CHAPTER
TWO
REVIEW OF LITERATURE
Amylases are enzymes that break down starch or glycogen into sugars. The recent
discoveries of starch degrading enzymes have led to increased application of amylases in various
industrial processes. Starch is a polysaccharide consisting of two molecules-amylose and
DP\ORSHFWLQH $P\ORVH LV IRUPHG IURP FKDLQV RI JOXFRVH OLQNHG E\ Į-1,4 branches and
DP\ORSHFWLQHLVIRUPHGIURPĮ-1,4 linked chains of glucose with 1,6 linked branch points.
)LJXUH6WUXFWXUHRIVWDUFKVKRZLQJĮ-DQGĮ-6) linkages
7KH Į-amylase (1,4-Į-D-JOXFDQ JOXFDQRK\GURODVH (& K\GURO\VHV Į-1,4 glucosidic
linkages in starch and related substrates. These starch degrading amylolytic enzymes are of great
significance in biotechnological applications ranging from food, fermentation, textile, paper,
pharmaceutical to sugar industries. The amylase can be derived from several sources such as
plants, animals and microbes however; enzyme from fungal and bacterial sources has dominated
applications in industrial sectors. The major advantage of using microorganisms for production
11
of amylases is in economical production capacity and microbes are also easy to manipulate to
obtain enzymes of desired characteristics. Two types of amylolytic enzymes have been
recognized called as liquefying and saccharifying. The main difference between them is that the
saccharifying enzyme produces a higher yield of reducing sugar than liquefying enzyme. The
enzyme production is dependent on the type of strain, composition of media and methods of
cultivation. Generally fungi secrete alpha amylase (dextrinizing enzymes) although a few fungi
have been known to secrete alpha amylase and beta amylase (saccharifying enzymes). The
application of an amylase in industrial reactions depends on its unique characteristics, such as its
action pattern, substrate specificity, major reaction products, optimal temperature, and optimal
S+%DFWHULDOĮͲamylase preferred for application in starch processing and textile industries due
to its action at higher temperature (75-105oC) and neutral to alkaline pH.
2.1 Mode of fermentation
2.1.1 Solid state fermentation
Alpha amylase production by a strain of Bacillus subtillis MAFE 118079 have been
demonstrated in solid state fermentation utilizing rice bran as substrate. A maximum amylase
production of 2311.1 U/g was showed with pH-7, when SSF was carried out at 37oC for 48 hr
using a substrate with 75% initial moisture. The effect of different carbon and nitrogen source
were also analyzed. The results showed a 1% increase in amylase production with soluble starch
and glucose [Rameshkumar A and Sivasudha T, 2005 ]. In a similar study reported using
Bacillus subtilis MTCC 1305 in SSF, substrates including wheat bran (WB), corn flour (CF), rye
straw (RS), wheat straw (WS) and rice bran (RB) were analyzed and the results showed a better
Į-amylase production foU ZKHDW EUDQ ,W KDV EHHQ IRXQG WKDW WKH Į-amylase production is the
highest at 80 hr incubation period, 55oC incubation temperatures, substrate: moisture ratio 1:1,
12
pH of 5.0 and 10% inoculum level. Glucose (0.05 g/g) has been found the best supplementary
carbon source. Supplementation of different nitrogen sources (0.02 g/g) showed decline in
enzyme production [Singh RK et al., 2010].
2SWLPL]DWLRQ RI WKHUPRVWDEOH Į-amylase production by Streptomyces erumpens MTCC
7317 in solid-state fermentation using cassava fibrous residue have been reported. Response
surface methodology (RSM) was used to evaluate the effect of incubation period (60 h), moisture
holding capacity (60%) and temperature (50oC) on enzyme production. Varying the inoculum
concentration (5-25%) of S. erumpens showed that 15% inoculum (v/w, 2.5 x 10 6 CFU/ml) was
WKH RSWLPXP IRU Į- amylase production. The results also showed that, beef extract was most
suitable nitrogen source for enzyme production. The maximum hydrolysis of soluble starch
(85%) and cassava starch (70%) was obtained with the application of 5ml crude enzyme (17185
units) after 5 h of incubation [Kar S et al., 2010].
3URGXFWLRQ RI Į-amylase from Penicillium chrysogenum under solid state fermentation
using agricultural by-products such as corncob leaf (CL), rye straw (RS), wheat straw (WS) and
wheat bran (WB) was investigated. Optimal moisture levels of substrates were 75, 65, 65 and 55
% for CL, WS, WB and RS substrates, respectively. Optimal particle size and inoculum
concentration IRUWKHSURGXFWLRQRIĮ-amylase were: >1 mm, 20 %; >1 mm, 20 %; 1 mm, 20 %
and >1 mm, 30 % for CL, WS, WB and RS, respectively. Maximum enzyme production of 160
U/ml was reported under the optimum conditions with wheat bran as substrate [Balkan B and
Ertan F, 2007].
A new isolate of Aspergillus VS0. ZDV XWLOL]HG IRU WKH SURGXFWLRQ RI Į-amylase by
SSF. Agricultural residual substrates like wheat bran, rice bran and green gram husk were
13
studied and the highest enzyme production was obtained with wheat bran as a substrate. The
results showed that a maximum enzyme production of 164 U/g was obtained under the optimum
conditions of 120 hr of incubation period, 30°C of temperature, 70% initial moisture content, 5.0
pH and 5% inoculum level [Chimata MK et al., 2010@ ,QYHVWLJDWLRQV RQ WKH SURGXFWLRQ RI Įamylase by Aspergillus niger and B. subtilis using wheat bran and rice bran as substrate have
also been reported [Nimkar MD et al., 2010]. Production and characterization of fungal amylase
produced under solid state fermentation by Aspergillus sp. JGI 12 have been reported. Substrates
including coconut oil cake, groundnut oil cake and rice bran were found to be effective and their
different combinations; wheat bran : groundnut oil cake: rice bran (1:2:2) resulted in higher
enzyme titre. The enzyme thus produced was used for characterization and was found to be
thermostable and active at wide range of pH [Alva S et al.,2007].
An investigation on the production of amylases by Aspergillus tamarii in solid state
fermentation under high initial glucose concentrations were carried out. They reported a
resistance to catabolite repression of the cultures grown on wheat bran under solid state, even at
high concentration of glucose (10%) and thus showed the potential of solid state systems to
overcome the adverse effects of high sugar concentrations in the media. The ability to prevent
catabolite repression appears to be related with the content moisture of solid state systems: less
content moisture of cultures means less catabolite repression caused by glucose [de Souza DF
and Peralta RM, 2001].
14
A study on the production of amylase by Aspergillus niger LPB 28 under solid state
fermentation using cassava starch and sugar cane bagasse present was reported. Different ratios
of cassava starch to sugar cane bagasse (1/1; 1.5/1; 1/1.5; 1/2; 2/1; 1/3; 3/1) were also tested and
IRXQG WKDW WKH UDWLRV RI ZDV WKH EHVW IRU RSWLPXP SURGXFWLRQ RI Į-amylase and
amyloglucosidase. The maximum yield of 1732.95 U/g of cassava starch was achieved under the
optimum process parameters of such as incubation period (60 hours), moisture level was 90%,
inoculation rate (105 spores/g of dried material), pH 4.0 and fermentation temperature (30oC).
The results also reported an enhanced production by the nitrogen source (KNO3) [Spier MR et
al., 2006]. Another study reported the enhanced production of amylase using vegetable wastes by
Aspergillus niger SK01 isolated from marine water. Out of the 155 fungal isolates, the strain
A.niger SK01 was screened to be the better strain for the production of alpha amylase using
vegetables as substrate. They showed a maximum enzyme activity at 0% moisture content, 70 oC
temperature and pH 9 [Namasivayam SKR and Nirmala D, 2011]. In a similar study, four fungal
isolates from soil were screened for alpha amylase production and the isolate Aspergillus niger
was found to have best activity among all the four isolates. SSF was carried out under the
optimum growth conditions of 28°C and pH 6.2 using four substrates namely wheat bran, rice
husk, vegetable waste (potato, tomato, brinjal) and banana peel. Alpha amylase produced using
all the four substrates was having good activity but wheat bran as a substrate was the best giving
an activity of 0.08U/ml/min followed by vegetable waste (0.06U/ml/min), banana peels
(0.05U/ml/min) and rice husk (0.045U/ml/min) [Khan JM and Yadav SK , 2010].
Solid state fermentation of Aspergillus oryzae for the production of glucoamylase on the
surface of rice husk, wheat bran, rice bran, cotton seed powder, corn steep solids, bagasse
powder, coconut oil cake, and groundnut oil cake as substrates has been reported. Optimum
15
glucoamylase production was observed on wheat bran supplemented with 1%, (w/w) starch,
0.25%, (w/w) urea at pH 6, 100%, (v/w) initial moisture and 30oC after incubation 120 hrs
[Zambare V, 2010].
Evolutionary operation (EVOP) factorial design technique has been explored in order to
economically produce amylase and protease in a single bioreactor by modified solid-state
fermentation. Maximum yields of amylase and protease were achieved, using wheat bran as a
substrate by a locally isolated strain of Aspergillus awamori Nakazawa MTCC 6652. The highest
secretion of amylase and protease were measured to be 9420.6 and 1930 U/g, respectively, at
37°C. pH and relative humidity were found to be optimum at 4 and 85%, evaluated through
EVOP method [Sangeeta Negi and Rintu Banerjee, 2006].
2.1.2 Submerged fermentation
2SWLPL]DWLRQ RI WKH JURZWK DQG Į-amylase production of Bacillus subtilis IP 5832 in
shake flask and laboratory fermentor batch cultures were examined. The results showed that, 0.5
VWDUFKZDVQHFHVVDU\IRUPD[LPXPĮ-amylase production, inducing 1.55 IU/ml of amylase to
be secreted after 8 hr of cultivation in shaking flasks and a 60 % higher activity (2.5 IU/ml) was
obtained, when fermented in 2L laboratory fermentor [Bozici N et al., 2011]. In a similar study
using Bacillus sp. was investigated using wheat bran, soybean meal and CaCO3 medium. Using
response surface methodology (RSM) the optimum values of pH, temperature and inoculums
size were reported to be 11.35, 35.16oC and 2.95% respectively. Under the optimum conditions
the enzyme activity was reported to increase 4.64 fold in comparison to the basal medium
[Zambare VP, 2011@2SWLPL]DWLRQSURGXFWLRQDQGSDUWLDOSXULILFDWLRQRIH[WUDFHOOXODUĮ-amylase
from Bacillus sp. marini have been investigated. The best enzyme activity of 8000 U of amylase
16
per litre of culture broth was observed at pH 7 and temperature 40°C, 5.5% NaCl concentration,
starch as carbon source and yeast extract as nitrogen source, which is 3 fold higher than before
optimization [Ashwini K et al., 2011].
Į-Amylase production by Penicillium fellutanum isolated from mangrove rhizosphere
soil was investigated. It was reported that the production medium without addition of sea
water and with provision of maltose as carbon source, peptone as nitrogen source,
incubated for 96 hr, maintained with pH of 6.5 at 30 o&ZDVIRXQGRSWLPDOIRUĮ-amylase
by P. fellutanum [Kathiresan K and Manivannan S, 2006].
Submerged fermentation of amylase enzyme by Aspergillus flavus using cocos nucifera
meal was discussed. Cocos nucifera meal medium supplemented with dextrose has shown the
highest amylase production at pH 6.0 and temperature of 30 oC with protein content of 201µg/ml;
98.4µg/ml and dry biomass of 1.28µg/ml [Pandey A et al., 2000].A novel submerged culture
system of A.kawachii NBRC 4308 using barley was reported for the simultaneous production of
glucoamylase and acid-stable alpha amylase. The culture supernatant showed a glucoamylase
activity of 150.8 U/ml and an acid stable a-amylase activity of 7.7 U/ml [Cui YQ van der Lans
RGJM et al., 1998].
A study on the influence of fermentation conditions and scale was reported on the
submerged fermentation of Aspergillus awamori. The hairy length of the pellets was shown to
increase initially and then decreased. At a comparable specific energy dissipation rate, in the
larger scale fermenters, pellets had a larger hairy length than in the smaller. Both counting the
number of pellets during the fermentations and observing pellets under agitated but non growth
conditions indicated that formation and breakage of pellets hardly occurred under the studied
17
conditions [Cui YQ et al., 1998]. In a similar work using Aspergillus awamori the aspects of
using complex media for submerged fermentation have been studied on wheat bran. The results
showed a dominated adhesion growth when the spore concentration was higher than 1.3x105
spores ml-1 and a dominated wheat bran free pellets with concentration lower than 1.8x104 spores
ml-1. In between these two values, the broth suspension consisted of wheat bran free pellets,
clean wheat bran particles, and adhesion colonies. Solid substrate suppressed the growth in free
filamentous mycelial form and was not completely utilized by the organism [Kammoun R et al.,
2008].
Optimization of parameters and culture medium for alpha amylase production by
Aspergillus oryzae CBS 819.72 grown on gruel was reported using a statistical design. The
optimisation of temperature, agitation and inoculum size was attempted using a Box±Behnken
design under the response surface methodology. Nutrients with positive influence on enzyme
formation-KH2PO4, urea, glycerol, (NH4)2SO4, CoCl2, casein hydrolysate, soybean meal
hydrolysate, MgSO4 were selected from the nineteen analyzed using a Plackett±Burman design.
The optimized nutrients concentration was obtained using a Taguchi experimental design and
showed a maximum activity of 151.1 U/ml which was 73.2% higher than the initial activity
[Shoji H et al., 2007].
Nitrogen supplements effect on amylase production by Aspergillus niger using cassava
whey medium was studied. They reported a maximum biomass yield of A. niger of 2.75 g/l
with maximum amylase activity of 643 U/ml, when yeast extract was employed as a
nitrogen supplement and the lowest biomass yield of 0.77 g/l and amylase activity of 206
U/ml when sodium nitrate (NaNO3) was used as nitrogen supplement [Oshoma CE et al.,
2010]. In a similar study, the effect of varying pH, temperature and nitrogen sources of the
18
PHGLXPIRUWKHSURGXFWLYLW\RIĮ- amylase from Aspergillus niger utilizing Ipomoea batatas was
investigated. Maximum amylase activity of 475 U/mg was reported after7 days at pH 7.0, room
temperature 28oC and in the presence of ammonium nitrate as nitrogen source [Sundar R et al.,
2012].
A study on production and stabilization of amylases had been carried out using
Aspergillus niger isolated from boiled rice. Maximum enzyme production was showed when
0.2% (w/v) peptone was used as nitrogen source at a temperature and pH of 30oC and 4.5
respectively for 7 days. The results demonstrated that 30% sorbitol, 30% sucrose and 5% PEG
improved the thermostability of the enzyme [Monga M et al., 2010].
(QKDQFHPHQWRIĮ-Amylase Activity
3URGXFWLRQRIĮ-amylase by the existing microorganisms can be improved considerably
by mutagenesis. The study of enhanced amylase production by a recombinant strain of Bacillus
subtilis ATCC 31784 using a dual exponential feeding strategy had shown that the organism
KDUERULQJ WKH SODVPLG S& ZLWK D WKHUPRVWDEOH Į-amylase gene could prevent substrate and
product inhibitions due to acetate accumulation. A dual feeding stream was developed for
feeding the less soluble tyrosine and tryptophan separately in ammonium water as a second feed
VWUHDPDQGDILQDOĮ-amylase activity of 41.4 U/ml and the overall biomass yield of 0.39 g cell/g
glucose were shown [Huang H et al., 2004]. Studies on strain improvement for the production of
Į-amylase using strong mutagens from Bacillus subtilis had been reported using UV radiation.
The results revealed that the higher enzyme production occurred in Bacillus subtilis with a UV
exposure time of 21 hr. both normal and UV exposed Bacillus subtilis showed maximum enzyme
production at neutral pH [Rani PS and Rukmini A., 2011].
19
+LJK OHYHO SURGXFWLRQ RI WKHUPRVWDEOH Į-amylase has been achieved from Sulfolobus
solfataricus in high-cell density culture of the food yeast Candida utilis. The very poorly
expressed genes of S. solfataricus were resynthesized based on codons preferentially found in the
highly expressed C.utilis glyceraldehyde-3-phosphate dehydrogenase (GAP) gene and the
expression of this synthetic gene under the control of the GAP promoter yielded biologically
DFWLYH Į-amylase, accounting for more than 50% of the soluble protein. This improved the
SURGXFWLRQ OHYHO RI WKH Į-amylase by more than 2x104 fold. The results also suggest that
SUHPDWXUH WHUPLQDWLRQ RI WKH WUDQVFULSWV LV UHVSRQVLEOH IRU WKH ORZ SURGXFWLRQ OHYHO 7KH Įamylase-producing C. utilis cells were grown up to 92 grams dry cell weight per liter in a
V\QWKHWLF PHGLXP \LHOGLQJ JO Į-amylase which accounts for up to 27% of total cell
proteins [Miura Y et al., 1999].
Strain improvement and genetic characterization of Aspergillus flavus FCBP-23 has been
demonstrated for its ability to reveal extra cellular alpha-amylase activity. The selected strains
were subjected to UV irradiation (5-40 min exposure) and ethyl methane sulphonate (EMS)
treatment (50-300 microg ml-1) for hyper activity of an alpha-amylase enzyme. On comparison
with the parental strain, mutant strains Af-UV-5.3 and Af-Ch-5.7 exhibited 79 and 110% more
enzyme activity. They reported a difference in amplicon patterns by randomly amplified
polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis of native as well as mutant
derivatives, which suggested that the mutation imparted changes in the genetic makeup of the
mutants probably involved enzyme production control [Shafique Set al., 2009]. A similar work
was carried out by the same group on amylolytic Aspergillus niger FCBP-198, to modify its
alpha-amylase activity. After UV and EMS treatments, the UV mutant-An-UV-5.6 and the
chemical mutant- An-Ch-4.7 were found to be efficient with enzyme activities of 76.41 units ml-1
20
and 89.38 units ml-1 in comparison with the parental enzyme activity of 34.45 units ml-1. Genetic
relationships of the mutants were analyzed with RAPD-PCR and the results showed that
genotypes of An-Ch-4.7 and An-Ch-4.2 were distinctly classified into one category, while the
isolates An-UV-5.6, An-UV-5.1 and A. niger FCBP-198 have the nearest genetic relationship
[Shafique S et al., 2010].
Į-Amylase production in recombinant Aspergillus oryzae during fed-batch and
continuous cultivations were investigated. Comparison of growth and enzyme production by the
three strains- a wild-type,
a transformant strain of the wild-type strain containing
DGGLWLRQDOFRSLHVRIWKHĮ-amylase gene and a morphological mutant of the transformant
VWUDLQ UHYHDOHG WKDW FKDQJHV LQ WKH PRUSKRORJ\ RI WKH IXQJXV LQIOXHQFH WKH Į-amylase
production during submerged growth [Spohr A et al., 1998].
Į-Amylase production by Bacillus subtilis entrapped in calcium alginate gel capsules
increased the yield and operational stability by tailoring the capsules characteristics. Capsules
prepared from 2% (w/v) sodium alginate and 3.5% (w/v) CaCl2 resulted in a 2.5-fold higher Įamylase production in comparison to the freely suspended cells. They reported that the
immobilized system retained more than 85% of its initial efficiency after 15 fermentation
batches, producing more than 1,450,000 units of extracellular Į-amylase during this period
[Konsoula Z et al., 2006]. Biosynthesis of extracellular amylase by encapsulated Bifidobatrium
bifidumin No.1, 791 in batch culture showed a maximum enzyme activity of 90 U/ml. In
repeated batch fermentation process, the encapsulated cells preserve their ability to produce
enzyme consistently over 21 cycles and the activity remain between 90 and 95 U/ml throughout
the cycles. The optimum pH and temperature of the enzyme were 7.5 and 75oC respectively. The
enzyme was strongly inhibited by Cu2+ and Mg2+ but less affected by Ca2+, Mn2+. In 1M and 5M
21
NaCl solutions, the enzyme retained 70% and 47% of the original activity after 24hr of
incubation at 4oC, respectively [Reyed RM, 2007].
Immobilization of Thermomucor indicae-seudaticae was investigated for the production
of thermostable and neutral glucoamylase in submerged fermentation. Thermomucor indicaeseudaticae ZDVLPPRELOL]HGLQDOJLQDWHț-carrageenan, agarose, agar, polyacrylamide and loofah
(Luffa cylindrica) sponge, and used for the production of glucoamylase. The results showed a
higher enzyme production with alginate-immobilization and a higher mycelial growth with
loofah immobilization [Bo L et al., 2007].
The media composition and culture conditions for tKH SURGXFWLRQ RI Į-amylase
production from free and immobilized cells of Aspergillus niger have been optimized. The
optimum pH, temperature and incubation period for enzyme production was shown to be 5.0,
35°C and 5th day for immobilized cells and 5.0, 30°C and 5th day for free cells respectively. The
enzyme produced had the pH optima ranged at 4-6 and temperature optima ranged at 30-40°C.
Starch was recorded to be the best carbon source and peptone at 0.03% was ideal nitrogen source
for enzyme production [Gupta A et al., 2010]. A study has been reported on the production of
gOXFRDP\ODVH DQG Į-amylase by Aspergillus niger immobilized in calcium alginate gel beads.
The immobilized mycelium produced lower enzyme activities than immobilized spores and in
repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with
immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each
successive batch were possible [Gao-Xiang Liet al., 1984].
22
Į-Amylase with Specific Features
For utilization in industrial process it is desirable to have enzymes active at high
WHPSHUDWXUHV,VRODWLRQDQGLGHQWLILFDWLRQRIDEDFWHULDOVWUDLQSURGXFLQJWKHUPRVWDEOHĮ-amylase
ZDVUHSRUWHG7KHKLJKHVWĮ-amylase producing [7.0 ±0.21 Um/l at 24 hr] was isolated from soil
receiving bakery waste and identified as Bacillus licheniformis ,QWKHDEVHQFHRI DGGLWLYHVĮamylase retained 37.6% of its initial activity at 90oC for 30 min and 10.4% of its activity for 1 hr,
whereas at 80oC and pH 7.0 it retained 68.8% of its initial activity for 30 min and 59.1% of its
initial activity for 1 hr. Half life of the enzyme was 21 min at pH 7.0 and 90 oC [Vaseekaran S et
al., 2010]. Properties of an amylase from thermophilic Bacillus sp. strain SMIA-2 cultivated in
liquid culture have been reported. The optimum temperature and pH of this enzyme were
reported to be 90oC and 8.5 respectively. The enzyme was stable for 1 hr at temperatures ranging
from 40-50oC while at 90oC, 66% of its maximum activity was lost. The enzyme was strongly
inhibited by Co2+, Cu2+ and Ba2+, but less affected by Mg2+, Na+ and K+. In the presence of 2.0
M NaCl, 63% of amylase activity was retained after 2 hr incubation at 45oC. The amylase
exhibited more than 70% activity when incubated for 1 hr at 50oC with sodium dodecyl sulphate
[De Carvalho RV et al., 2008].
6WXGLHV RQ WKH Į-amylase producing thermophilic bacterium Bacillus sphaericus
were carried out in a laboratory scale fermenter and the optimum conditions were found to
be pH 7 and 50oC [Al-Qodah Z et al., 2007]. Thermophilic and hyperthermophilic bacterial
population of three Iranian hot-VSULQJV ZHUH VFUHHQHG WR GHWHFW WKH WKHUPRVWDEOH Į- amylase
producing strain. They isolated a Bacillus sp. which produced maximum enzyme level at 70 °C
in the presence of soluble starch (1%) at pH 6. The addition of calcium (10 mM) and peptone
WRWKHPLQHUDOPHGLXPLPSURYHGWKHJURZWKDQGĮ-amylase synthesis whereas the addition
23
of glucose (1%) to the culture greatly diminished its syntheses. The extracted enzyme retained
100% activity when incubated for 45 minutes at 100°C [Fooladi J and Sajjadian A, 2010].
$QRWKHUZRUNGHPRQVWUDWHGWKHVLPXOWDQHRXVSURGXFWLRQRIWKHUPRVWDEOHĮ-amylase and neutral
proteases using the thermophilic strain Bacillus caldolyticus DSM 405. According to the
UHSRUWHG UHVXOWV WKH XVH RI SHD SXOS LQ WKH PHGLXP LQFUHDVHG WKH Į-amylase activity by 160%
with 97% reduction in medium costs in comparison to control medium [Jamrath T et al., 2011].
Screening and phenotypic characterization of thermostable amylases producing yeasts
and bacteria strains from some Cameroonian soils have been reported. Among the one hundred
and nineteen amylases producing strains, the yeast strain designated as 04LBA3 and the
bacterial strains designated as 04BBA15 and 04BBA19 showed very high amylolytic potential
and were characterized as Schwanniomyces
alluvius,Bacillus
amyloliquefaciens and
Lactobacillus fermentum respectively. Cluster analysis on the basis of amylolytic activity
and thermostability of crude amylase extract showed similarities between strains of same
geographic origin. These results concluded that amylase activity and thermostabilty can serve as
indicators for micro-organisms traceability [Fossi BT et al., 2009].
Culture conditions for the production of thermostable amylase by Penicillium rugulosum
were investigated. Maximum production of amylase was observed on 3rd day at an initial pH of
7.0. The results showed an improved amylase production in the presence of galactose and
peptone whereas the presence of EDTA and metal ions Mn2+ and Fe2+ inhibited the enzyme
activity [Tiwari KL et al., 2007].
Amylases production of the thermophilic fungus Thermomyces lanuginosus IISc 91was
investigated. The enzyme was shown to be stable for more than 7 hr at 50°C with a maximum
24
activity at the pH and temperatures of 5·6 and 65°C respectively. At 65°C, Į-amylase was nearly
8-times more stable in the presence of calcium. Addition of calcium increased the melting
temperature of Į-amylase from 66°C to 73°C. Upon incubation at 94°C, Į-amylase was
progressively and irreversibly inactivated, and converted into an inactive 72 kDa trimeric species
[Mishra RS and Maheshwari R, 1996].
Growth profile and amylolytic activity of the thermophilic fungus Aspergillus fumigatus
isolated from soil samples at 50°C have been reported. They reported a maximum growth and
amylase production after 96 hr incubation at 30°C in a mineral medium containing 1% starch and
1.5% organic nitrogen concentration. Use of sorghum starch as carbon source and a combination
of inorganic (NH4Cl) and organic (soybean meal) nitrogen sources led to the synthesis of higher
concentration of amylase in culture fluid. The results also indicated a slight repression in
amylase activity by Fe2+ and Mn2+ but not inhibited by Cu2+, Co2+ and Hg2+ at a concentration of
2 mM [Nwagu TN and Okolo BN, 2011].
The thermophilic Į-Amylase produced by Aspergillus niger van Tieghem has been
reported to be purified and characterized. The enzyme was purified from the culture filtrate by
DEAE-Sephadex chromatography and was established by gel electrophoresis and confirmed by
sedimentation studies. This enzyme was shown to exhibit a lower energy of activation, increased
tolerance to lower pH and enhanced affinity to starch. While Ag+, Pb2+, Hg+, Al3+ and EDTA
inhibited the activity of the enzyme, Ca2+ enhanced its activity [Ramasesh N et al., 1982].
Investigations on the characterization of amylase from a thermophilic Aspergillus niger isolated
from municipal compost soil have been reported. The highest yield of amylase was obtained with
the agronomic waste rice bran and the addition of soluble starch and peptone as carbon and
nitrogen supplements, respectively, enhanced the enzyme production. The optimum pH,
25
temperature and incubation period for amylase production by the isolate were found to be 6.0,
30°C and 120 hr, respectively. The partially purified enzyme was optimally active at 70°C and
pH 5.0 and 9.0 [Kumari S et al., 2012].
$Q DFLG WROHUDQW Į-amylase was reported using Pseudomonas sp. 2 isolated from
rhizosphere. Vitamins B12 and the amino acids cysteine (30 mg), tyrosine (40 mg) and alanine
(30 mg) were found to stimulate the enzyme production. The enzyme was partially purified by
ion exchange DEAE-cellulose column Chromatography followed by ammonium sulphate
precipitation, which yielded an enzyme with 45.2 fold purity than the crude enzyme. The
molecular weight of the enzyme was reported to be approximately 62 kDa [Varalakshmi KN et
al., 2012].
A thermophilic and acidophilic amylase with unusual characteristics has been produced
by a thermophilic acidophilic Bacillus sp. 11 1S. The pH optimum for activity was 2.0, and
substantial activity was noted in the pH range of 1.5 to 3.5. The optimal temperature was 70°C,
but the activity decreased markedly in lower reaction temperatures. Unlike most of the amylases,
the activity or stability of the enzyme was not likely to depend on Ca2+. About 34% of glucosidic
linkages of soluble starch were hydrolyzed at 65°C and pH 2.0, in 24 hr, and the major
hydrolytic products were maltotriose and maltose [Uchino F, 1982].
A new breed of Aspergillus niger Tx-78 was investigated for its ability to produce acid
VWDEOHDQGDFLGDFWLYHĮ-amylase, which was purified with alcohol precipitation, CM52 and DE52
cellulose ion-exchange chromatography, and its purity was tested with SDS-PAGE and got its
molecular weight at 74 ku. The most suitable reaction temperature and pH were 70Ԩ and 4.0
respectively and the ions Ca2+, and Ba2+ were found to increase its activity [Bo L et al., 2008].
26
The influence of fermentation components on extracellular acid amylase by Aspergillus
awamori has been evaluated based on Taguchi method. Under the optimized media
composition of soluble starch ± 3%; CSL ± 0.5%; KH 2PO4± 0.125%; MgSO4.7H2O ±
0.125%; casein ± 1.5 % a t p H 4 . 0 and temperature at 31°C 48% increase in the enzyme
activity was reported [Prakasham RS et al., 2007].
Statistical optimization and partial characterization of amylases produced by halotolerant
Penicillium sp. was carried out in submerged fermentation. Maximum enzyme production was
obtained after two days of incubation at pH 4 in a medium containing 10% (w/v) NaCl, 1% (w/v)
MgSO4 .7H2O and 1% (w/v) of starch. Partial characterization of the crude enzyme revealed the
presence of two enzymes with pH-optima at 9 and 11. Furthermore, the optimum temperature
was 30°C at pH 11 and from 30°C to 90°C at pH value of 9. The two enzymes were stable in
wide pH range with the maximum stability at pH 9 to 11 after 5 days and at temperature range of
40-60°C [Gouda M and Elbahloul Y, 2008].
Amylase from moderate halophiles isolated from wild ass excreta has been investigated.
The 24 isolates were able to grow optimally at pH 5-6, 30-Û&WHPSHUDWXUHDQG-15% NaCl
(w/v), no growth was reported below 5% NaCl indicated moderate halophilic nature of the
isolates. Out of the nine moderate halophiles producing extracellular amylase, Bacillus
macquariensis was the best amylase producer. The organism secretes maximum amylase at pH
WHPSHUDWXUH Û& VWDUFK DQG SHSWRQH DV QLWURJHQ VRXUFH DIWHU KU >Khunt M et al.,
2011]. A study on the production and biochemical characterization of Į-amylase from the
moderate halophile Halomonas meridian has been reported. The highest amylase production was
achieved by growing H. meridian cultures in media with 5% salts and starch, in the absence of
glucose until the end of the exponential phase and the optimal temperature and salinity for
27
activity were 37oC and 10% NaCl, respectively. The amylase exhibited maximal activity at pH
7.0, which is relatively stable in alkaline conditions. The results showed enzyme activity at
salinity as high as 30% salts [Coronado MJ et al., 2000].
.LQHWLFVRIĮ-amylase Production
$ VWXG\ RQ RSWLPL]DWLRQ RI WKH LQGXVWULDO SURGXFWLRQ RI EDFWHULDO Į-amylase and the
analysis of kinetic data for enzyme production by two strains of Bacillus amyloliquefaciens
(strain 267 and strain 267CH) has been demonstrated. Application of logistic, Luedeking-Piret
and the modified Luedeking-Piret mathematical models WRWKHNLQHWLFGDWDUHYHDOHGWKDWĮamylase production in both cases occurred through both growth- and non-growth
associated mechanisms and that the amount of enzyme produced through non-growth
associated mechanism exceeded that produced through growth associated mechanism by
3.5 and 2.3 fold by strains 267 and 267CH, respectively. In the bioreactor, increasing
aeration from 1 to 2 vvm increased the overall specific growth rate, the production rate,
the specific production rate, and the specific substrate consumption rate and also
shortened the time necessary for maximum production of both biomass and enzyme. The
result also showed an increase in enzyme production with increase in biomass and even after
reaching the maximum biomass it still continued to increase [Mohammad F et al., 2007].
$QRWKHU VWXG\ HYDOXDWHG WKH NLQHWLF SDUDPHWHUV RI Į-amylase producing thermophile Bacillus
sphaericus. The growth and enzyme production optimum conditions were pH 7 and 50oC.
The kinetic study of cellular growth indicates µ max, Ks, Td, Yx/s and kd were 0.53 hr-1,
1.1 g/l, 1.98 hr, 0.44 g cell/g starch and 0.4 g/l/hr, respectively.
The optimum starch
concentration for the enzyme production was 32 g/l and higher concentrations show
substrate inhibition with inhibition constant Ki 190 mg/l. The kinetic parameters of -amylase
28
activation Vmax, and Km were 263 mole mg-1 enzyme min-1 and 0.97 mg/ml, respectively
[Abe J et al., 1988].
Morphology and physiology of Į-amylase production has been investigated for the batch
cultivation of Aspergillus oryzae. The kinetic parameter k in the cube-root law is derived from
the growth kinetics with no mass transfer limitation, k ȝhȝh - hyphal length). Based on an
R[\JHQ EDODQFH WKH DFWLYH JURZWK OD\HU LQ WKH SHOOHW LV HVWLPDWHG WR EH WR ȝP DQG
consequently, up to 50% of the biomass is limited by oxygen for large pellets. A constitutive,
ORZ Į-DP\ODVH SURGXFWLRQ ZDV REVHUYHG DW KLJK JOXFRVH FRQFHQWUDWLRQ DQG WKH VSHFLILF Įamylase production was significantly higher for filamentous growth than for pellets [Carlsen M
et al., 1996]. Another study was reported on the kinetics of alpha-amylase secretion by
Aspergillus oryzae. Methionine incorporation and protein secretion kinetics in Aspergillus oryzae
have been studied by pulse and pulse-chase experiments. Pulse experiments confirmed the
mechanism of methionine uptake and pulse-chase experiments were carried out to investigate the
alpha-amylase secretion kinetics. No unglycosylated alpha-amylase was detected neither
intracellularly nor extracellularly demonstrating that glycosylation was not the rate controlling
step in the secretory pathway. The pulse chase experiments indicated that there are two pools of
intracellular alpha-amylase: a fast secreted and a slow secreted and the secretions of the two
pools were described with a kinetic model fitted to the pulse chase experiments [Santerre
Henriksen AL et al., 1999].
29