Advances in Microbial Amylases

Biotechnol. Appl. Biochem.
(2000) 31, 135152 (Printed in Great Britain) 135

REVIEW
Advances in microbial amylases
Ashok Pandey*
1
, Poonam Nigam, Carlos R. Soccol*, Vanete T. Soccol, Dalel Singh
and Radjiskumar Mohan*
*Laboratorio de Processos Biotecnologicos, Departamento de Engenharia Quimica, Universidade Federal do Parana (UFPR),
CEP 81531-970 Curitiba-PR, Brazil, School of Applied Biological and Chemical Sciences, University of Ulster,
Coleraine BT52 1AS, Northern Ireland, U.K., Departamento de Patologia BaT sica, Universidade Federal do Parana (UFPR),
CEP 81531-970 Curitiba-PR, Brazil, and Department of Microbiology, Haryana Agriculture University, Hisar-125 004, India
This reviewmakes a comprehensive survey of microbial
amylases, i.e. -amylase, -amylase and glucoamylase.
Amylases are among the most important enzymes and
are of great signicance in present-day biotechnology.
Although they can be derived from several sources,
such as plants, animals and micro-organisms, the
enzymes from microbial sources generally meet
industrial demands. Microbial amylases could be po-
tentially useful in the pharmaceutical and ne-chemical
industries if enzymes with suitable properties could be
prepared. With the advent of new frontiers in bio-
technology, the spectrum of amylase application has
widenedin many other elds, such as clinical, medicinal
and analytical chemistries, as well as their widespread
application in starch saccharication and in the textile,
food, brewing and distilling industries. In this review,
after a brief description of the sources of amylases, we
discuss the molecular biology of amylases, describing
structures, cloning, sequences, and protoplast fusion
and mutagenesis. This is followed by sections on their
production and nally the properties of various
amylases.
Introduction
Amylases [a term that refers here -amylase, -amylase and
glucoamylase (GA)] are among the most important enzymes
in present-day biotechnology. The amylase family of
enzymes is of great signicance due to its wide area of
potential application. Interestingly, the rst enzyme pro-
duced industrially was an amylase from a fungal source in
1894, which was used as a pharmaceutical aid for the treat-
ment of digestive disorders [1]. Amylases nd potential
application in a number of industrial processes such as in
the food, fermentation, textiles and paper industries.
Microbial amylases have successfully replaced the chemical
hydrolysis of starch in starch-processing industries. They
would be potentially useful in the pharmaceutical and ne-
chemicals industries if enzymes with suitable properties
could be prepared [2].
With the advent of new frontiers in biotechnology, the
spectrum of amylase application has expanded into many
other elds, such as clinical, medicinal and analytical
chemistries. Recently Witczak [3] presented a review on the
biological relevance of thio-sugars as potential new thera-
peutics, which are gaining substantial attention. The new
developments, especially in the synthetic and medicinal
chemistry of thio-sugars are critically important for carbo-
hydrate drug design. Enzyme-controlled mechanisms, in-
volving enzymes such as amylase and lipase, have contributed
to the understanding of the biological processes. There are
several processes in the medicinal and clinical areas that
involve the application of amylases [414]. Sutton et al. [4]
assessed 13 analytes, including amylase, for serumevaluation.
Lepp et al. [5] developed a liquid-stable reagent containing -
amylase for the new Olympus AU6004 chemistry analyser.
Chiu and Chandler [6] described a revised amylase method
for the DuPont Dimension2 clinical chemistry system. The
application of a liquid-stable reagent, based on for the Ciba
Corning Express clinical chemistry system has been de-
scribed by Becks et al. [7]. Giri et al. [8] developed a process
for the detection of higher oligosaccharides, which involved
application of -amylase. The method was claimed to be
more efcient than the silver nitrate test, a method that is
limited in its sensitivity. Menzel et al. [9] developed
biosensors with an electrolyte isolator semiconductor
capacitor (EIS-CAP) transducer for process monitoring.
Amongst other enzymes this contained -amylase and GA
and was used for sugar syrup analysis. -Amylase was used to
prepare a hybrid membrane using chitosan as a dispersant
in the solgel process [10]. -Amylase was also used as an
enzyme thermistor for the biochemical analysis of cyclo-
dextrins [11]. GA has been used as chiral selector in
chromatography [13].
Abbreviation used: GA, glucoamylase.
1
Present address and address for correspondence: Biotechnology Division,
Regional Research Laboratory, Trivandrum-695 019, India.
#2000 Portland Press Ltd
136 A. Pandey and others
Table 1 Microbial sources of -amylase, -amylase and GA
Source Reference
-Amylase
Aeromonas caviae [15]
Alicyclobacillus acidocaldarius [16]
Alteromonas haloplanetis [17]
Anaerobic bacterium [18]
Archaeobacterium pyrococcus woesei [19]
Aspergillus sp. [20]
A awamorei [21,22]
A. flavus [23]
A. fumigatus [24]
A. kawachi [25]
A. niger [26,27]
A. oryzae [28,29]
A. usanii [30]
Bacillus sp. [3133]
B. acidocoldarius [34]
B. amyloliquefaciens [3538]
B. brevis [3941]
B. circulans [42]
B. coagulans [43]
B. flavothermus [44]
B. globisporus [45]
B. licheniformis [39,42,4649]
B. megaterium [50]
B. stearothermophilus [51]
B. subtilis [5254]
Chloroflexus aurantiacus [55]
Clostridium acetobutylicum [56]
C. butricum [57]
C. thermohydrosulfuricum [19]
C. thermosulfurogenes [58,63]
Eubacterium sp. [59]
Filobasidium capsuligenum [60]
Halobacterium halobium [61]
H. salinarium [49]
Humicola insolens [66]
H. lanuginosa [66]
H. stellata [66]
Lactobacillus brevis [64]
L. cellobiosus [65]
Malbrachea pulchella var. sulfurea [66]
Micrococcus luteus [67]
M. varians [68]
Micromonospora vulgaris [69]
Mucor pusillus [66]
Myceliophthora thermophila [70]
Myxococcus coralloides [71]
Nocardia asteroides [72]
Penicillium brunneum [73]
Pseudomonsa stutzeri [74]
Pycnoporus sanguineus [75]
Pyrococcus woesei [76]
Rhizopus sp. [77]
Scytalidium sp. [78]
Schizophyllum commune [79]
Talaromyces thermophilus [66]
Thermus sp. [81]
Thermoactinomyces sp. [78]
T. vulgaricus [81]
Thermococcus profundus [82]
Thermomonospora viridis [83]
Thermonospora curvata [84]
T. vulgaris [85]
Thermomyces lanuginosus [86]
Thermotoga maritima [87]
-Amylase
Bacillus cereus [88]
B. circulans [89]
B. megatarium [78,90]
B. polymyxa [88]
Clostridium thermosulfurogenes [91,92]
C. thermocellum [93]
Pseudomonas sp. [88]
Rhizopus japonicus [88]
Streptomyces sp. [88]
GA
Acremonium zonatum [94]
Amylomyces rouxii [94]
Arxula adeninivorans [95]
Aspergillus sp. [96]
A. awamori [94,97100]
A. candidus [94]
A. foetidus [94]
A. niger [94,101111]
A. oryzae [112,113]
A. phoenicus [114]
A. saitri [94]
A. terreus [115,116]
Bacillus firmus/lentus [117]
B. stearothermophillus [94]
Candida famata [94]
C. fennica [94]
Cephalosporium eichhorniae [94]
C. charticola [94]
Chalara paradoxa [94]
Clostridium sp. [94]
C. acetobutylicum [94]
C. thermohydrosulfuricum [118]
C. thermosaccharolyticum [119]
C. thermosulfurogenes [94]
Collectotrichum gloesporoides [120]
Coniophora cerebella [94]
Endomyces sp. [94]
Endomycopsis capsularis [94]
E. fibuligera [121,122]
Flavobacterium sp. [94]
Fusidium sp. [123]
Halobacter sodamense [94]
Humicola lanuginosa [94]
Lactobacillus brevis [124]
Monscus kaoling [94]
Mucor rouxianus [94]
M. javanicus [94]
Neurospora crassa [94]
N. sitophila [125]
Paecilomyces globosus [94]
P. varioti [94]
Penicillium italicum [94]
P. oxalicum [94]
Piricularia oryzae [94]
Rhizoctania solani [94]
Rhizopus sp. [121,126]
R. delemar [127,128]
R. javanicus [94]
R. niveus [35,94]
R. oligospora [94]
R. oryzae [127,128]
Rhizooctonia solani [109]
Saccharomyces diastaticus [129]
Schizophyllum commune [94]
Schwanniomyces alluvius [94]
S. occidentalis [130]
Thermomyces lanuginosus [94]
Torula thermophilla [94]
Trichoderma reesei [131]
T. viride [94]
Microbial amylases 137
Sources of amylases
Amylases have most widely been reported to occur in
micro-organisms, although they are also found in plants and
animals. Two major classes of amylases have been identied
in micro-organisms, namely -amylase and GA. In addition,
-amylase, which is generally of plant origin, has also been
reported from a few microbial sources. -Amylases (endo-
1,4--D-glucan glucohydrolase, EC 3.2.1.1) are extracellular
enzymes that randomly cleave the 1,4--D-glucosidic linkages
between adjacent glucose units in the linear amylose chain.
These are endoenzymes that split the substrate in the in-
terior of the molecules and are classied according to their
action and properties. For example, amylases that produce
free sugars are termed saccharogenic and those that
liquefy starch without producing free sugars are known as
starch-liquefying. -Amylase (-1,4-glucan maltohydrolase,
EC 3.2.1.2) is usually of plant origin, but a few microbial
strains are also known to produce it. It is an exo-acting
enzyme that cleaves non-reducing chain ends of amylose,
amylopectin and glycogen molecules. It hydrolyses alternate
glycosidic linkages, yielding maltose (-anomeric form). Since
-amylase is unable to by-pass -1,6-glycosidic linkages
in amylopectin, it results in incomplete degradation of the
molecule, yielding 5060% maltose and a -limit dextrin. GA
(synonyms amyloglucosidase, glucogenic enzyme, starch
glucogenase and -amylase ; exo-1,4--D-glucan glucano-
hydrolase, EC 3.2.1.3) hydrolyses single glucose units from
the non-reducing ends of amylose and amylopectin in a
stepwise manner. Unlike -amylase, most glucoamylases are
also able to hydrolyse the 1,6--linkages at the branching
points of amylopectin, although at a lower rate than 1,4-
linkages. Thus glucose, maltose and limit dextrins are the
end products of GA action.
-Amylase
-Amylase may be derived from several bacteria, yeasts and
fungi. Bacterial amylase, however, is generally preferred over
fungal amylase due to several characteristic advantages that
it offers. Table 1 enumerates important microbial sources of
-amylase [1587]. Strains of Aspergillus sp. and Bacillus sp.,
mainly Bacillus amyloliquefaciens and B. licheniformis, are
employed for commercial applications. Thermostable -
amylases are generally preferred as their application mini-
mizes contamination risk and reduces reaction time, thus
providingconsiderableenwergysaving. Hydrolysiscarriedout
at higher temperatures also minimizes polymerization of D-
glucose to iso-maltose.
-Amylase
Unlike other members of the amylase family, only a few
attempts have been made to study -amylases of microbial
origin, as -amylases have generally been obtained fromplant
sources. As is evident from the Table 1, bacterial strains
belonging to Bacillus, Pseudomonas (aerobic) and Clostridium
(anaerobic) sp., actinomycete strains belonging to Strepto-
myces sp. and fungal strains belonging to Rhizopus sp. have
been reported to synthesize -amylase [8893].
GA
Pandey [94] has reviewed the main features of GA research,
providing insight into various aspects, including the sources
of the enzyme. GAcan be derived froma number of sources,
such as plants, animals and micro-organisms. Commercial
need, however, is met by GA obtained from microbial
sources. Filamentous fungi apparently constitute the
major source of GA among all microbes. Table 1 lists some
micro-organisms used as the sources of GA [95131].
Molecular biology of amylases
Gene structure, cloning and sequencing
Genetic engineering has been used extensively for cloning of
amylase-producing strains, mainly -amylase and GA, in
order to achieve desirable characteristics in the cloned host.
The purpose of gene cloning can be, amongst others, the
expression of thermostable enzymes, higher enzyme pro-
ductivity and co-expression of two enzymes by the same
organism.
A great deal of work has been done on the cloning of -
amylase genes in different microbes, mostly in Escherichia coli
or Saccharomyces cerevisiae (Table 2). Liebl et al. [87]
described the gene structure of the -amylase from Thermo-
toga maritima MSB8. It is a chromosomal -amylase gene,
designated amyA, and was predicted to code for a 553-amino
acid preprotein with signicant amino acid sequence. The T.
maritima -amylase appeared to be the rst known example
of a lipoprotein -amylase. Following the signal peptide, a 25-
residue putative linker sequence rich in serine and threonine
residues was found. The amylase gene was expressed in E.
coli. Suganuma et al. [30] studied the N-terminal sequence of
the amino acids of the -amylase from Aspergillus usanii. The
sequence of the rst 20 amino acids was identical to the -
amylase from A. niger. Kim et al. [132] described a gene
encoding a new -amylase of Bacillus licheniformis, which was
cloned and expressed in E. coli. The genomic DNA of B.
licheniformis was double-digested with EcoRI and BamHI and
ligated the pBR322. The transformed E. coli carried the
recombinant plasmid pIJ322 containing a 3.5-kb fragment of
B. licheniformis DNA. The puried enzyme encoded by pIJ322
was capable of hydrolysing pullulan and cyclodextrin as well
as starch. Iefuji et al. [133] described the cloning and
sequencing of a raw-starch-digesting and thermostable -
Table 2 Expression of cloned genes for microbial amylases: some examples
Source of gene Recombinant host References
-Amylase
Thermotoga maritima Escherichia coli [87]
Alicyclobacillus acidocaldarius E. coli [16]
Bacillus lichenformis E. coli [132]
B. subtilis Saccharomyces
cerevisiae
[138]
B. amyloliquefaciens S. cerevisiae [140]
B. stearothermophilus E. coli [141]
B. amyloliquefaciens S. cerevisiae [140]
B. subtilis Xanthomonas
campestris
[142]
B. subtilis E. coli [134]
B. lichenformisl E. coli [41]
B. lichenformisl B. brevis [41]
S. cerevisiae S. cerevisiae [166]
Saccharomycopsis fibuligera S. cerevisiae [167]
GA
Aspergillus awamori S. cerevisiae [138]
A. awamori S. cerevisiae [152]
Schwanniomyces occidentalis S. cerevisiae [130]
S. cerevisiae S. cerevisiae [154]
Arxula adeninivorans S. cerevisiae [95]
Saccharomyces diastaticus S. cerevisiae [155]
S. diastaticus S. cerevisiae [140]
Rhizopus S. cerevisiae [168]
A. niger S. cerevisiae [169]
A.oryzae S. cerevisiae [140]
A. terreus E. coli [171]
S. diastaticus Schizosaccharomyces
pombe
[172]
S. cerevisiae Saccharomyces pombe [173]
S. fibuligera S. cerevisiae [173]
Clostridium sp. E. coli [174]
Lactobacillus amylovorus E. coli [145]
amylase from Cryptococcus sp. S2. An open reading frame of
the cDNA specied 611 amino acids, including a putative
signal peptide of 20 amino acids. The N-terminal region of
the enzyme (from the N-terminus to position 496) shared
49.7% similarity with that of an -amylase from A. oryzae
while the C-terminus had a sequence similar to the C-
terminal region of GA from A. niger. Marco et al. [134]
inserted a B. subtilis -amylase gene into a plasmid, which was
transferred to E. coli. During the cloning, a 3h region encoding
171 C-terminal amino acids was replaced by a nucleotide
sequence that encoded 33 amino acid residues not present
in the indigenous protein. The transformed protein
produced substantial amylolytic activity. Matsuura et al.
[135] and Tada et al. [136] have described the amino acid
sequence and three-dimensional structure of -amylase.
To develop a yeast strain that could produce ethanol
directly from starch, -amylase cDNA was introduced into
the haploid Saccharomyces diastitcus secreting GA by using a
linearized integrating vector [137]. The integrating vector
contained a LEU2 gene and KpnI was used to cut the LEU2
gene tomake the linearized vector. One of the transformants
exhibited 100% mitotic stability after 100 generations of cell
multiplication. To improve its ethanol fermentability, the
haploid transformant was rare-mated with a polyploid
industrial strain with no amylase activity. The resulting
hybrid RH51 gave high ethanol yields. Birol et al. [138]
cloned three S. cerevisiae strains (YPG\AB, YPG\MM and
YPB-G). YPB-G secreted a bifunctional fusion protein that
contained the B. subtilis -amylase and Aspergillus awamori
GA. When these strains were used for ethanol production
from different substrates such as glucose and starch,
YPG\AB showed the most efcient utilization of starch for
ethanol production. The superior performance of the
YPG\AB as compared with YPB-G was found to correlate
with its higher level of -amylase activity. Shiba et al. [139]
investigated the effect of ethanol concentration on cloned -
amylase gene expression in recombinant S. cerevisiae strain
20B-12 containing one of the two plasmids, pNA3 and
pNA7. Both the plasmids contained -amylase gene under
the control of the SUC2 and PGK promoters, respectively.
When the ethanol concentration was 25 g\l, the gene
expression was twice as high as when ethanol concentration
was 20 g\l.
Steyn and Pretorius [140] cloned an -amylase-
encoding gene (AMY) from B. amyloliquefaciens and a
GA-encoding gene (STA2) from S. diastaticus into a yeast-
integrating shuttle vector (Yip5), generating recombinant
plasmids pSP1 and pSP2. The STA2 and AMY genes were
jointly cloned into Yip5, generating plasmid pSP3. Subse-
quently, the dominant selectable marker APH1, encoding
resistance to Geneticin 418, was cloned into pSP3 resulting
pSP4. For enhanced expression of Geneticin 418, the APH1
gene was fused to the GAL10 promoter and terminated by
the URA3 terminator, resulting in pSP5. Plasmid pSP5 was
converted to a circular minichromosome (pSP6) by the
addition of ARS1 and CEN4 sequences. Laboratory strains of
S. cerevisiae transformed with plasmids pSP1pSP6 stably
produced several -amylases and GAs.
Miranda and Berglund [141] described a B. stearo-
thermophilus -amylase expressed in E. coli y the use of a
food-grade polymer. Stripecke et al. [142] studied the role of
a cloned -amylase gene in fermentation by Xanthomonas
campestris, which was prepared by transforming a hybrid
plasmid pAP1 containing the -amylase gene from B. subtilis
into amylolytic and non-amylolytic X. campestris cells. Juge et
al. [143] described -amylase production by heterologous
gene expression in A. niger. The cDNA encoding -amylase
isozyme 1 (AMY1) and its signal peptide was placed under
the control of the A. nidulans glyceraldehyde-3-phosphate
dehydrogenase (GPD) promoter and the A. nidulans trpC
gene terminator. The recombinant AMY1 was puried to
homogeneity. -Amylase was from a plant source (barley)
and this has been claimed to the rst plant protein efciently
secreted and correctly possessed by A. niger using its own
signal sequence.
Apparently, no attention has been paid to the cloning of
genes for -amylase of microbial origin, although a few
reports are available on its expression in E. coli, mostly from
Bacillus sp. and at least one from Thermoanaerobacterium
[144148]. To express the cloned -amylase cDNA in E. coli
under control of the tac promoter, Yoshigi et al. [149]
constructed a plasmid pBETA92, which consisted of 6312 bp.
Later, these authors [150] constructed plasmid pB927, which
was used for the expression of a 7-fold increase in
production of -amylase with increased thermostability in
E. coli. The intragenic amino acid replacements were found
to have simple additive effects on the thermostability of the
enzyme.
Like -amylase, the number of GA-encoding genes also
varies between strains. In a recent work, James and Lee
[151] reviewed the GA multigene family. A. awamori and A.
niger, the two most frequently used strains for GA synthe-
sis, generally produced two forms of GA but from only
one structural gene. Rhizopus oryzae too possesses only one
structural gene. As is evident fromTable 2, most of the work
on GA cloning has been done in S. cerevisiae. Many of such
studies were intended to develop an improved (cloned)
yeast strain for industrial production of ethanol. Zhang et al.
[152] developed a mathematical model for the aerobic
culture of a recombinant yeast, which was formulated to
stimulate plasmid loss and recombinant protein production
amongst other things. The yeast strain was S. cerevisiae
containing the GA gene from A. awamori.
Erratt and Nasim [153] described a protocol for the
genetic engineering of a yeast strain (brewers yeast), which
carried the S. cerevisiae var. diastaticus gene. These authors
cloned the STA1 gene from S. cerevisiae using a modied
Yep13 plasmid vector and obtained the rst patent for the
gene-cloning procedure. Nakamura et al. [154] constructed
a starch-fermenting yeast, S. cerevisiae SR93, by integrating a
GA-producing gene (STA1) into the chromosome of S.
cerevisiae SH1089. The GA was constitutively produced by
the recombinant yeast. The GA gene of the yeast Arxula
adeninvorans Ls3 was cloned from a genomic library and
sequenced. The gene was localized to chromosome 2 from
Ar. adeninvorans and comprised 1875 bp. The rst 16 N-
terminal amino acids represented the signal sequence for
entering the endomembrane system. When the amino acid
sequence of this GA was compared with GA from other
fungal sources, it showed homology with the GA from R.
oryzae (32.6%), Sachharomycopsis buligera (23.1%), A. niger
(22.1%) and S. diastaticus (15.4%). No homology was
detected with the GA of Schwaninomyces occidentalis. By
using the GAL1 promoter from S. cerevisiae within an
autonomously replicating plasmid, it was possible to express
the isolated Arxula GA gene in S. cerevisiae.
When a new allelic variant of the STA2 gene from S.
diastaticus, designated as STA2(K) and coding for a secreted
GA, was cloned, differences were revealed both in the
structural gene and in the promoter region (as compared
with other STA genes). STA2(K) showed peculiar charac-
teristics, such as a 1.1-kb natural deletion in its promoter
located 189 nucleotides upstream of the translation start
codon and an Asn Asp single amino acid change within the
putative active site of the encoded GA [155]. Chen et al.
[156] expressed Asn-182 Ala A. awamori GA in S.
cerevisiae, which showed increased thermostability.
Suominen et al. [157] described the fusion of poly(aspartic
acid) tails of different lengths (5, 7 and 10 Asp residues) to
the GA of A. awamori into the N-terminus of the full-length
mature GA (amino acids 1616). Fusion proteins were
designated as GAND5, GAND7 and GAND10. Three other
fusion proteins, designated GACD0, GACD5 and GACD7
(0, 5 and 7 Asp residues, respectively) were also constructed.
All of the charged tails showed the general sequence Met-
Ala-Asp
n
-Tyr, where n was 0, 5, 7 or 10. The modied genes
were expressed in S. cerevisiae and the proteins were
secreted in the culture medium.
A few attempts have also been made to clone the
bacterial GA gene. The GA gene from Clostridium sp. G0005
was apparently the rst bacterial GA gene to be cloned
[151]. James et al. [158] cloned the GA gene from
Lactobacillus amylovorus in E. coli. GA from the wild and
recombinant sources showed similar characters.
Protoplast fusion and mutagenesis
Protoplast fusion and mutagenesis have been used widely by
several workers as a tool of protein engineering to achieve
strains with higher enzyme productivity or desired
characters. Svensson and Sogaard [159] reviewed the effects
of mutation on the structure and function of GA and related
enzymes. Linardi et al. [160] described an intraspecic
protoplast fusion of amylase-producing strains of Candida
fennica. Although protoplast fusion did not signicantly
stimulate the synthesis of GA in the fusants, the production
of -amylase was increased (32%) by one of the auxotrophic
mutants of the yeast.
Mutagenesis has mainly been employed to obtain GA
hyper-producing strains. It can be effected with the help of
chemicals such as N-methyl-Nh-nitro-N-nitrosoguanidine
(NTG), or by radiation, such as UV radition. Suntornsuk and
Hang [161] reported strain improvement of R. oryzae for GA
production using NTG as well as UV radiation. One mutant,
3N4, produced more GA than the parent strain. The
mutagenesis of a GA-producing strain of A. awamori resulted
in a mutant that exhibited an 80% increase in enzyme
productivity [162]. Flory et al. [163] carried out random
mutagenesis of A. awamori to generate thermosensitive
mutants, which then were expressed in S. cerevisiae.
Hulseweh et al. [130] carried out site-directed mutagenesis
to dene the active site of the Sch. occidentalis GA. The
mutated GAM1 genes were expressed in S. cerevisiae and
transformants were evaluated. Mutants were transcribed
and translated similarly to wild-type GA. Therefore, all
effects on enzymic activity could be traced to single amino
acid substitutions. Asp-470 was shown to be essential for
enzyme activity. Replacement of Asp-470 by glycine led to a
complete loss of activity. Substitution of Trp-468 by Ala
affected predominantly the -1,6 activity and not -1,4
activity of the enzyme. Chen et al. [156] also reported that
site-directed mutagenesis obtained thermostable GA. Table
2 shows some other studies of expression of cloned genes
for amylase production [164174].
Production of amylases
Although amylases can be produced by several micro-
organisms, it remains a challenging task to obtain a strain
capable of producing commercially acceptable yields. Selec-
tion of a suitable strain is the most signicant factor in the
amylase production process. Sometimes a single strain can
produce more than one enzyme, i.e. -amylase as well as
GA. For example, the strains of A. niger can produce as many
as 19 enzymes, whereas -amylase can be produced in
reasonably good titres by as many as 28 strains [175].
Commercial production of amylases is carried out in various
steps, essentially because the environmental factors required
for the optimum growth of the micro-organism being
employed for production may differ from those required for
the production of enzymes. These parameters include
nutrient supplementation, pH of the medium, osmotic
relationship, degree of aeration, temperature and the
control of contamination during fermentation. Maintaining
the purity of the medium is also a very important factor,
especially when the fermentation is carried out under
aerobic conditions. Although the details of the specic
fermentation processes adopted by different manufacturers
vary, there remain two main methods for amylase pro-
duction, submerged fermentation and solid-state fermen-
tation. Solid-state fermentation has gained renewed interest
from researchers for the production of these enzymes in
view of its several economic and engineering advantages
and has been often employed to produce amylases
[110,175179]. Selvakumar et al. [180] reviewed microbial
synthesis of starch-saccharifying enzymes in solid cultures.
Since thermostability is a feature of most of the enzymes
sold in bulk for industrial application, thermophilic micro-
organisms are of special interest for the production of
thermophilic amylases. Recent research on thermostable
amylases, especially -amylase, has concentrated on the
enzymes of thermophiles and extreme thermophiles. Be-
tween the thermophilic and mesophilic groups lies the much
less common and largely unexplored facultative thermo-
philes. Facultative thermophiles generally grow in the
mesophilic temperature range. Whereas their growth may
be optimum at 45 mC, they are capable of growing well at
higher temperatures, thus covering both the mesophilic and
thermophilic ranges. However, not much is known about the
processes for enzyme production involving such organisms.
Comparative analysis of the structure and physico-chemical
properties of these two groups (mesophilic and thermo-
philic) may provide some information about the molecular
basis of the high-temperature tolerance ability of thermo-
stable amylases.
Production of -amylase
Bacillus species are considered to be the most important
sources of -amylase and have been used for enzyme
production using SSF [43,52,181] or SmF [3537,44,
51,182,183]. Lonsane and Ramesh [184] reviewed the
production of bacterial thermostable -amylase in SSF by B.
amyloliquefaciens and B. licheniformis. They referred to the
SSF process as the potential tool for achieving economy in
enzyme production and starch hydrolysis.
Bajpai et al. [182] and Omidiji et al. [51] developed
simple and cheap media based on cheese whey, corn steep
liquor and soya bean meal for -amylase production. It was
claimed that the mediumcould be exploited for the industrial
production of -amylase. Salva and Moraes [183] studied the
effects of different carbon sources on -amylase production.
Whereas lactose, dextran and soluble starch were found
suitable for enzyme production, the highest enzyme yield
was obtained when glucose was used. El Helow and El
Gazaerly [54] compared -amylase production in three
different nutritional media. Different patterns of enzyme
induction were obtained when beet pulp, corn cob, rice
husk, wheat bran and wheat straw were used separately to
partially replace the nutrient contents of the selected
medium. -Amylase was maximally expressed in the pres-
ence of corn cob or wheat bran. Syu and Chen [36] also
investigated the effects of different carbon sources (glucose,
maltose, xylose and starch) on -amylase production. Higher
cell density and higher specic growth rate were obtained
from glucose but higher enzyme activity and higher specic
enzyme activity were obtained from starch. Using a dened
synthetic medium, Hiller et al. [37] demonstrated the effect of
lactose and nitrogen on cell physiology and -amylase pro-
duction. Results showed cell-growth and -amylase-
production patterns to be similar regardless of the limiting
nutrient and suggested stationary phase gene control of -
amylase production as opposed to a direct response to
nutrient limitation. Kelly et al. [44] described the production
of -amylase in a bi-phasic process in which high -amylase
activities were achieved. Oxygen-transfer conditions, and
especially the dissolved oxygen tension, were reported as
vital factors for -amylase production [35]. High aeration
rates were found to be essential for good yields of enzyme.
Control of dissolved oxygen tension, however, was not
found to be advantageous. Babu and Satyanarayana [43] also
achieved maximum -amylase yield in aerated reactors.
In view of the apparent advantages offered by cell
recycling in bioprocesses, Gron et al. [185] investigated -
amylase production in a cell-recycling bioreactor incor-
porating a membrane-ltration module for cell separation.
The reactor gave increased enzyme yield and volumetric
productivity compared with conventional continuous fer-
mentation. Coupling of fermentation and microltration for
-amylase production was also studied by Morcel and
Biedermann [186]. Compared with a batch process, con-
tinuous fermentation with cell recycling led to a reduction in
-amylase concentrations but to a doubling of volumetric
productivities. Tari et al. [187] also demonstrated operation
strategies of a two-stage bioreactor for -amylase pro-
duction. They claimed that this would improve enzyme
production.
During laboratory studies on enzyme production, as a
practice, single-stage inoculum is used for fermentation
processes. Generally, it is carried out in a routine way
without being given critical attention. Keeping this in mind,
Milner et al. [35] studied one-stage and two-stage inocula for
-amylase production. Signicantly better results were
obtained using two-stage inoculum.
Since lamentous fungi are generally considered to be
the most prolic producers of extracellular enzymes,
attempts have been made to study -amylase production
using them. The thermophilic fungus Thermomyces lanuginosa
was reported to be an excellent producer of -amylase
[86,188]. Increased production of the enzyme could be
obtained by manipulating the growth conditions and medium
composition. Sudo et al. [189] compared acid-stable -
amylase production in SmF and SSF and examined the reason
why A. kawachii IFO 4308 produced larger amounts of acid-
stable -amylase in SSF than in SmF. Some of the SSF
characteristics were given as the major reasons for higher
enzyme production in SSF. A new source of -amylase was
identied in Pycnoporus sanguineus. Cultivation in SSF resulted
in 4-fold-higher enzyme production than in SmF [15,75]. de
Souza et al. [77] tested more than 800 Rhizopus strains for -
amylase production. One of the strains showed great ability
to produce a thermostable -amylase in SSF. Torrado et al.
[29] studied pH regulation in solid-state cultures using the
initial ratio between oxidized and reduced sources of
nitrogen. On the basis of these, they suggested a model for
-amylase production. Krishna and Chandrasekaran [52]
cultivated Aeromonas caviae (CBTK 185) on banana waste.
The results indicated the excellent scope for utilizing this
strain and banana waste for commercial production of -
amylase in SSF.
The cell-immobilization technique has also been
employed for -amylase production. Ivanova et al. [190]
compared various immobilization techniques including en-
trapment in gels such as calciumalginate, -carrageenan, agar
and their combinations with polyethylene oxide, adsorption
on cut disks of polymerized polyethylene oxide and xation
on formaldehyde-activated acrylonitrile\acrylamide mem-
branes for -amylase production. Among these, agar, -
carrageenan, agar\polyethylene oxide gels and the mem-
branes were found to be suitable. Membrane-immobilized
cells of B. licheniformis produced 176% more enzyme than
free cells. Ariga et al. [41] studied continuous production of
-amylase by PVA-encapsulated recombinant E. coli and B.
brevis. In all the experiments with B. brevis, the enzyme was
released fromthe encapsulated cells without cell leakage and
cell growth outside the capsules. In the case of immobilized
E. coli cells, it was necessary to add glycine to the medium for
enzyme to be released. Stefanova et al. [40] used agar-gel-
immobilized cells of B. brevis for the production of thermo-
stable -amylase. Dobreva et al. [46] used polymer mem-
brane to immobilize B. licheniformis cells for the production
of -amylase. The enzyme yields were affected by the
reactive chemical groups of the carriers and the spacer size.
Formaldehyde-activated polysulphone membranes were the
most suitable for effective immobilization. A 62% increase
over the control experiment was obtained in repeated batch-
mode operation. Lin et al. [38] described the design of a
continuously rotating bioreactor for immobilized cells of B.
amyloliquefaciens to produce -amylase. They claimed that
the volumetric productivity of the enzyme in this bioreactor
could be much higher than in a shake ask, if the reactor was
operated properly. The cells of a bacterial strain of Halo-
bacterium salinarium were immobilized in calcium alginate
beads and on a polyvinyl alcohol lm with an aim to produce
halophilic -amylase [62].
Production of -amylase
As mentioned previously, -amylases are usually of plant
origin, and not much work has been done on the production
of -amylase using micro-organisms. Some of the micro-
organisms reported to produce -amylases include Bacillus
polymyxa, B. cereus, B. megatarium, Streptomyces sp., Pseudo-
monas sp. and Rhizopus japonicus [1,88].
Ray et al. [90] compared the production of -amylase
from starch waste by a hyper-amylolytic strain of B.
megaterium B6 mutant UN12 in SmF and SSF. The starchy
wastes used as substrates were from arrowroot, arum,
maize, potato, pulse, rice, rice husk, tamarind, kernel,
cassava, water chestnut, wheat and wheat bran. Arum and
wheat bran gave the highest yields. They also used
immobilized cells of B. megatarium B6 for enzyme pro-
duction. Among the tested methods, ionotropic gelation was
found to be most suitable for -amylase production [191].
Production of GA
Extensive work has been carried out on the production
of GA in solid cultures using A. niger [94,101111,
180,191195]. The study included screening of a number
of agro-industrial residues such as wheat bran, rice bran,
rice husk, gram our, wheat our, corn our, tea waste,
copra waste etc. [101,107,177,192]. Apart from the sub-
strate particle size, which showed profound impact on
fungal growth and activity, substrate moisture and water
activity also inuenced the enzyme yield signicantly
[94,105,111,191].
Strains of A. awamori too have been used frequently for
GA production [100,196,197]. Queiroz et al. [100] con-
ducted a study on GA production by A. awamori in order to
dene the rheological changes of broth. They proposed two
kinds of mathematical correlation; one between the biomass
concentration and the rheological parameter consistency
index (K) from the Power law, and another between either
the specic growth rate or the specic GA production rate
and K. Wang and Webb [197] studied the effect of cell
concentration on the rheology of GA fermentation broth of
A. awamori using a Bohlin CS rheometer. By controlling the
cell morphology, spore aggregation was prevented com-
pletely. They also described the experimental results in
terms of the Power-law model.
Elegado and Fujio [198] screened 39 Rhizopus new
isolates and nine authentic Rhizopus strains (grown on wheat
bran in SSF) for their soluble-starch-digestive GA (SSGA)
and raw-starch-digestive GA (RSGA) activities. Results
showed that these strains could be classied into four
groups, based on their SSGA and RSGAproduction and ratio
of SSGA to RSGA. Chiarello et al. [128] compared raw-
starch-degrading GA production by the strains of R. oryzae
MUCL 28627, R. oryzae MUCL 28168 and R. delemar ATCC
34612. R. oryzae MUCL 28627 presented the highest
specic activity values. Soccol et al. [126] screened 19
Rhizopus strains for their ability to grow on raw cassava.
Only three strains grew signicantly and GA production was
higher on raw cassava than on cooked cassava.
GA has also been produced using some bacterial and
yeast cultures such as Lactobacillus brevis [124], B. coagulans
[199], S. cerevisiae [154], Saccharomycopsis buligera [200], C.
fennica, C. famata [201] and Endymycopsis buligera [202].
Design and conguration of fermenter, its mode of
operation and fermentation inuence the enzyme
production strongly. Different types of bioreactor were
used to evaluate GA production. These included asks,
trays, rotary reactors and columns (vertical and horizontal)
[103,108,192,195]. Fermentation techniques included SmF,
SSF, semi-solid and an aqueous two-phase system[203205].
Enzyme production in trays occurred in maximum quantities
in 36 h compared with 96 h typically required in asks [192].
Ariff and Webb [98] studied GA production using freshly
suspended cells of A. awamori in batch and continuous
operations. They used an air-lift fermenter and a stirred-tank
fermenter. In comparison with freely suspended cell fermen-
tations, neither batch nor continuous fermentation of
immobilized cells improved GA production signicantly.
Interestingly, contrary to the general ndings, Fujio and
Morita [204] reported 4.6-fold lower GA yield by Rhizopus
sp. A-11 from conventional SSF on wheat-bran medium than
that in SmF, which used metal-ion-supplemented medium.
Ramadas et al. [203] also noted a similar trend when a fungal
strain of A. niger produced higher GA titres in SmF (102
units\ml) than in SSF (66 units\ml) in shorter period (66 h in
comparison with 96 h). The production in the fed-batch
process was higher in comparison with the batch process. In
an aqueous two-phase system of poly(ethylene glycol) and
salt, however, both the GA productivity and yield were
twice as high as those in the control fermentation.
Purication and properties of amylases
Enzyme application in pharmaceutical and clinical sectors
requires high-purity amylases. Thus, it is signicant to
develop economic processes for their purication to obtain
chemically pure enzymes with maximum specic activity.
Traditionally the purication of amylases from fermentation
media has been done in several steps, which include
centrifugation of the culture (a step of extraction may be
required for solid media), selective concentration of the
supernatant, usually by ultraltration, and selective pre-
cipitation of the enzyme by ammonium sulphate or organic
solvents such as ethanol in the cold. Then the crude enzyme
is subjected to chromatography (usually afnity or ion-
exchange chromatography) and gel ltration.
-Amylase
Bacterial -amylase As bacterial -amylases have generally
been produced from the strains belonging to genus Bacillus,
several attempts have been made at their purication and
characterization, from both mesophilic and thermophilic
strains. Uguru et al. [206] described a thermostable extra-
cellular -amylase from B. subtilis, which was puried 24-fold
to a specic activity of 2200 units\mg per litre. Bolton et al.
[207] puried an -amylase to homogeneity using a com-
bination of ammonium sulphate precipitation, ion-exchange
chromatography and gel ltration. The enzyme was inhibited
by diethyl pyrocarbonate. An extracellular thermostable -
amylase produced by B. licheniformis was puried by two-
phase separation in a poly(ethylene glycol)\dextran system
followed by gel ltration and ion-exchange chromatography
[208]. Thermostability was Ca
2
+
-dependent. The puried
enzyme was inhibited strongly by N-bromosuccinimide and
EDTA. Marco et al. [134] described a genetically modied -
amylase. Its molecular mass (48000 Da) was lower than the
molecular mass calculated from the derived amino acid
sequences of the parent strain (57700 Da). The enzyme was
inhibited by Hg
2
+
, Fe
3
+
and Al
3
+
and stimulated by Mn
2
+
and
Co
2
+
. Katoh et al. [209] puried an -amylase, produced by
recombinant cells, by specic elution from anti-peptide
antibodies.
The fundamental characteristics of extracellular
amylases are expected to reect the pH and temperature of
the environment in which they are grown [210]. This was
reected with B. avothermus grown at pH 6.0 and 55 mC
whose -amylase showed pH and temperature optima at
5.56.0 and 60 mC [210]. Another example was an alkalophi-
lic strain of Bacillus sp. GM8901, which best grew at pH 10.5
and 50 mC and produced an -amylase that showed
pH and temperature optima at 11.012.0 and 60 mC [211].
Another strain of Bacillus sp. grew best at pH 8.5 and the -
amylase produced by it had a pH optimum of 9.0 [212].
Industrial autofocusing has also been used as a tech-
nological purication technique to obtain chemically pure
amylases. Dobransky et al. [213] used this technique to
purify -amylase produced by different bacterial strains of B.
subtilis. A natural pH gradient in autofocusing is usually
created automatically by the puried supernatant. Irrespec-
tive of the undulations or the steps in the pH gradient, the
proteins reach an appropriate pI along the pH gradient.
The peaks are sufciently separated and the void volume is
far enough from the peak containing most of the -amylase
activity. In this method, large amounts of the starting material
(up to 1000 litres with a protein concentration of about
3 g\100 ml) can be employed. It must be emphasized that
this method should not be used in cases where the highest
activity of the enzyme corresponds with the main peak of the
proteins [213].
Satoh et al. [214] puried an intracellular -amylase
fromStreptococcus bovis [148], which was induced by maltose
and soluble starch and produced about 80% maltotriose
from soluble starch. The enzyme showed different charac-
teristics when compared with the extracellular enzyme from
the same strain and there was no homology between them.
An extracellular -amylase from a different strain of S. bovis
JB1 showed 40 and 27% homologies with -amylases of
Bacillus sp. and Clostridium acetobutylicum, respectively, and
produced maltose, maltotriose and maltotetraose from
amylose [215]. Ilori et al. [64,67] puried extracellular -
amylase from Micrococcus luteus and Lactobacillus brevis.
Purication was by 28- and 70-fold by gel ltration and ion-
exchange chromatographies, respectively. Enzyme from
both the sources was inhibited by EDTA, KCN and citric
acid but was activated by Mg
2
+
, Ca
2
+
, Na
+
and K
+
. A
Bidobacterium adolescentis extracellular -amylase was
inhibited signicantly by EDTA, glucose, maltose, Cu
2
+
, Zn
2
+
and N-bromosuccinimide but was activated by -mercapto-
ethanol [216].
A few reports also deal with the properties of the
amylases produced by facultative thermophiles [207]. It
would be interesting to know if they offer any technological
advantages in their application. In the rst report of its kind
on the characterization of -amylase from a thermophilic
strain of the genus Thermus, Egas et al. [217] puried the
enzyme by ion-exchange chromatography. Ca
2
+
enhanced
the thermostability at temperatures above 80 mC. EDTA
inhibited the enzyme activity, which was reversed by the
addition of Ca
2
+
or Sr
2
+
ions. The enzyme was also inhibited
by Hg
2
+
, Cu
2
+
and p-chloromercuribenzoic acid. Shaw et al.
[80] also puried the -amylase from Thermus sp. by afnity
adsorption on starch granules. The enzyme activity was
inhibited strongly by Cu
2
+
and Fe
2
+
but Ca
2
+
did not stimulate
its activity. A thermostable -amylase from an archae-
bacterium Thermococccus profundus was puried to hom-
ogeneity by ammonium sulphate precipitation, DEAE
Toyopearl chromatography and gel ltration on Superdex
200 HR. The thermostability of the enzyme was enhanced by
Ca
2
+
[82]. However, the thermostability or activity of -
amylase from another archaebacterium, Pyrococcus furiosus,
was not dependent on Ca
2
+
[218]. The -amylase from
Alicyclobacillus acidocaldarius showed an acidophilic nature
[16]. A maltotriose- and maltotetraose-producing -
amylase, which was stable at alkaline pH (up to 12) and high
temperatures (up to 55 mC) was puried from a strain of
Chloroexux aurantiacus, a thermophilic, green, photo-
synthetic bacterium [55].
Fungal -amylase In fungi, detailed studies on -amylase
purication have largely been limited to a few species of
mesophilic fungi [219223]. Although the growth and
amylase production from several thermophilic fungi have
been described, extended data concerning their physico-
chemical properties have been published with regard to only
a few species, such as Mucor pusilus [224], Talaromyces
emersonii [225] and Thermomyces lanuginosa [86]. Thermo-
stable -amylase from T. lanuginosa [86] had a pH optimum
about 1 pH unit lower than the -amylase from A. oryzae
[226] and 1 pH unit higher than that from M. pusilus [222],
but it resembled -amylases from T. emersonii [223] and A.
awamori [227].
Yeast -amylase There are only a very few reports
describing the purication of -amylase from yeasts. A raw-
starch-digestive thermostable -amylase from yeast Crypto-
coccus sp. was puried in just one step by using an -
cyclodextrinSepharose 6B column [133]. The enzyme
Table 3 Properties of some microbial - and -amylases
Source
No. of
forms
K
m
value
for starch
Molecular
mass (Da) Optimum pH
Optimum
temperature (mC) Reference
Bacterial -amylase
Alicyclobacillus acidocaldarius 1 160000 3.0 75 [16]
Alteromonas haloplanctis 1 49340 [230]
Bacillus sp. 3 110000140000 5.5 90 [231]
Bacillus sp. 1 97000 11.012.0 60 [211]
Bacillus sp. 2 150000, 42000 9.0 70 [212]
B. flavothermus 1 2.2 mg/ml 5.56.0 60 [207]
B. licheniformis 1 0.9 g/l 58000 6.06.5 90 [208]
B. licheniformis
a
1 64000 6.08.0 50 [132]
B. lentus 1 42000 6.1 70 [232]
B. megaterium 1 59000 [50]
B. subtilis 1 54780 5.6 80 [206]
B. subtilis 1 5.46.4 50 [183]
B. subtilis
a
1 3.85 mg/ml 57700 6.5 50 [134]
Bifidobacterium adolescentis 1 2.4 g/l 66000 5.5 50 [216]
Chloroflexus aurantiacus 1 210000 7.5 71 [55]
Clostridium acetobutylicum 1 3.6 g/l 84000 5.6 45 [56]
C. perfringenes 1 76000 6.5 30 [233]
Cytophaga sp. 1 59000 4.56.5 5060 [234]
Lactobacillus amylovorus 1 140000 5.5 6065 [235]
L. brevis 1 0.27 mg/ml 75900 6.5 55 [67]
L. plantarum 3 2.38 g/l 50000 5.5 65 [236]
Micrococcus luteus 1 56000 6.0 30 [64]
M. varians 3 0.5 mg/ml 1400056000 7.0 45 [68]
Myxococcus coralloides 1 2i10
3
g/l 22500 8.0 45 [71]
Pyrococcus furiosus 1 66000 100 [218]
P. woesei 1 70000 130
b
[76]
Streptococcus bovis 1 0.88 mg/ml 77000 5.06.0 50 [215]
Thermococcus profundus 1 42000 5.56.0 80 [82]
Thermus sp. 1 59000 5.56.5 70 [80]
T. filiformis 1 5.0 mg/ml 60000 5.56.0 95 [217]
Fungal and yeast -amylase
Aspergillus sp. 1 2.5 mg/ml 56000 5.5 40 [220]
A. flavus 1 0.5 g/l 52500 6.0 55 [223]
A. flavus 1 75000 7.0 30 [219]
A. fumigatus 1 0.42 mg/ml 65000 5.5 40 [222]
A. oryzae 1 50000 5.25 44 [237]
A. oryzae 1 0.22% 52000 45 50 [221]
Cryptococcus sp. 1 66000 [133]
Filobasidium capsuligenum 1 2 mg/ml 56000 5.6 45 [228]
Lipomyces kononenkoae 1 0.8 g/l 76000 4.55.0 70 [229]
Nocardia asteroides 2 65000, 56000 6.9 50 [72]
Rhizopus sp. 2 5.0 mg/ml 64000 4.05.6 6065 [75]
Scytalidium sp. 1 87000 6.5 50 [238]
Thermoactinomyces vulgaris 1 53000 4.86.0 62.5 [239]
Thermomyces lanuginosus 1 4.65.2 60 [86]
Thermotoga maritima
a
1 61000 7.0 8590 [87]
-Amylase
Bacillus circulans 1 0.9 mg/ml 64000 7.0 50 [89]
B. polymyxa 1 53000 5.5 45 [241]
Clostridium thermosulfurogenes 1 180000 6.0 70 [92]
Hendersonula toruloidea 1 0.3 mg/ml 60000 6.0 60 [240]
Syncephalastrum racemosum 1 5.0 60 [242]
a
Expressed in E. coli.
b
Hyper-active.
showed some similarities with the -amylase from A. oryzae
and with the GA from A. niger. Tsiomenko et al. [228]
puried and characterized an -amylase from basidiomycete
yeast Filobasidium capsuligenum. The enzyme was not
inuenced by Ca
2
+
. Prieto et al. [229] puried a novel -
amylase from the yeast Lipomyces kononenkoae to hom-
ogeneity by ammonium sulphate treatment, afnity binding
on cross-linked starch, and DEAEBiogel Achromatography.
Table 3 shows the properties of some other -amylases
[230239].
Table 4 Properties of some microbial GAs
Source No. of forms Molecular mass (Da) Optimum pH
Optimum
temperature (mC)
Raw starch
digestion Reference
Aspergillus awamori 1 83700 4.5 60 + [246]
A. awamori 2 110000, 86000 - [99]
A. awamori var. kawachi 3 5700090000 3.84.5 + [256]
A. niger 2 74000, 96000 4.2, 4.5 60, 65 + [257]
A. niger 4 61000112000 4.4 60 + [100]
A .niger 6 5840099500 3.55.0 +
a
[247]
A. niger 2 99000112000 4.55.0 0 [247]
A. niger 3 7000090000 3.54.0 5060 0 [258]
A. oryzae 3 3800076000 4.5 50, 60 +
a
[259]
A. phoenicus 1 4.5 60 0 [260]
A. saitri 1 90000 4.5 0 [261]
A. terreus 1 70000 5.0 60 0 [262]
Cephalosporium charicola 1 5.4 60 0 [263]
Coniphora cerebella 1 4.04.5 0 [244]
Corticum rolfsii 1 4.5 4050 0 [245]
Clostridium 1 77000 4.5 65 0 [264]
C. thermosaccharolyticum 1 75000 5.0 70 + [119]
Endomyces sp. 55000 0 [265]
Endomycopsis sp. 53000 0 [266]
E. capsularis 4.5 4050 0 [267]
Humicola lanuginosa 2 4.9, 6.6 65, 70 0 [243]
Monascus kaoligang 2 48000, 68000 4.5 50 0 [252]
Mucor rouxianus 2 49000, 59000 4.7 55 + [246]
Paecilomyces varioti 1 69000 5.0 0 [253]
Penicillium oxalicum 2 84000, 86000 4.6 5560 + [246]
Piricularia oryzae 1 94000 4.5 5055 0 [268]
Rhizopus sp. 3 5860074000 4.550 + [269]
R. delemer 1 100000 4.5 40 0 [270]
R. niveus 5 4.56.0 + [251]
Schizophyllum commune 1 66000 5.0 40 + [255]
Termomyces lanuginosa 1 37000 70 0 [254]
a
Raw-starch digestion only by one form.
-Amylase
-Amylases frommicrobial sources generally possess greater
thermal resistance than plant -amylases. -Amylases have
higher pH optima than -amylases and they do not require
Ca
2
+
for stabilization or enhancement of activity. Odibo et al.
[240] puried and characterized a -amylase from
Hendersonula toruloidea by ammoniumsulphate fractionation,
ion-exchange chromatography on DEAEcellulose and gel
ltration on Sephadex G-75. The enzyme activity was
inhibited strongly by Hg
2
+
, Zn
2
+
and Cu
2
+
but was activated
by Na
+
. An extracellular -amylase was puried by ad-
sorption on raw corn starch to 22.5-fold from a new isolate
of B. polymyxa [241]. The degradation of raw starch by the
enzyme was greatly stimulated by pullulanase addition. A
thermostable -amylase was puried with ammonium sul-
phate, DEAEcellulose column chromatography and gel
ltration using Sephadex G-200 [92]. Maltose was identied
as the major hydrolysis product of starch and it had -
anomeric form. Another thermotolerant -amylase was
puried from a strain of Bacillus circulans S31 that was not
activated by any metal ions [89]. Ray and Chakraverty [242]
partially puried a -amylase from Syncephalastrum race-
mosum. The enzyme activity remained unaffected in the
presence of metal ions and was not increased in the presence
of exogenous thiols, indicating the absence of thiols at the
active site of the enzyme.
GA
Pandey presented a comprehensive review on the puri-
cation and properties of GA [94]. Table 4 cites some
examples. It is evident that GAs are generally active on the
acidic side of neutrality, with optimum pH values of around
4.55.0. The optimum temperature for GA activity from
most sources has generally been found to be between 40 and
60 mC. However, there are a few reports describing the
extreme nature of GA. GA I and GA II from a thermophilic
fungus Humicola lanuginosa showed pH optima of 4.9 and 6.6,
respectively [243]. GA II was stable up to pH 11.0. The GAs
from Coniophora cerebella [244] and Corticum rolfsii [245]
were stable up to pH 9.0 whereas that of Mucor rouxianus
showed pH stability up to 8.0 [246].
A GA preparation from solid cultures of A. niger was
puried 32.4-fold with a nal specic activity of 49.25
units\mg of protein, which contained four different forms
(GA-I, GA-Ih, GA-II and GA-IIh), each with different charac-
teristics. This was the rst report on four forms of GA
produced by A. niger in SSF [102]. The enzymes were
glycoproteins with carbohydrate contents of 16%. Enzyme
activity was inhibited strongly by Hg
2
+
whereas Mn
2
+
and Fe
2
+
were stimulatory. Hata et al. [205] compared the properties
of two GAs produced in SmF and SSF by A. oryzae. Enzyme
produced in SSF could digest raw starch but that in SmF
could not. GAs obtained in SmF and SSF exhibited different
characteristics. Medda et al. [96] studied the raw-starch
adsorption and elution behaviour of GA I of black Aspergillus
sp. The enzyme was completely adsorbed on to raw starch
at pH 3.4, which was the isoelectric point of the enzyme. GA
of A. awamori var. kawachi had three forms, GA I, GA Ih
and GA II, with molecular masses of 90000, 83000 and
57000 Da, respectively [256]. GAI and GAIh were optimally
active at pH 3.8, whereas for GA II the optimum was at
pH 4.0. The pH-stability ranges were 210, 2.56.5 and 47,
respectively, for isoforms. The isoelectric points were 3.55,
3.45 and 3.28, and, at 60 mC, GA I was stable, while GA Ih and
GA II were unstable. GA I could hydrolyse raw starch
whereas the other two could not. The N-terminal amino
acids were Ala for GA I, Ala+Ile for GA Ih and Ile for GA II.
Acommercial preparation of GAfromA. niger consisted
of at least six species, ve forms of which were characterized
fully [247]. They were apparently different in molecular
mass. The optimum pH varied between 3.5 and 5.0, and
when enzymological characteristics were compared, one of
the species was peculiar and quite different from others.
Lineback et al. [248] reported two forms of GAfromA. niger.
The molecular mass of GA I was 99000 Da and that of GA
II was 112000 Da. Both forms of GA contained covalently
linked carbohydrate (containing D-mannose, D-glucose and
D-galactose residues) and were therefore glycoenzymes.
Both showed identical amino acid composition. However,
the two GAs differed in carbohydrate content, with GA II
containing as much as double the number of carbohydrate
units per molecule as GA I. The carbohydrateprotein
linkage in the GA was essentially glycosidic to the hydroxyl
group of L-serine and L-threonine residues. There are several
other reports that have described the characterization of
two forms of GA from A. niger (e.g. [94]).
Ueda and Kano [249] puried the GA system of
Rhizopus sp. By fractionation, two kinds of GA were
obtained; one with strong debranching activity and the other
with weak debranching activity. Moriyam et al. [250] studied
the inuence of dielectric constants and ligand binding on the
thermostability of GA from Rhizopus niveus. Investigations
were carried out (mainly at 60 mC and pH 4.5) in relation to
kinetics, in both the presence and absence of various ligands.
With substrate analogues, such as glucose, lactose and
gluconolactones, and with glycerol, the thermostability of
the enzyme greatly increased, whereas with dioxane, ethanol
and glycol it decreased. From the model equations, binding
constants of the ligands were estimated in order to conrm
the validity of the assumptions. GA of R. niveus was also
studied by Saha and Ueda [251] for raw-starch adsorption,
elution and digestion behaviour. The preparation consisted
of ve forms, which were separated by preparative iso-
electric focusing. All of them were strong in debranching
activity, raw-starch adsorption and digestion. Among them,
two were major, GA C and GA D, having isoelectric points
of 8.45 and 9.1, and the carbohydrate contents were 14.9
and 12.7%, respectively.
Two forms of GA were isolated and puried from a
culture of Monascus kaoang nov. sp. F-I, which were
homogeneous in nature. GAI and GAII exhibited pH optima
of 4.5 and 4.7, respectively. The molecular mass of GA I was
48000 Da and that of GA II was 68000 Da [252]. Takeda et
al. [253] studied the purication and substrate specicity of
GA of Paecifomyces varioti AHU 9417. The enzyme was
homogeneous; its molecular mass was estimated to be
69000 Da and the pH optimum was 4.5. The GA from
Piricularia oryzae, the pathogenic fungus of rice blast disease,
was optimally active at pH 6.5 and at temperatures of
5060 mC. Its molecular mass was 94000 Da [246]. The GA
of Thermomyces lanuginosus was established to be hom-
ogenous [254]. This was a glycoprotein with an average
molecular mass of about 57000 Da and a carbohydrate
content of 1012%. Shimazaki et al. [255] puried an
extracellular GAfromSchizophyllum commune by ammonium
sulphate precipitation, acid-clay treatment and Sephadex G-
100 gel ltration, which gave a single band.
A bacterial GA was puried and characterized from the
anaerobic thermophilic bacterium Clostridium thermo-
saccharolyticum by Specka et al. [119]. The enzyme, which
consisted of a single subunit, had an apparent molecular mass
of 75000 Da. The puried enzyme attacked -1,4 as well as
-1,6 glycosidic linkages in various -glucans. The enzyme
showed a pH optimum of 5.0 and a temperature optimum of
70 mC, and attacked polysaccharides preferentially. Table 3
lists some properties of GAs from some other sources
[256270].
Conclusions
Amylases are among the most important enzymes used for
industrial purposes, and now in the light of biotechnology
they are considered useful for biopharmaceutical appli-
cations. They are useful tools in medicinal and clinical
chemistry. Although amylases can be derived from a number
of sources, microbial sources, especially for -amylase and
GA, are signicant for commercial production. Since starch
is the only natural substrate to be hydrolysed by amylases, it
is desirable to isolate efcient microbial strains producing
high amylase titres active on raw starch. Starch-hydrolysis
processes may be improved by the use of new thermostable
GA that is mutually compatible with -amylase, which would
lead to the whole process being performed in a single step,
providing economic benets. Microbial strains with efcient
dual activity, i.e. liquecation and saccharication, e.g.
amylolytic yeasts, need to be developed. Microbial strains
with efcient -amylase activity should be developed as the
enzyme could be used to produce maltose syrup. 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 micro-
organisms. Finally, it is hoped that amylases will continue to
provide new opportunities in biotechnology as biocatalysts
and that new applications will emerge in the bio-
pharmaceutical sector.
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Received 23 July 1999\7 November 1999; accepted 21 January 2000

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