increased production of -amylase from Thermomyces lanuginosus by the addition of Tween 80. Enz Microb Technol 1998;23:24952. [36] Ueno S, Miyama M, Ohashi Y, Izumiya M, Kusaka I. Secretary enzyme production and conidiation of Aspergillus oryzae in submerged liquid culture. Appl Microbiol Biotechnol 1987;26:2736.

209

[37] Shah SH, Wainwright SJ, Merrett MJ. The interaction of sodium and calcium chloride and light on growth potassium nutrition and proline accumulation in callus cultures of Medicago sativa L. New Phytol 1990;116: 3745. [38] Hashemi M, Razavi SHJ, Shojaosadati SA, Mousavi SM, Khajeh K, Safari M. Development of a solid-state fermentation process for production of an alpha amylase with potentially interesting properties. J Biosci Bioeng 2010;110(3):3337. [39] McMahon HEM, Kelly CT, Fogarty WM. Effect of growth rate on amylase production by Streptomyces sp, IMD 2679. Appl Microbiol Biotechnol 1997;48:5049. [40] Pham PL, Taillandier P, Delmas M, Strehaiano P. Optimization of the culture medium for xylanase production by Bacillus sp. using statistical experimental designs. World J Microbiol Biotechnol 1998;14:18590. [41] Wenster-Botz D. Experimental design for fermentation media development: statistical design or global random search? J Biosci Bioeng 2000;90: 47383. [42] Prakasham RS, Subba Rao C, Rao SR, Sarma PN. Enhancement of acid amylase production by an isolated Aspergillus awamori. J Appl Microbiol 2007;102:20411. [43] Vihinen M, Mantsala P. Microbial amylolytic enzymes. Cri Rev Biochem Mol Biol 1989;24:329418. [44] Schwermann B, Pfau K, Liliensiek B, Schleyer M, Fischer T, Bakker EP. Purication, properties and structural aspects of a thermoacidophilic -amylase from Alicyclobacillus acidocaldarius ATCC 27009. Insight into acidostability of proteins. Eur J Biochem 1994;226:98191. [45] Liping Z, Yan X, Jianzhong J. The application and study of acid -amylase. Liquor Making 2002;29(3):1922. [46] Zeng Q, Wei C, Jin J, Wu C, Huang B. Cloning of the gene encoding acid-stable alpha-amylase from Aspergillus niger and its expression in Pichia pastoris. Afr J Food Sci 2011;5:66875. [47] Aguilar G, Morlon-Guyot J, Trejo-Aguilar B, Guyot JP. Purication and characterization of an extracellular -amylase produced by Lactobacillus manihotivorans LMG 18010T, an amylolytic lactic acid bacterium. Enz Microb Technol 2000;27:40613. [48] Mehta M, Satyanarayana T. Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolyzing -amylase from an extreme thermophile Geobacillus thermoleovorans. J Mol Catal B: Enzym 2013;8586:22938. [49] Sharma A, Satyanarayana T. Cloning and expression of acidstable, high maltose-forming, Ca2+ -independent a-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis. Extremophiles 2012;16:51522. [50] Hamilton LM, Kelly CT, Fogarty WM. Production and properties of the raw starch-digesting -amylase of Bacillus sp. IMD 435. Proc Biochem 1999;35:2731. [51] Asoodeh A, Chamani J, Lagzian M. A novel thermostable, acidophilic a-amylase from a new thermophilic Bacillus sp. Ferdowsicous isolated from Ferdows hot mineral spring in Iran: purication and biochemical characterization. Int J Biol Macromol 2010;46:28997. [52] Dey G, Palit S, Banerjee R, Maiti BR. Purication and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313. J Indust Microbiol Biotechnol 2002;28:193200. [53] Liu W, Shi P, Chen Q, yang P, wang G, Wang Y, Luo H, Yao B. Gene cloning, overexpression, and characterization of a xylanase from Penicillium sp. CGMCC 1699. Appl Biochem Biotechnol 2010;162:112. [54] Ballschmiter M, Futterer O, Liebl W. Identication and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl Environ Microbiol 2006;72:220611. [55] Liu B, Wang Y, Zhang X. Characterization of a recombinant maltogenic amylase from deep sea thermophilic Bacillus sp. WPD616. Enz Microb Technol 2006;39:80510. [56] Paoli P, Fiaschi T, Cirri P, Camici G, Manao G, Cappugi G, Raugei G, Moneti G, Ramponi G. Mechanism of acylphosphatase inactivation by Woodwards reagent K. Biochem J 1997;328:85561. [57] Chauthaiwale J, Rao M. Chemical modication of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group. Biochim Biophys Acta 1994;1204:1648. [58] Dong G, Vieille C, Savchenko A, Zeikus JG. Cloning, sequencing, and expression of the gene encoding extracellular -amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl Environ Microbiol 1997;63:356976. [59] Khajeh K, Hosseinkhani S, Dabirmanesh B. Purication and characterization of a thermostable phytate resistant -amylase from Geobacillus sp. LH8. Int J Biol Macromol 2010;46:2736. [60] Oikawa A, Maeda A. The role of calcium in Taka amylase A. II. The exchange reaction. Can J Biochem 1957;46:46368. [61] Sajedi RH, Taghdir M, Naderi-Manesh H, Khajeh K, Ranjbar B. Nucleotide sequence, structural investigation and homology modeling studies of a Ca2+ -independent -amylase with acidic pH-prole. J Biochem Mol Biol 2007;40:31524. [62] Hmidet N, Bayoudh A, Berrin JG, Kanoun S, Juge N, Nasri M. Purication and biochemical characterization of a novel -amylase from Bacillus licheniformis NH1 Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli. Proc Biochem 2008;43:499510.

210

A. Sharma, T. Satyanarayana / Process Biochemistry 48 (2013) 201211 [90] Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C. Probing structural determinants specifying high thermostability in Bacillus licheniformis -amylase. J Mol Biol 2000;301:104157. [91] Igarashi K, Hatada Y, Ikawa K, Araki H, Ozawa T, Kobayashi T, Ozaki K, Ito S. Improved thermostability of a Bacillus a-amylase by deletion of an arginineglycine residue is caused by enhanced calcium binding. Biochem Biophys Res Commun 1998;248:3727. [92] Igrashi K, Hatada Y, Hagihara H, Saeki K, Takaiwa M, Uemura T, Ara K, Ozaki K, Kawai S, Kobayashi T, Ito S. Enzymatic properties of a novel liquefying a amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences. Appl Environ Microbiol 1998;64:32829. [93] Nielsen JE, Borchert TV. Protein engineering of bacterial -amylases. Biochim Biophys Acta 2000;1543:25374. [94] Shaw A, Bott R, Day AG. Protein engineering of aamylase for low pH performance. Curr Opin Biotechnol 1999;10:34952. [95] Rivera MH, Lopez-Munguia A, Soberon X, Saab-Rincom G. Alpha-amylase from Bacillus licheniformis mutants near to the catalytic site: effects on hydrolytic and transglycosylation activity. Protein Eng 2003;16:50514. [96] Katsuya Y, Mezaki Y, Kubota M, Matsuura Y. Three-dimensional structure of resolution. J Mol Biol 1998;281:88597. Pseudomonas isoamylase at 2.2 A [97] Hasegawa K, Kubota M, Matsuura Y. Roles of catalytic residues in -amylase as evidenced by the structures of the product-complexed mutants of a maltotetraose-forming amylase. Protein Eng 1999;12:81924. [98] Kusunoki M Y, Harada W, Kakudo M. Structure and possible catalytic residues of taka-amylase A. J Biochem 1984;95:697702. [99] Takase K, Matsumoto T, Mizuno H, Yamane K. Site-directed mutagenesis of active site residuesin Bacillus subtilis -amylase. Biochim Biophys Acta 1992;1120:2818. [100] Vihinen M, Ollikka P, Niskanen J, Meyer P, Suominen I, Karp M, Holm L, Knowles J, Mantsala P. Site-directed mutagenesis of a thermostable amylase from Bacillus stearothermophilus: putative role of three conserved residues. J Biochem 1990;107:26772. [101] Priyadharshini R, Gunasekaran P. Site-directed mutagenesis of the calciumbinding site of a-amylase of Bacillus licheniformis. Biotechnol Lett 2007;29:14939. [102] Ghollasi M, Khajeh K, Naderi-Manesh H, Ghasemi A. Engineering of a Bacillus -amylase with improved thermostability and calcium independency. Appl Biochem Biotechnol 2010;162:44459. [103] Machius M, Declerck N, Huber R, Wiegand G. Kinetic stabilization of Bacillus licheniformis -amylase through introduction of hydrophobic residues at the surface. J Biol Chem 2003;278:1154653. [104] Liu Y-H, Lu F-P, Li Y, Wang JL, Gao C. Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations. Appl Microbiol Biotechnol 2008;80:795803. [105] Nielsen JE, Borchert TV, Vriend G. The determinants of -amylase pH activity proles. Protein Eng 2001;14:50512. [106] Verhaert RM, Beekwilder J, Olsthoorn R, Van DJ, Quax WJ. Phage display selects for amylases with improved low pH starch binding. J Biotechnol 2002;96:10318. [107] Hirata A, Adachi M, Sekine A, Kang YN, Utsumi S, Mikami B. Structural and enzymatic analysis of soybean beta amylase mutants with increased pH optimum. J Biol Chem 2004;279:728795. [108] Matsumura M, Yasumura S, Aiba S. Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein. Nature 1986;323:3568. [109] Matsumura M, Signor G, Matthews BW. Substantial increase of protein stability by multiple disulphide bonds. Nature 1989;342:2913. [110] Pantoliano MW, Whitlow M, Wood JF, Dodd SW, Hardman KD, Rollence ML, Bryan PN. Large increases in general stability for subtilisin BPN through incremental changes in the free energy of unfolding. Biochemistry 1989;28:720513. [111] Serrano L, Day AG, Fersht AR. Step-wise mutation of barnase to binase: a procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. J Mol Biol 1993;233:30512. [112] Rubin-Pitel SB, Zhao H. Recent advances in biocatalysis by directed enzyme evolution. Comb Chem High Throughput Screen 2006;9:24757. [113] Sen S, Dasu VV, Mandal B. Development in directed evolution for improving enzyme functions. Appl Biochem Biotechnol 2007;143:21223. [114] Schelert J, Dixit V, Hoang V, Simbahan J, Drozda M, Blum P. Occurence and characterizaton of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of gene disruption. J Bacteriol 2004;186: 42737. [115] Albers SV, Driessen AJ. Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome. Archaea 2007;2:1459. [116] Worthington P, Hoang V, Perez-Pomares F, Blum P. Targeted disruption of the -amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus. J Bacteriol 2003;185:4828. [117] Richardson TH, Tan X, Frey G, Callen W, Cabell M, Lam D, Macomber J, Short JM, Robertson DE, Miller C. A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable alpha amylase. J Biol Chem 2002;277:265017. [118] Jones A, Lamsa M, Frandsen TP, Spendler T, Harris P, Sloma A, Xu F, Nielson JB, Cherry JR. Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25. J Biotechnol 2008;134:32533. [119] Kyte J. Mechanism in protein chemistry. New York: Garland Publishing; 1995. p. 25883.

[63] Yamane K, Hirata Y, Furusato T, Yamazaki H, Nakayama A. Changes in the properties and molecular weights of Bacillus subtilis M-type and N-type amylases resulting from a spontaneous deletion. J Biochem 1984;96:184958. [64] Nakada T, Kubota M, Sakai S, Tsujisaka Y. Purication and characterization of two forms of maltotetraoseforming amylase from Pseudomonas stutzeri. J Agric Biol Chem 1990;54:73743. [65] Lin LL, Hsu WH, Chu WS. A gene encoding for an -amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli. J Appl Microbiol 1997;82:32534. [66] Marco JL, Bataus LA, Valencia FF, Ulhoa CJ, Astol-Filho S, Felix CR. Purication and characterization of a truncated Bacillus subtilis -amylase produced by Escherichia coli. Appl Microbiol Biotechnol 1996;44:74652. [67] Vihinen M, Peltonen T, Iitia A, Suominen I, Mantsala P. C-Terminal truncations of a thermostable Bacillus stearothermophilus -amylase. Protein Eng 1994;7:12559. [68] Feller G, DAmico S, Benotmane AM, Joly F, Van Beeumen J, Gerday C. Characterization of the C-terminal propeptide involved in bacterial wall spanning of -amylase from the psychrophile Alteromonas haloplanctis. J Biol Chem 1998;273:1210915. [69] Ali S, Youse F, Ghollasi M, Daneshjou S, Tavoli H, Ghobadi S, Khajeh K. Investigations on possible roles of C-terminal propeptide of a Ca-independent -amylase from Bacillus. J Microbiol Biotechnol 2012;22(8):107783. [70] Rodriguez Sanoja R, Morlon-Guyot J, Jore J, Pintado J, Juge N, Guyot JP. Comparative characterization of complete and truncated forms of Lactobacillus amylovorus -amylase and role of the C-terminal direct repeats in raw-starch binding. Appl Environ Microbiol 2000;66:33506. [71] Salimi A, Youse F, Ghollasi M, Daneshjou S, Tavoli H, Ghobadi S, Khajeh K. Investigations on possible roles of C-terminal propeptide of a Ca-independent -amylase from Bacillus. J Microbiol Biotechnol 2012;22(8):107783. [72] Suganuma T, Fujita K, Kitahara K. Some distinguishable properties between acid-stable and neutral types of -amylases from acid-producing koji. J Biosci Bioengi 2007;104(5):35362. [73] Guillen D, Santiago M, Linares L, Perez R, Morlon J, Ruiz B, Sanchez S, Rodrguez-Sanoja R. Alpha-amylase starch binding domains: cooperative effects of binding to starch granules of multiple tandemly arranged domains. Appl Environ Microbiol 2007;73:38337. [74] Florencio JA, Eiras-Stofella DR, Soccol CR, Raimbault M, Guyot JP, Fontana JD. Lactobacillus plantarum amylase acting on crude starch granules. Native isoforms and activity changes after limited proteolysis. Appl Biochem Biotechnol 2000;8486:72130. [75] Morlon-Guyot J, Mucciolo-Roux F, Rodrguez-Sanoja R, Guyot JP. Characterization of the L. manihotivorans -amylase gene. DNA Seq 2001;12:2737. [76] Nakamura LK, Crowell CD. Lactobacillus amylovorus, a new starchhydrolysing species from swine waste-corn fermentation. Dev Ind Microbiol 1979;20:53540. [77] Giraud E, Cuny G. Molecular characterization of the -amylase genes of Lactobacillus plantarum A6 and Lactobacillus amylovorus reveals an unusual 39-end structure with direct tandem repeats and suggests a common evolutionary origin. Gene 1997;198:14957. [78] Santiago M, Linares L, Sanchez S, Rodrguez-Sanoja R. Functional 9 characteristics of the starch binding domain of Lactobacillus amylovorus -amylase. Biol Bratis 2005;60:1114. [79] Sinnott ML. Catalytic mechanisms of enzymic glycosyl transfer. Chem Rev 1990;90:1171202. [80] Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure 1995;3:8539. [81] Nielsen JE, Beier L, Otzen D, Borchert TV, Frantzen HB, Andersen KV, Svendsen A. Electrostatics in the active site of an alpha-amylase. Eur J Biochem 1999;264:81624. [82] Boel E, Brady L, Brzozowski AM, Derewenda Z, Dodson GG, Jensen VJ, Petersen SB, Swift H, Thim L, Woldike HF. Calcium binding in -amylases: an X-ray resolution of two enzymes from Aspergillus. Biodiffraction study at 2.1-A chemistry 1990;29:62449. [83] Machius M, Declerck N, Humber R, Wiegand G. Activation of Bacillus licheniformis -amylase through a disorder order transition of the substrate binding site mediated by a calcium-sodium-calcium metal traid. Structure 1998;6:28192. [84] Larson SB, Greenwood A, Cascio D, McPherson DJ. Rened molecular struc resolution. J Mol Biol 1994;235: ture of pig pancreatic -amylase at 2.1 A 156084. [85] Brzozowski AM, Lawson DM, Turkenburg JP, Bisgaard-Frantzen H, Svendsen A, Borchert TV, Dauter Z, Wilson KS, Davies GJ. Structural analysis of native and ligand complexes. Biochem 2000;39:9099107. [86] Suvd D, Fujimoto Z, Takase K, Matsumura M, Mizuno H. Crystal structure of Bacillus stearothermophilus -amylase: possible factors determining the thermostability. J Biochem (Tokyo) 2001;129:4618. [87] Ramasubbu N, Paloth V, Luo Y, Barayer GD, Levine MJ. Structure of human resolution: implications for its role in the role salivary -amylase at 1.6 A cavity. Acta Crystallogr 1996;52:43546. [88] Declerck N, Joyet P, Trosset JY, Garnier J, Gaillardin C. Hyperthermostable mutants of Bacillus licheniformis a-amylase: multiple amino acid replacements and molecular modeling. Protein Eng 1995;8: 102937. [89] Declerck N, Machius M, Chambert R, Wiegand G, Huber R, Gaillardin C. Hyperthermostable mutants of Bacillus licheniformis a-amylase: thermodynamic studies and structural interpretation. Protein Eng 1997;10:5419.

A. Sharma, T. Satyanarayana / Process Biochemistry 48 (2013) 201211 [120] Sali A, Potterton L, Yuan F, Van Vlijmen H, Karplus M. Evaluation of comparative protein modeling by MODELLER. Proteins 1995;23:31826. [121] Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997;18:271423. [122] Koradi R, Billeter M, Wuthrich KMOLMOL. A program for display and analysis of macromolecular structures. J Mol Graph 1996;14:515. [123] Aehle W, Misset O. Enzymes for industrial applications. In: Rehm HJ, Reed G, editors. Biotechnology. 2nd ed. Germany: WileyVCH; 1999. p. 189216. [124] Nigam P, Singh D. Enzyme and microbial systems involved in starch processing. Enz Microb Technol 1995;17:7708. [125] Van der Maarel MJEC, Van der Veen B, Uitdehaag JCM, Leemhuis H, Dijhuizen L. Properties and applications of starch-converting enzymes of the -amylase family. J Biotechnol 2002;94:13755. [126] Van Dam HW, Hille JDR. Yeast and enzymes in bread making. Cereal Foods World 1992;37(2):24552. [127] Rao JLUM, Satyanarayana T. Improving production of hyperthermostable and high maltose-forming -amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications. Biores Technol 2007;98:34552. [128] Babu KR, Satyanarayana T. -Amylase production by thermophilic Bacillus coagulans in solid state fermentation. Proc Biochem 1994;30:3059. [129] Bai Y, Huang H, Meng K, Shi P, Yang P, Luo H, Luo C, Feng Y, Zhang W, Yao B. Identication of an acidic a-amylase from Alicyclobacillus sp. A4 and assessment of its application in the starch industry. Food Chem 2012;131: 14738. [130] Morgan FJ, Priest FG. Characterization of a thermostable -amylase from Bacillus licheniformis NCIB 6346. J Appl Bacteriol 1981;50:10714. [131] Amritkar N, Kamat M, Lali A. Expanded bed afnity purication of bacterial amylase and cellulose on composite substrate analoguecellulose matrices. Process Biochem 2004;39:56570. [132] Mamo G, Gessesse A. Purication and characterization of two raw-starchdigesting thermostable a-amylases from a thermophilic Bacillus. Enz Microb Technol 1999;25:4338. [133] Sanoja RR, Morlon-Guyot J, Jore J, Pintado J, Juge N, Guyot JP. Comparative characterization of complete and truncated forms of Lactobacillus amylovorus -amylase and role of the C-terminal direct repeats in raw-starch binding. Appl Environ Microbiol 2000;66(8):33506. [134] Matzke J, Schwermann B, Baker EP. Acidostable and acidophilic proteins: the example of the -amylase from Alicyclobacillus acidocaldarius. Comp Biochem Physiol 1997;118A:4759. [135] Kanno M. -Amylase production by Bacillus acidocaldarius, Bacillus stearothermophilus and their d-cycloserine resistant mutants. Agric Biol Chem 1986;50:26335. [136] Koch R, Zablowski P, Antranikian G. Highly active and thermostable amylases and pullulanases from various anaerobic thermophiles. Appl Microbiol Biotechnol 1987;27:1928.

211

[137] Ben Ali M, Mhiri S, Mezghani M, Bejar S. Purication and sequence analysis of the atypical maltohexaose-forming a-amylase of the B. stearothermophilus US100. Enz Microb Technol 2001;28:53742. [138] Ali MB, Mezghani M, Bejar S. A thermostable alpha amylase producing maltohexose from a newly isolated Bacillus sp US100: study of activity and molecular cloning of the corresponding gene. Enz Microb Technol 1999;24:5849. [139] Nagarajan DR, Rajagopalan G, Krishnan C. Purication and characterization of a maltooligosaccharide-forming alpha-amylase from a new Bacillus subtilis KCC103. Appl Microbiol Biotechnol 2006;73:5917. [140] Prieto JA, Bort BR, Martinez J, Randez-Gil F, Buesa C, Sanz P. Purication and characterization of a new -amylase of intermediate thermal stability from the yeast Lipomyces kononenkoae. Biochem Cell Biol 1995;73:419. [141] Savchenko A, Vieille C, Kang S, Zeikus G. Pyrococcus furiosus -amylase is stabilized by calcium and zinc. Biochemistry 2002;41:6193201. [142] Yabuki M, Ono N, Hoshino K, Fukui S. Rapid induction of -amylase by nongrowing mycelia of Aspergillus oryzae. Appl Environ Microbiol 1977;34:16. [143] Bhella RS, Altosaar I. Purication and some properties of the extracellular -amylase from Aspergillus awamori. Can J Microbiol 1985;31:14955. [144] Olutiola PO. -Amylolytic activity of Aspergillus chevalieri from mouldy maize seeds. Ind Phytopathol 1982;35:42833. [145] Alazard D, Baldensperger JF. Amylolytic enzymes from Aspergillus hennebergi (A. niger group): purication and characterization of amylases from solid and liquid cultures. Carbohydr Res 1982;107:2317. [146] Michelena VV, Castillo FJ. Production of amylase by Aspergillus foetidus on rice our medium and characterization of the enzyme. J Appl Bacteriol 1984;56:395400. [147] Schellart JA, Visser FMW, Zandstva T, Middlehover WJ. Starch degradation by the mold Trichoderma viride. I. The mechanism of degradation. Ant van Leeuw J Microbiol Serol 1976;42:229. [148] Perevozchenko II, Tsyperovich AS. Comparative study of the properties of alpha-amylases from Aspergillus molds. Prikl Biokhim Mikrobiol 1972;8:128. [149] Narayanan AS, Shanmugasundaram ERB. Studies on amylase of Fusarium vasinfectum. Arch Biochem Biophys 1967;118:317. [150] Zenin CT, Park YK. Purication and characterization of acid -amylase from Paecilomyces sp. J Ferment Technol 1983;61:109. [151] Mishra RS, Maheshwari R. Amylases of the thermophilic fungus Thermomyces lanuginosus. Their purication properties action on starch and response to heat. J Biosci 1996;21:65372. [152] Wanderley KJ, Torres FAG, Moraes LMP, Ulhoa CJ. Biochemical characterization of -amylase from the yeast Cryptococcus avus. FEMS Microb Lett 2004;231:1659. [153] Iefuji H, Chino M, Kato M, Iimura Y. Raw-starch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp. S-2: purication, characterization, cloning and sequencing. Biochem J 1996;318:98996.