Journal of Biology and today's world 2012, volume 1, issue 1, pages: 39-50

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Journal of Biology and today's world
ISSN 2322-3308

Review Article
Application of alpha-amylase in biotechnology
Mohsen Mobini-Dehkordi1,2 and Fahime Afzal Javan1,2

1
Department of Genetics, Faculty of Science, Shahrekord University
2
Research Institute of Biotechnology, Shahrekord University

Received: 7 November 2012 / Accepted: 20 November 2012 / Published: 29 November 2012

Copyright © 2012 Mohsen Mobini-Dehkordi et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.

Abstract
Alpha-amylases are digestive enzymes which hydrolyze glycosidic bonds in starch to glucose,
maltose, maltotriose and dextrin. They have diverse applications in a wide variety of
industries such as food, textile, paper, detergent as representing approximately 30% of the
world enzyme production. With the advent of new frontiers in biotechnology, the spectrum of
amylase application has expanded into many other fields, such as clinical, medicinal and
analytical chemistry. To improve the productivity of amylases can use classical strain
improvement by mutation and selection and or recombination Amylases are produced by
microorganisms. This study reviews the microbial sources and application of this enzyme in
industry and biotechnology.

Key words: Alpha-amylase, biotechnology, industrial enzyme.


Correspondence should be addressed to Fahime Afzal Javan, Department of Genetics, Faculty of Science,
Shahrekord University & Research Institute of Biotechnology, Shahrekord University. Email:
f_afzaljavan@yahoo.com; Tel: +98-915-114-0766.

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1. Introduction
The industrial enzyme producers sell enzymes for a wide variety of applications. The
world market for industrial enzymes is estimated to be 1.6 billion $US, split between food
enzymes (29%), feed enzymes (15%), and general technical enzymes (56%) [1]. Amylases
constitute a class of industrial enzymes representing approximately 30% of the world enzyme
production [2]. They have diverse applications in a wide variety of industries such as food,
fermentation, textile, paper, detergent and sugar industries. It can be used in field related with
biotechnology such as: removing environmental pollutant, conversion of starch to desired
substrate by many microorganisms, infiltration of waste contains starch and production
biochemical material with helping starch substrate. With the advent of new frontiers in
biotechnology, the spectrum of amylase application has expanded into many other fields, such
as clinical, medicinal and analytical chemistry. Interestingly, the first enzyme produced
industrially was an amylase from a fungal source in 1894, which was used as a
pharmaceutical aid for the treatment of digestive disorders [3]. Although amylases can be
derived from several sources, including plants, animals and microorganisms, microbial
enzymes generally meet industrial demands [4-5]. The microbial amylases have almost
completely replaced chemical hydrolysis of starch in starch processing industry [6]. Most
important genetic engineering proceed to introduce recombinant α-amylase can signify
transmission of α-amylase gene from bacillus species to other microbial host [7]. Some
recombinant strain had represented to enhance production of enzyme because of high
expression of gene [8]. Also gene transmission has done from bacterial source into yeast
Saccharomyces cerevisiae [9]. In the other hand, gene transmission has done from fungal
source into prokaryote and eukaryote hosts [10-11].

2. Details of study

2.1. Alpha-amylase
Amylases are one of the most important industrial enzymes that have a wide variety of
applications ranging from conversion of starch to α-Amylases (E.C. 3.2.1.1.) are starch-
degrading enzymes that catalyze the hydrolysis of internal α-1,4-O-glycosidic bonds in
polysaccharides with the retention of α-anomeric configuration in low molecular weight
products, such glucose, maltose and maltotriose units [12]. Most of the a-amylases are
metalloenzymes, which require calcium ions (Ca2+) for their activity, structural integrity and
stability. They belong to family 13 (GH-13) of the glycoside hydrolase group of enzymes.
These enzymes possess an 8 (α/β) or TIM barrel structure containing the catalytic site
residues and Contain of four highly conserved regions in their primary sugar syrups, to the
production of cyclodextrins for the pharmaceutical industry [13]. These enzymes account for
about 30 % of the world’s enzyme production. You can see a 3D image of this enzyme in
figure 1.

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Figure 1. 3D image of Alpha-amylase

2.2. Organisms producing amylase

Alpha-amylase has been derived from several fungi, yeasts, bacteria and actinomycetes,
however, enzymes from fungal and bacterial sources have dominated applications in
industrial sectors [14]. The major advantage of using microorganisms for the production of
amylases is the economical bulk production capacity and the fact that microbes are easy to
manipulate to obtain enzymes of desired characteristics [15]. Fungal sources are confined to
terrestrial isolates, mostly to Aspergillus species and to only one species of Penicillium,
P.brunneum [16]. The Aspergillus species produce a large variety of extracellular enzymes,
and amylases are the ones with most significant industrial importance [17]. Filamentous
fungi, such as Aspergillus oryzae and Aspergillus niger, produce considerable quantities of
enzymes that are used extensively in the industry. A. oryzae has received increased attention
as a favourable host for the production of heterologous proteins because of its ability to
secrete a vast amount of high value proteins and industrial enzymes, e.g. α-amylase [18].
Aspergillus niger has important hydrolytic capacities in the α-amylase production and, due to
its tolerance of acidity (pH <3), it allows the avoidance of bacterial contamination [19].
Filamentous fungi are suitable microorganisms for solid-state fermentation (SSF), especially
because their morphology allows them to colonize and penetrate the solid substrate [20]. The
fungal α-amylases are preferred over other microbial sources due to their more accepted
GRAS (Generally Recognized as Safe) status [21]. The thermophilic fungus Thermomyces
lanuginosus is an excellent producer of amylase. A large variety of bacteria employ
extracellular or intracellular enzymes able to convert starch or glycogen that can thus serve as
energy and carbon sources [22]. For commercial applications α-amylase is mainly derived
from the genus Bacillus [23]. It is estimated that Bacillus sp. enzymes make up about 50% of
the total global enzyme market [22]. Amylases, biosynthesized by the bacteria, show unique
characteristics such as thermophilic, thermotolerant, alkaline and acidophilic properties [24].
Thermostability is a desired characteristic of most of the industrial enzymes. Thermostable
enzymes isolated from thermophilic organisms have found a number of commercial
applications because of their stability. As enzymatic liquefaction and saccharification of
starch are performed at high temperatures (100–110°C), thermostable amylolytic enzymes

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have been currently investigated to improve industrial processes of starch degradation and are
of great interest for the production of valuable products like glucose, crystalline dextrose,
dextrose syrup, maltose and maltodextrins. B. subtilis, B. stearothermophilus, B.
licheniformis, B. amyloliquifaciens are known to be good producers of α-amylase [25-26].
A great deal of work has been done on the cloning of α-amylase genes in different microbes,
mostly in Escherichia coli or Saccharomyces cerevisiae [27-28].

2.3. Use of Alpha-amylase in biotechnology

2.3.1. Starch processing industries

The major market for a-amylases is in the starch industry, which is used for starch hydrolysis
in the starch liquefaction process that converts starch into fructose and glucose syrups. The
enzymatic conversion of all starch includes: gelatinization, which involves the dissolution of
starch granules, thereby forming a viscous suspension; liquefaction, which involves partial
hydrolysis and loss in viscosity; and saccharification, involving the production of glucose and
maltose via further hydrolysis. This process requires the use of a highly thermostable a-
amylase for starch liquefaction, which can act at temperatures around 70-100oC depending
upon the temperature [29]. Initially, the α-amylase of Bacillus amyloliquefaciens was used but
it has been replaced by the α-amylase of Bacillus stearothermophilus or Bacillus licheniformis
[30]. The enzymes from the Bacillus species are of special interest for large-scale
biotechnological processes due to their remarkable thermostability and because efficient
expression systems are available for these enzymes [31]. Thermostable a-amylases are
generally preferred as their application minimizes contamination risk and reduces reaction
time, thus providing considerable energy saving. Hydrolysis carried out at higher
temperatures also minimizes polymerization of D-glucose to isomaltose [32].

2.3.2. Food industries

With the development of modern biotechnology, the food industry has undergone great
changes. There are many reports about the genetic engineering enzymes that have been used
safely in the food industry. Amylases are extensively employed in processed-food industry
such as baking, brewing, preparation of digestive aids, production of cakes, fruit juices and
starch syrups [33]. These enzymes can be added to the dough of bread to degrade the starch in
the flour into smaller dextrins, which are subsequently fermented by the yeast. The addition of
α-amylase to the dough results in enhancing the rate of fermentation and the reduction of the
viscosity of dough, resulting in improvements in the volume and texture of the product.
Moreover, it generates additional sugar in the dough, which improves the taste, crust color
and toasting qualities of the bread. Besides generating fermentable compounds, α-amylases
also have an anti-staling effect in bread baking, and they improve the softness retention of
baked goods, increasing the shelf life of these products. Currently, a thermostable maltogenic
amylase of bacillus stearothermophilus is used commercially in the bakery industry [21, 30].

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2.3.3. Biofuel industries

Ethanol is the most utilized liquid biofuel. For the bioethanol production, starch is the most
used substrate due to its low price and easily available raw material in most regions of the
world. In this production, starch has to be solubilized and then submitted to two enzymatic
steps in order to obtain fermentable sugars [34, 35]. The conventional process for the
bioconversion of starch into ethanol involves saccharification, where starch is converted into
sugar using an amylolytic microorganism or enzymes such as glucoamylase and α-amylase,
followed by fermentation, where sugar is converted into ethanol using an ethanol fermenting
microorganism such as yeast Saccharomyces cerevisiae [36]. In order to obtain a new yeast
strain that can directly produce ethanol from starch without the need for a separate
saccharifying process, protoplast fusion was performed between the amylolytic yeast
Saccharomyces fibuligera and S. cerevisiae [37]. Among bacteria, α-amylase obtained from
thermoresistant bacteria like Bacillus licheniformis or from engineered strains of Escherichia
coli or Bacillus subtilis is used during the first step of hydrolysis of starch suspensions [38].
In recent years, the many research projects have been performed for designing of microbial
strains with more ethanol production capacity and enhancement of process ethanol efficiency.
In addition, Saccharomyces cerevisiae has more specific characters biotechnologically,
genetically, and physiologically than other microorganisms and considers the best microbial
strain for bioethanol production. One of the best methods for bioethanol efficiency increment
is the isolation of yeast mutant cells that resistant to high concentration of ethanol and capable
to produce more bioethanol. Based on recent reports, the suitable yeast mutant strains have
been isolated for more bioethanol productivity [39]. These mutant strains could produce 7%
(W/V) bioethanol and tolerate up to 12% (V/V) exogenous ethanol but could not grow in the
presence of other alcoholic compounds such as 2-propanol and 1-butanol [40]. In addition,
Saccharomyces cerevisiae is a safe microorganism and classify to GRAS group. There are
accepted biosafety rules in developed countries such as Japan about integration of other safe
yeast genes to Saccharomyces cerevisiae genome and designing of recombinant
Saccharomyces spp. by auxotrophic markers. Finally, new recombinant strains of
Saccharomyces cerevisiae have been designed by over expression of self-gene(s) for
bioethanol efficiency enhancement [41].

2.3.4. Detergent industries

Detergent industries are the primary consumers of enzymes, in terms of both volume and
value. The use of enzymes in detergents formulations enhances the detergents ability to
remove tough stains and making the detergent environmentally safe [42]. Amylases are the
second type of enzymes used in the formulation of enzymatic detergent, and 90% of all liquid
detergents contain these enzymes. These enzymes catalyze the hydrolysis of glucosidic
linkages in starch polymers, commonly found in foods such as pasta, fruit, chocolate, baby
food, barbecue sauce and gravy. As colored stains, their removal is of interest in both
detergent and dishwashing contexts. Removal of starch from surfaces is also important in
providing a whiteness benefit, since it is known that starch can be an attractant for many types
of particulate soils. The suitability of any hydrolytic enzymes for inclusion in detergent
formulation is dependent on its stability and compatibility with detergent components. The

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use of amylases in detergent formulations is problematic since the enzyme must over stability
and an optimal level of activity in commercially utilized formulations, in the presence of
proteases [43]. To save energy, the temperature used in household laundering and automated
dishwashers has been reduced in recent years. These often results in problems with efficient
cleaning and stain removal that enzyme technology can help overcome [44]. Examples of
amylases used in the detergent industry are derived from Bacillus or Aspergillus [45].

2.3.5. Textile industries

In textile industry, strength of the textile is improved by warping the starch paste to textile
weaving. It also prevents the loss of string by friction, cutting and generation of static
electricity on the string by giving softness to the surface of string due to laid down warp.
After weaving the cloth, the starch is removed and the cloth goes to scouring and dyeing. The
starch on cloth is usually removed by application of α-amylase. The α-amylases remove
selectively the size [46]. Amylase from Bacillus strain was employed in textile industries for
quite a long time [47].

2.3.6. Paper industries

The use of α-amylases in the pulp and paper industry is for the modification of starch of
coated paper, i.e. for the production of low-viscosity, high molecular weight starch. The
coating treatment serves to make the surface of paper sufficiently smooth and strong, to
improve the writing quality of the paper. In this application, the viscosity of the natural starch
is too high for paper sizing and this can be altered by partially degrading the polymer with α-
amylases in a batch or continuous processes [48]. Starch is a good sizing agent for the
finishing of paper, improving the quality and erasebility, besides being a good coating for the
paper. The size enhances the stiffness and strength in paper [49]. Cold active α-amylase is
also very useful for paper industry as it reduces the viscosity of starch for appropriate coating
of paper [50].

2.3.7. Clinical and medicinal applications

α-amylases would be potentially useful in the pharmaceutical and fine chemicals industries if
enzymes with suitable properties could be prepared [51]. Interestingly, the first enzyme
produced industrially was an amylase from a fungal source in 1894, which was used as a
pharmaceutical aid for the treatment of digestive disorders [3]. Synthetic and natural
biodegradable polymers have been a major focus of interest in pharmaceutical research. The
biodegradable polymers are used to control the drug release rate from parenteral controlled
delivery systems [52]. For drugs with limited solubility or for some drugs for which solubility
can be influenced by variation of gastro-intestinal pH, a system is required to accelerate drug
release. Polysaccharide biodegradable matrices are of interest since the degradation of a
natural product like starch occurs naturally in the human body [53]. Alpha-starch (pre-
gelatinized starch) and cross-linked starch have been used as hydrogels. It has been reported
that increasing the degree of cross-linking of starch decreases the drug release rate. The
release rate is also dependent on a-amylase activity contained in the dissolution media. The

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addition of α-amylase to cross-linked amylose (CLA) tablets can modulate the release kinetics
of drugs [54].

2.3.8. Elimination of environmental pollutants

Starch is a polysaccharide that is widely distributed in nature as a reserve of stored energy in

many species of plant, and occurs extensively in waste materials produced from the
processing of plant raw materials [55]. Starch-processing waste is produced in large quantities
and causes pollution problems. To purify starch pollutant materials can use microbial
amylolytic enzyme or microorganisms producing amylolytic enzyme [56]. For this, can use
strains produced high level enzyme such as yeast [57-58].

2.3.9. Molecular applications

Reporter gene assays have become essential for the study of gene regulatory elements and
gene expression [59]. In molecular biology, the presence of amylase can serve as an
additional method of selecting for successful integration of a reporter construct in addition to
antibiotic resistance. Insertion of foreign DNA into this gene result in a loose of amylolytic
activity in the host cell that can be assayed using a simple and inexpensive iodine staining
procedure [60].

3. Conclusion
The use of α-amylase in starch based industries has been prevalent for many decades
and a number of microbial sources exist for the efficient production of this enzyme, but only a
few selected strains of fungi and bacteria meet the criteria for commercial production. The
search for new microorganisms that can be used for amylase production is a continuous
process. More recently, many authors have presented good results in developing α-amylase
purification techniques, which enable applications in pharmaceutical and clinical sectors
which require high purity amylases. Amylases are among the most important enzymes used
for industrial purposes, and now in the light of biotechnology they are considered useful for
biopharmaceutical applications. They are useful tools in medicinal and clinical chemistry.
Utilization of low-value agro-industrial residues as substrates should be focused upon for
enzyme production, as this would reduce the cost of production and help to solve their
disposal and pollution problems. Since thermostability is considered a useful and important
feature of amylases for industrial application, attempts should be made to develop enzymes
from thermophilic and extremely thermophilic microorganisms. It is hoped that amylases will
continue to provide new opportunities in biotechnology as biocatalysts and that new
applications will emerge in the biopharmaceutical sector.

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References

[1] Outtrup, H. Jorgensen, S.T. 2002. The importance of Bacillus species in the production of
industrial enzymes. In Applications and systems of Bacillus and relatives. Edited by R.
Berkley. Blackwell Science Inc., Malden, Mass. pp. 206–218.

[2] Calik, P. Ozdamar, TH. 2001. Carbon sources affect metabolic capacities of Bacillus
species for the production of industrial enzymes theoretical analyses for serine and neutral
proteases and alpha-amylase. Biochemical Engineering Journal, 8:61-81.

[3] Pandey, A. Nigam, P. R.Sccol, C. T.Soccol, V. Singh, D. Mohan, R. 2000. Advances in

microbial amylases. Journal of Biotechnology, 31:135-152.

[4] Sumitani, J. Nagae, H. Kawaguchi, T. Arai, M. 1998. Bacillus animal type alpha-amylase:
Cloning and sequencing of the gene, and comparison of the deduced amino acid sequence
with that of other amylases. Journal of Fermentation And Bioengineering, 85:428-432.

[5] Ibrahim, C.D. 2008. Development of applications of industrial enzymes from Malaysian
indigenous microbial sources. Bioresource Technology, 99:4572-4582.

[6] Veille, C. Zeikus, G.J. 2001. Hyperthermophilic enzymes: sources, use, and molecular
mechanisms for thermostability. Microbiol. Mol. Biol. Rev, 65:1–43.

[7] Karakas, B. Inana, M. Certe, M. 2010. Expression and characterization of Bacillus subtilis
PY22 _alpha amylase in Pichia pastoris Journal of Molecular Catalysis B: Enzymatic,
64:129–134.

[8] Niu, D. Shi, G. Wang. Z. 2009. Genetic improvement of alpha-amylase producing

Bacillus licheniformis by homolog-mediated alpha-amylase gene amplification. Chin
journal of biotechnologh, 25:375-380.

[9] Afzaljavan, F. (2011). Amplification, sequencing, and cloning of Bacillus subtilis alpha-
amylase gene in commercial Saccharomyces cerevisiae strain, M.S thesis of Genetics,
Shahrekord University, Iran.

[10] Iefuji, H. Chino, M. Kato, M. Iimura, Y. 1996. Raw-starch-digesting and thermostable a-

amylase from the yeast Cryptococcus sp. S-2 : purification, characterization, cloning and
sequencing Biochem. J, 318:989-996.

[11] Xu Dong, L. Yan, X. 2009. Molecular cloning and characterization of an alpha-amylase

with raw starch digestibility from Bacillus sp.YX-1. Annals of Microbiology, 59:91-96.

[12] Brayer, G.D. Luo, Y. Withers, S.G. 1995. The structure of human pancreatic alpha-
amylase at 1.8 A resolution and comparisons with related enzymes. Protein Sci. 4:1730-
1742.

[13] Svensson, B. 1994. Protein engineering in the α-amylase family; catalytic mechanism,
substrate specificity, and stability. Plant Mol. Biol. 25:141–157.

46 | P a g e
Journal of Biology and today's world 2012, volume 1, issue 1, pages: 39-50

[14] Tanyildizi, M.S. Ozer, D. Elibol, M. 2005. Optimization of α-amylase production by

Bacillus sp. using response surface methodology Process Biochem, 40;2291–2296.

[15] Lonsane, B.K. Ramesh, M.V. 1990. Production of bacterial thermostable a-amylase by
solid state fermentation: a potential tool for achieving economy in enzyme production and
starch hydrolysis. In: Advances in applied microbiology, vol. 35. San Diego: California
Academic Press, pp:1-56.

[16] Kathiresan, K. Manivannan, S. 2006. α-Amylase production by Penicillium fellutanum

isolated from mangrove rhizosphere soil. Afr. J.Biotechno,. 5:829-832.

[17] Hutcheon, G.W. Vasisht, N. Bolhuis, A. 2005. Characterisation of a highly stable alpha-
amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles, 9:487-495.

[18] Jin, B. van Leeuwen, H.J. Patel, B. Yu, Q. 1998. Utilisation of starch processing
wastewater for production of microbial biomass protein and fungal α-amylase by
Aspergillus oryzae. Bioresour. Techno, 66:201-206.

[19] Djekrif-Dakhmouche, S. Gheribi-Aoulmi, Z. Meraihi, Z. Bennamoun, L. 2006.

Application of a statistical design to the optimization of culture medium for α-amylase
production by Aspergillus niger ATCC 16404 grown on orange waste powder. J Food
Process Eng, 7:190–197.

[20] Rahardjo, Y. Weber, F.J. Haemers, S. Tramper, J. Rinzema, A. 2005. Aerial mycelia of
Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation
system. Enzyme Microb.Technol. 36:900–902.

[21] Gupta R, Gigras P, Mohapatra H, Goswami V.K, Chauhan B. 2003. Microbial α-

amylases: a biotechnological perspective. Process Biochem, 38:1599-1616,

[22] Schallmey M, Singh A, Ward OP. 2004. Developments in the use of Bacillus species for
industrial production. Canadian Journal of Microbiology, 50:1-17.

[23] Mahmood, A.U. Greenman, J. Scragg, A.H. 1998. Orange and potato peel extracts:
Analysis and use as Bacillus substrates for the production of extracellular in continuos
culture. Enzyme and Microbial Technology, 22:130-137.

[24] Kandra, L. 2003. Alpha-Amylases of medical and industrial importance. Journal of

Molecular Structure (Theochem), 666-667:487-498.

[25] Asgher, M. Asad, M.J. Rahman, S.U. Legge, R.L. 2007. A thermostable α-amylase from
a moderately thermophilic Bacillus subtilis strain for starch processing. J Food Process
Eng, 79:950- 955.

[26] Declerck, N. Machius, M. Joyet, P. Wiegand, G. Huber, R. Gaillardin, C. 2002.

Engineering the thermostability of Bacillus licheniformis alpha-amylase. Biologia, 57:203-
211.

[27] Murakami, S. H. Nishimoto, Y. Toyama, E. Shimamoto, S. Takenaka, J.Kaulpiboon, M.

Prousoontorn, T. Limpaseni, P. Pongsawasdi, Aoki, K. 2007. Purification and
characterization of two alkaline, thermotolerant α-amylases from Bacillus halodurans 38C-

47 | P a g e
Journal of Biology and today's world 2012, volume 1, issue 1, pages: 39-50

2-1 and expression of the cloned gene in Escherichia coli. Biosci. Biotechnol. Biochem,
71:2393–240.

[28] Mobini-Dehkordi, M. Afzaljavan, F. Saffar, B. 2011. Identifying of native bacillus strain

producing alpha amylase and clonig of alpha amylase gene in E.coli, First national
conference biological sciences. P:787-789.

[29] Regulapati, R. Malav, PN. Gummadi, SN. 2007. Production of thermostable alpha
amylase by solid state fermentation- A review, 2(1):1- 11.

[30] Ogasahara, K. Imanishi, A. Isemura, T. Studies on thermophilic aamylase from Bacillus

stearothermophilus . I. 1970. Some general and physico-chemical properties of
thermophilic a-amylase. J Biochem, 67:65-75.

[31] Prakash, O. Jaiswal, N. 2010. alpha-Amylase: An Ideal Representative of Thermostable

Enzymes. Appl Biochem Biotechnol, 162(7):2123-4.

[32] Maarel, M. Veen, B. Uitdehaag, J. Leemhuis, H. Dijkhuizen, L. 2002. Properties and

applications of starch-converting enzymes of the alpha-amylase family. Journal of
Biotechnology, 94:137-155.

[33] Zeman, N. McCrea, J. 1985. Alpha-amylase Production Using a Recombinant DNA

Organism. Cereal food world, 30:777-780.

[34] Sanchez, O.J. Cardona, C.A. 2008. Trends in biotechnological production of fuel ethanol
from different feedstocks. Bioresour Technol, 99:5270-5295.

[35] Moraes, L.M.P. Filho, S.A. Ulhoa, C.J. 1999. Purification and some properties of an α-
amylase glucoamylase fusion protein from Saccharomyces cerevisiae. World J. Microbiol.
Biotechnol, 15:561-564.

[36] Öner, E.T. 2006. Optimization of ethanol production from starch by an amylolytic
nuclear petite Saccharomyces cerevisiae strain. Yeast, 23:849-856.

[37] Chi, Z. Chi, Z. Liu, G. Wang, F. Ju, L. Zhang, T. 2009. Saccharomycopsis fibuligera and
its applications in biotechnology. Biotechnol Adv, 27:423-43.

[38] Saxena, R.K. Dutt, K. Agarwal, L. Nayyar, P. A. 2007. highly thermostable and alkaline
amylase from a Bacillus sp. PN5. Bioresour Technol, 98:260-265.

[39] Mobini-Dehkordi, M. Nahvi, I. Zarkesh-Esfahani, H. Ghaedi, K. Tavassoli, M. Akada, R.

2008. Isolation of a novel mutant strain of Saccharomyces cerevisiae by ethyl methyl
sulfonate mutagenesis approach as a high producer of bioethanol. J Biosci Bioengin.
105:403-408.

[40] Mobini-Dehkordi, M. Nahvi, I. Zarkesh, H. Ghaedi, K. Akada, R. 2011. Characterization

of an interesting novel mutant strain of commercial Saccharomyces cerevisiae. Iranian J
Biotechnol. 9:109-114.

[41] Mobini-Dehkordi, M. Nahvi, I. Zarkesh, H. Ghaedi, K. Akada, R. 2011. Overexpression

of FLO1 gene in an industrial strain of Saccharomyces cerevisiae by self-cloning method
for increment of bioethanol production. Under Review.

48 | P a g e
Journal of Biology and today's world 2012, volume 1, issue 1, pages: 39-50

[42] Hmidet, N. Bayoudh, A. Berrin, J.G. Kanoun, S. Juge, N. Nasri, M. 2008. Purification
and biochemical characterization of a novel alpha-amylase from Bacillus licheniformis.
NH1: cloning, nucleotide sequence and expression of amyN gene in Escherichia coli.
Process Biochemistry, 43:499-510.

[43] Mitidieri, S. Martinelli, A.H.S. Schrank, A. Vainstein, M.H. 2006. Enzymatic detergent
formulation containing amylase from Aspergillus niger: a comparative study with
commercial detergent formulations, Bioresour. Technol, 97:1217–1224.

[44] Kirk, O. Borchert, T.V. Fuglsang, C.C. 2002. Industrial enzyme applications. Curr Opin
Biotechnol, 13:345-351,

[45] Ramasesh N, Sreekantiah K.R, Murthy K.R, Ramasesh V.S. 1982. Purification and
characterization of thermophilic alpha-amylase of Aspergillus niger. Van Thieghem
Starke, 34, 274–279.

[46] Feitkenhauer, H. 2003. Anaerobic digestion of desizing wastewater: influence of

pretreatment and anionic surfactant on degradation and intermediate accumulation.
Enzyme Microb. Technol. 33:250–258.

[47] Haq, I. Ali, S. Javed, M.M. Hameed, U. Saleem, A. Adnanf-Qadeer, M.A. 2010.
Production of alpha amylase from a randomly induced mutant strain of bacillus
amyloliqefaciens and its application as a desizer in textile industry. Pak. J. Bot. 42(1): 473-
484.

[48] Fryer, P.J. Asteriadou, K.A. 2009. Prototype cleaning map: a classification of industrial
cleaning processes. Trends in Food Science and Technology, 20:255-262.

[49] Bruinenberg P.M, Hulst A.C, Faber A, Voogd R.H. 1996. A process for surface sizing or
coating of paper. European Patent Application, EP 0 690 170 A1.

[50] Kuddus, M. 2010. Microbial cold-active α-amylases: From fundamentals to recent

developments. Current research, technology and education topics in applied microbiology
and microbial biotechnology, 1265-1276.

[51] Fogarty W.M, Kelly C.T. 1980. In Economic Microbiology, Microbial Enzymes and
Bioconversions. Academic Press, pp. 115–170.

[52] Dumoulina, Y. Cartiliera, L. Mateescub, M. 1999. Cross-linked amylose tablets

containing a-amylase: an enzymatically-controlled drug release system, Journal of
Controlled Release, 60:161–167.

[53] Kost, J. Shefer, S. 1990. Chemically-modified polysaccharides for enzymatically-

controlled oral drug delivery. Biomaterials, 11:695–698.

[54] Dumoulin, Y. Cartilier, L. Mateescu, M. 1999. Cross-linked amylase tablets containing

alpha-amylase: an enzymatically-controlled drug release system. Journal of Controlled
Release, 60:161-167.

[55] Barbesgard, P. Heldt-halsen, H. Diterichsen, B. 1999. On the safty of the aspergillus

oryzae. Applied microbiology and biotechnology, 9:569-572.

49 | P a g e
Journal of Biology and today's world 2012, volume 1, issue 1, pages: 39-50

[56] Wu, H. Mulchandani, A. Chen, W. 2008. Versatile microbial surface-display for

environmental remediation and biofuels production. Trends in Microbiology, 14,4:181-188.

[57] Jurado-Alameda, E. Bravo-Rodriguez, V. Bailón-Moreno, R. Nuñez Olea, J. Altmajer-

Vaz, D. 2003. Bath-substrate-flow method for evaluating the detersive and dispersant
performance of hard-surface detergents. Industrial & Engineering Chemistry Research,
42:4303-4310.

[58] Mobini-Dehkordi, M. Afzaljavan, F. Saffar, B. 2011.Cloning of bacterial alpha amylase

gene to saccharomayces cerevisiae for environmental applications. Third national
conference of Biosafty andgenetic engineering, p: 68.

[59] Aubel, D. Morris, R.P. Rimann, M. Kaufmann, H. Thompson, C.J. Bailey, J.E.
Fussenegger, M. 2001. Design of a novel mammalian screening system for the detection of
bioavailable, non-cytotoxic streptogramin antibiotics. J. Antibiot, 54:44–54.

[60] Ikuta, N. Souza, M. Valencia, F. Castro, M. Schenberg, A. Pizzirani, A. Astolfi-filho, S.

1990. The alpha-amylase gene as a marker for gene cloning: direct screening for
recombinant clonies. Nature, 8:241-242.

50 | P a g e