Vol. 13(46), pp. 4338-4346, 12 November, 2014
DOI: 10.5897/AJB2013. 13424
Article Number: E08AE0648573
ISSN 1684-5315 African Journal of Biotechnology
Copyright © 2014
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJB

Full Length Research Paper

Production of amylase enzyme from mangrove

fungal isolates
K. Sahoo1*, N. K. Dhal1 and R. Das2
1
Environment and Sustainability Department, CSIR-Institute of Mineral and Materials Technology, Bhubaneswar-
751013, Odisha, India.
2
Depertment of Botany, Utkal University, Bhubaneswar-751004, Odisha, India.
Received 27 October, 2013; Accepted 21 October, 2014

The mangrove ecosystem serves as a bioresource for various industrially important microorganisms.
The use of fungi as a source of industrially relevant enzymes led to an increased interest in the
application of microbial enzymes in various industrial processes. Fungal colonies were isolated from
sediments of five different mangrove ecosystems of Odisha such as Bhitarakanika, Dhamra, Mahanadi,
Devi and Budhabalanga areas. Forty (40) fungal colonies were isolated and screened for amylase
activity out of which five strains were found to be more active. Among the isolates IFamy value was
maximum; that is, 5.2 for MSF-9. The amylase enzyme activity of MSF-9 was maximum at pH-5.0, 1%
NaCl, 1% substrate and Inositol as carbon source. The most potent fungi was identified through
morphological, microscopical and 18S rDNA sequence methods and identified as Penicillium citrinum-
JQ249898. This strain can be better utilized in large scale industries for enzyme production. Hence
further study is suggested on enzyme purification for various value-added product formation.

Key words: Mangrove, fungi, IFamy, Penicillium citrinum, microscopical study.

INTRODUCTION

Marine fungi is an important target group for various The enzyme has found numerous applications in
useful products of industrial importance such as commercial processes, including thinning and liquefaction
enzymes, sugars, antibiotics, alcohols, beverages, food of starch in alcohol, brewing and sugar industries. α-
products etc (Gupta et al., 2007). Study based on Amylases are hydrolytic enzymes that are widespread in
screening of fungal resources of mangroves for enzyme nature, being found in animals, microorganisms and
and their application has been done to accomplish plants (Octávio et al, 2000). In fungi, detailed studies on
environment friendly technological development (Maria et α-amylase purification have largely been limited to a few
al., 2005). Microbial enzymes have completely replaced species of fungi (AbouZeid, 1997; Khoo et al., 1994). The
the chemical hydrolysis of starch in starch process major advantage of using microorganisms for production
industry (Pandey et al., 2000). Amylases are among the of amylases is in economical bulk production capacity
most important enzymes in present-day biotechnology. and microbes are also easy to manipulate to obtain

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
 Sahoo et al. 4339

enzymes of desired characteristics (Lonsane and (Wood and Bhat, 1988). The mixture was incubated for 10 min at
Ramesh, 1990). 30°C. The amount of reducing sugars released was determined by
dinitrosalicylate method (Miller, 1959) at 540 nm and is expressed
In the present study fungal strains were isolated from in units (one unit is the amount of enzyme releasing 1 mg of
five different mangrove ecosystems of Odisha, India. The glucose per mL per minute).
screening of the fungal isolates and enzyme assay of the
most active strain will be carried out. Finally the identi-
fication of potent fungal strain was done by morpho- Optimization of culture conditions
logical, microscopical and molecular study. The factors such as pH, salinity, carbon sources and different
substrate conc. concentrations and affecting production of amylase
MATERIALS AND METHODS were optimized by varying the parameters one at a time. The
experiments were conducted in 250 mL Erlenmeyer flask containing
Collection of sediment 150 mL of production medium for each parameter. After sterilization
by autoclaving, the flasks were cooled and inoculated with culture
Sediment samples were collected from five different mangrove and maintained under various culture conditions such as pH (3.0,
ecosystems such as Bhitarakanika, Dhamra, Mahanadi, Devi and 5.0, 7.0, 9.0 and 11.0), carbon source (D-glucose, lactose, dex-
Budhabalanga areas of Odisha, India. Sediment samples were trose, sucrose and inositol at 1%), salinity (0, 0.5, 1, 1.5, 2.0 and
collected with an air dried Beckman’s grab. The sediment collected 2.5% of NaCl) and substrate conc. (0, 0.5, 1, 1.5, 2.0 and 2.5% of
were aseptically transferred to labeled polythene bags and kept in starch). After 72 hr (expect for incubation period effect), 4 mL of the
an ice-chest box before transferring to the laboratory. The collected culture filtrate was assayed in triplicate to study the enzyme activity.
sediments were air dried for 7-10 days at room temperature in the
laboratory for further study.
Statistical analysis

Isolation of microorganism All experiments were carried out in triplicates, and repeated three
times. The samples collected from each replicate were tested for
Isolation of fungi has been was done through serial dilution amylase production and activity. Means and standard errors of
technique by taking one gram of air-dried sediment samples. The amylase activity and production were calculated, respectively. and
air-dried sediment sample was serially diluted with 50% sea water significant differences were calculated by determining standard
and plated on the Potato Dextrose Agar (PDA) media (Himedia) by error. The significance level of the individual parameters was done
spread plate method with the addition of 100 mg/L of ampicillin to through The data were statistically analyzed using one way analysis
avoid unwanted growth of bacteria and the culture plates were of variance (ANOVA) and Tukey’s multiple comparision test with the
incubated at 28°C for 36-48 h. The total viable count (TVC) was aid of the software, Graph pad 5.0; data with p-values lesser than
estimated from the mixed culture plate. The pure culture was 0.05 were considered significant.
obtained by repeated sub-culturing of the fungal isolates from the
mix culture plates. Then these pure cultures were maintained in
fresh PDA slants and preserved at 4°C for further study. Identification of potent strains

Morphological analysis
Amylase enzyme assay
The fungal isolates were identified by studying the morphological
Screening of isolates
and microscopical study. Morphological characterization was
Primary screening was done by using 1% of starch soluble in the carried done by studying the upper and lower surface of the culture
media plate. The results were recorded by using Gram’s iodide in plate.
the agar plate and the strains showing hydrolysis were selected and
index value (IFamy) has been determines for the isolates. Microscopical analysis

Production medium The microscopical analysis has been done by using Lactophenol
cotton blue (LPCB) staining method and were observed under
The production medium used for growth of the fungal isolate was compound microscope. The fungal spores were also observed
soluble starch 50 g, yeast extract 0.5 g, KH2PO4 10 g, (NH4)2SO4 under the SEM to observe the spore surface and structure.
10.5 g, MgSO4 0.3 g, CaCl2 0.5 g, FeSO4 0.013 g, MnSO4 0.004 g,
ZnSO4 0.004 g, CoCl 0.0067 g, 50% seawater. The pH was
adjusted to 6.5 and the media were sterilized in an autoclave for 15 Molecular taxonomy of the potent isolates
min at 121°C. The media were inoculated with a loop-full of fungal
spore suspension and incubated at 30°C in an orbital shaker set at Phylogenetic (18S rDNA) analysis of the strain the potent the most
100 rpm for 72 hrs. The media were centrifuged at 5,000 g for 15 amylase-producing strain possessing maximum IFAMY value was
min to obtain crude enzyme solution. carried out using following procedures

Enzyme assay Genomic DNA isolation

Amylase assay has been was carried out by taking the reaction Genomic DNA was isolated by using approximately 50mg of
mixture (4 mL) which consisted of 1 mL of centrifuged enzyme mycelium (wet weight) from a fresh culture plate as described by
solution and 2 mL of soluble starch in phosphate buffer, pH 6.5 Hapwood et al. (1985).
4340 Afr. J. Biotechnol.

6%  5%  1%
14% 
74% 

Figure 1. Comparative study of fungal diversity of mangrove ecosystems of Orissa.

PCR amplification of the 18S rDNA mangrove sediments of Orissa (Figure 1).
Genomic DNA from the fungal cultures was used to perform PCR
reaction. The 500bp of rDNA fragment was amplified using high
fidelity PCR polymerase. 10 µL of the amplifications were analyzed Amylase screening
on 1% agarose gel.
Maximum index value IFamy was observed in case of
MSF-9 that is, 5.2 whereas in MSF-3, MSF-7, MSF-13
18S rDNA sequencing and MSF-28 it was 3.09, 2.68, 3.63 and 4.75,
The purified fragments were directly sequenced bidirectionally. The
respectively (Figure 2).
data was analyzed using applied biosystem DNA editing and
assembly software and sequence comparisons were obtained using
the micro Seq Software. Amylase assay

Effect of pH
Sample identification

A distance matrix was generated using the Jukes-Cantor corrected The most potent amylase-producing fungal culture (MSF-
distance model. Identification was done on the pair wise alignment 9) was inoculated into the flask containing amylase assay
algorithms and phylogenetic tree. media at different pH such as 3.0, 5.0, 7.0, 9.0 and 11.0.
The enzyme activity was maximum at pH-5.0, that is,
Phylogenetic analysis
80.68±0.09U mL-1 whereas minimum activity; that is,
22.67±0.33 U mL-1 was observed at pH-3.0. At pH-7.0
Sequence similarity search was made for the 18S rDNA sequence and 9.0 the enzyme activity was 67.44±0.23 U mL-1 and
of the fungal strain MSF-9 by applying its sequence to BLAST 58.58±0.41 U mL-1 respectively. The enzyme activity was
search for in NCBI. The Evolutionary tree was inferred by using first increased up to pH 5.0 and then gradually decreased
neighbor-joining method (Thompson et al., 1997). The CLUSTAL X with the increase of pH (Figure 3).
package was used for multiple alignment and identification of the
strains.

Effect of NaCl
RESULTS AND DISCUSSION
The isolate MSF-9 was inoculated into the flask
Diversity analysis containing amylase assay broth at different NaCl
concentrations such as 0, 0.5, 1, 1.5, 2 and 2.5%. The
The total fungal diversity was found to be maximum that enzyme activity was maximum at 1% NaCl; that is,
is, 74% at Bhitarakanika, 14% at Dhamra, 6% at 36.89±0.26 U mL-1, whereas minimum activity
Mahanadi, 5% at Devi and least 1% at Budhabalanga (6.76±0.063 U mL-1) at 0% NaCl. At various concen-
 Sahoo et al. 4341

Figure 2. Screening for amylase enzyme activity of mangrove fungal isolates.

Figure 4. Effect of NaCl conc. (in %) on amylse enzyme activity of

Figure 3. Effect of pH on amylse enzyme activity of MSF-9 strain. MSF-9 strain.

trations of NaCl such as 0.50, 1.50, 2 and 2.5%, the

enzyme activity was 17.84±0.14 U mL-1, 26.66±0.18 U 98.25±0.23 U mL-1, whereas minimum activity
mL-1, 18.13±0.38 U mL-1 and 13.06±0.30 U mL-1 respec- (12.64±0.711 U mL-1) at 0% substrate. At various
tively. The enzyme activity first increased up to 1% NaCl concentrations of 0.50, 1.50, 2 and 2.5%, the enzyme
and then gradually decreased (Figure 4). activity was 49.02±0.54 U mL-1, 65.42±0.45 U mL-1,
54.86±0.69 U mL-1 and 45.51±0.34 U mL-1 respectively.
The enzyme activity first increased up to 1% starch
Effect of substrate concentration
soluble and then gradually decreased (Figure 5).
The selected strain MSF-9 was inoculated into the flask
containing amylase assay broth at different substrate Effect of carbon sources
levels such as 0, 0.5, 1, 1.5, 2 and 2.5%. The enzyme
activity was maximum at 1% starch soluble; that is, The amylase enzyme activity of MSF-9 was recorded at
4342 Afr. J. Biotechnol.

Identification

Morphological and microscopic analysis

Morphological study of the fungal isolates was carried out

by growing the isolates on PDA growth media. Among
the forty different isolates, twenty isolates such as MSF-1
to MSF-20 were found commonly in all the five study
areas. The twenty strains were further characterized and
screened for the presence of amylase enzyme. Out of the
20 isolates, five strains (MSF-3, MSF-7, MSF-9, MSF-13
and MSF-28) were found to possess more activity after
screening and were observed under microscope by using
cotton blue stain (Figure 2). The growth patterns of the
potent fungal isolate; that is, MSF-9 was carried out in
different media such as PDA, CDA (Czapek Dox Agar)
and MA (Mycological Agar). It was showing very good
Figure 5. Effect of substrate conc. (starch) on amylse enzyme growth in CDA medium than other two media (Table 1
activity of MSF-9 strain.
and 2). The mycellial structure observed under micro-
scope and identified (Figure 7).

SEM Study

The dried aerial mycelium with spore of the potent fungal

strain (MSF-9) was studied by cover slip culture method
in the SEM (Zeiss). The spores were attached like
woolen balls and the round balls like structures forms a
chain which observed at different magnifications from low
to high; that is, 4.21K×10, 000, 8.78 K×10,000, 23.98
K×10,000 and 29.32 K×10,000 at 2.00 KV. From the
spore structures it was assumed to be the member of the
genus Penicillium (Figure 8).

Molecular identification of the potent most productive

isolate (MSF-9)

The potent most productive fungal isolate, MSF-9, was

identified by molecular methodology. Partial 18S rDNA
Figure 6. Effect of carbon sources on enzyme activity.
sequence having up to 555 bp was amplified by PCR.
The similarity pattern of the target sequence was
compared with other sequences in NCBI database. The
similarity pattern of the target isolate was compared with
different carbon sources such as D-glucose, Dextrose, other strains and it was showing showed 97% significant
Sucrose, Inositol, and Lactose. The enzyme activity of homology with Penicillium citrinum GZU-BCECYN60-2 by
MSF-9 was found to be minimum in lactose using the universal marker; that is, F-27 and R-1492. The
(127.58±0.754 U mL-1) but maximum in inositol band pattern of the PCR product of MSF-9 resembles
(140.58±0.22 U mL-1). In case of control, D-glucose, 97% with of the strain Penicillium citrinum (Table 4).
dextrose and sucrose, the activity was 24.84±0.05 U mL-
1
, 128.664±0.39 U mL-1, 126.74±0.079 U mL-1, and Phylogenetic tree
131.042±0.079 U mL-1 respectively (Figure 6).
Significant relationship between individual of variables The evolutionary history was inferred using the Neighbor-
such as pH, NaCl conc. concentration, substrate concen- Joining method (Saitou and Nei, 1987). Phylogenetic
tration and carbon sources was observed through the analyses were conducted in MEGA4 (Tamura et al.,
one way analysis of variance (ANOVA) and Tukey’s 2007). From the tree it can be confirmed that the isolate
multiple comparison tests (P<0.05). belongs to Penicillium citrinum (Figure 10).
 Sahoo et al. 4343

Table 1. Morphological and microscopic characteristics of the fungal isolates.

Culture Morphological characteristics

MSF-3 Black colony with whitish margin, smooth, reverse light yellow
MSF-7 Colonies growing rapidly up to 9cm in four days, whitish, yellowish green
MSF-9 Pale yellowish brown colony, white periphery, reverse light brownish, medium to small in size
MSF-13 White at first becoming pinkish later on, small to medium colony
MSF-28 Greenish, slow growing, reverse pale yellow in colour

Table 2. Identification of fungal Isolates showing maximum amylase

enzyme activity.

Given names ID No. Identified strains

MSF-3 Aspergillus niger
MSF-7 3724.10 Trichoderma viride
MSF-9 3728.10 Penicillium citrinum
MSF-13 3731.10 Paecilomyces variotii
MSF-28 4108.10 Eurotium amstelodmi

MSF-3

(1) (2)
MSF-7

(1) (2)
MSF-9

(1) (2)

Figure 7. Pure culture ((1)) and respective microscopic photo ((2)) of

MSF-3, MSF-7, MSF-9, MSF-13 and MSF-28 fungal isolates
4344 Afr. J. Biotechnol.

(a) (b)

Figure 8. SEM photos of the fungal isolate (MSF-9) having magnification at 23.98 K×10,000 (a) and
8.78 K × 10, 000 (b).

Table 3. Growth characteristics of MSF-9 in different media.

Media Used Growth characteristics

PDA Good growth (Upper part- Light brownish, lower part- off white or light orange, pigment- No)
CDA Very good (Upper part- Light brownish, lower- yellowish black, Pigment- yellow to black soluble)
MA Slow growth (Upper part- Light brownish, lower- yellowish black, Pigment- No)
PDA; CDA; MA, Mycological agar

DISCUSSION reports concerning the optimization of media composition

for fungal strains in amylase production (Quang et al.,
Distribution of fungal species within the mangrove habitat 2000). The potent fungal strain That is, Penicillium
may reflect the physical conditions and/or habitat pre- citrinum from the present study was found to have better
ference such as temp., salinity, humidity, organic activity in all the different optimized culture conditions.
contents (Das et al., 2008; Ravikumar et al., 2004). The The maximum amylase production for the strain was
diversity was maximum at Bhitarakanika that is, 74% and found to have maximum enzyme activity at pH-5.0, 1%
least 1% at Budhabalanga mangrove sediments which NaCl, 1% of substrate (starch) and Inositol as carbon
may be due to the clayey sediment which indicates the source. Similar finding has been done by Kathiresan and
presence of more humus content. Manivannan (2006) for fungi. Similar type of study was
Selections of new microorganisms for enzyme produc- conducted by Gupta et al. (2010) where maximum
tion are increasing all around the world. So the study site enzyme production has been observed for the fungal
is mainly focused on mangrove areas of Odisha, India strain Aspergillus niger. The pH change observed during
which are very less explored. The major advantage of the growth of microbes also affects product stability in the
using microorganisms for production of amylases is in medium (Gupta et al., 2003). Most of the earlier studies
economical bulk production capacity and microbes are revealed the optimum pH range between 5.0 and 7.0 for
also easy to manipulate to obtain enzymes of desired the growth of some fungal strains such as P. fellutanum,
characteristics (Lonsane and Ramesh, 1990). Starch- however, Aspergillus oryzae released amylase only in
degrading amylolytic enzymes are of great significance in alkaline pH above 7.2 (Yabuki et al., 1977).
biotechnological applications ranging from food Identification of fungi was carried out by the usual
fermentation, textile to paper industries (Lin et al., 1997; methods such as cotton blue staining and conidia or
Pandey et al., 2000). The selection of potent strains for spore structure attachment etc. The two genus such as
enzyme production was done by screening and calcula- Aspergillus sp., and Penicillium sp. were found to be
ting the index values which indicates the utilization of dominant in the present study area, which were expected
substrate by the mangrove isolates. During screening of to furnish optimal conditions for the discovery of new
amylase enzyme activity, the fungal isolate Penicillium metabolites from mangrove associated fungi. The fungal
citrinum has maximum IFamy value 5.2 and selected for strains were identified as A. niger, Trichoderma viride,
enzyme assay purpose. The media optimization is an Penicillium citrinum, Paecilomyces variotii, Eurotium
important aspect to be considered in the development of amstelodmi by microscopic and morphological
fermentation technology. However, there are only a few observation (Table 3 and Figure 7). Penicillium citrinum
 Sahoo et al. 4345

Table 4. Alignment view and distance matrix of the MSF-9 isolate.

Max. Total Query E Max ident

Accession Description
score score coverage (%) value (%)
GU565136 Penicillium citrinum GZU-BCECYN60-2 795 795 85 0 97
HQ407424 Eupenicillium brefeldianum TZ-16 728 728 85 0 94
HM214448 Penicillium janthinellum Zh9A 728 728 85 0 94
GU981580 Eupenicillium brefeldianum CBS:235.81 728 728 85 0 94
AF033443 Penicillium fuscum NRRL 721 728 728 85 0 94
AF033435 Eupenicillium brefeldianum NRRL 710 728 728 85 0 94
HM469409 Penicillium sp. 6 JJK-2011 725 725 86 0 94
FJ613818 Fungal endophyte sp. ZY-2009 725 725 85 0 94
AM262422 Eupenicillium sp. SS-1627 725 725 83 0 94
AF481123 Penicillium sp. NRRL 28214 725 725 86 0 94

1000bp
900bp 
800bp 
700bp 
600bp  555 bp
500bp 
 

400bp 
 

300bp 
 
200bp 
 
100bp 

A B
Figure 9. Agarose gel electrophoresis of the genomic DNA (A) and 18S rDNA PCR Product (B) of
MSF-9 (M- 100bp Marker).

strain was found to grow luxuriantly in CDA media and mangrove isolate P.citrinum produced more amylase
the SEM study indicates the general Penicillium. The enzyme. Hence this potent fungal strain can also be used
molecular study that is, 18S rDNA analysis showed that in large scale industry for various useful product
the MSF-9 isolate has 96% similarity with Penicillium formation.
citrinum GZU-BCECYN60-2 (Table 4, Plate 35). The
phylogenetic analysis showed the similarity pattern of P. Conflict of Interests
citrinum (MSF-9) with other species (Figures 9 and 10). It
can be concluded from the present study that the The author(s) have not declared any conflict of interests.
4346 Afr. J. Biotechnol.

Eupenicillium brefeldianum CBS:235.81

Eupenicillium brefeldianum NRRL 710
Penicillium janthinellum Zh9A
Eupenicillium brefeldianum TZ-16
Fungal endophyte sp. ZY-2009
Penicillium sp. 6 JJK-2011
Eupenicillium sp. SS-1627
Penicillium fuscum NRRL 721
Penicillium sp. NRRL 28214
IMMT
Penicillium sp. GZU-BCECYN60-2

0.005
Figure 10. Evolutionary relationships of 11 taxa of MSF-9.

ACKNOWLEDGEMENTS Lonsane BK and Ramesh MV (1990). Production of bacterial

thermostable α-amylase by solid state fermentation: a potential tool
The authors are thankful to the Director, CSIR- IMMT, for achieving economy in enzyme production and starch hydrolysis.
In: Advances in Appl. Microbiol. 35:1-56.
Bhubanes war for providing necessary facilities to carry Maria GL, Sridhar KR, Raviraja NS (2005). Antimicrobial and enzyme
out the work. The corresponding author is very much activity of mangrove endophytic enzyme of southwest coast of India.
thankful to Dr. P. N. Chowdhry, NCFT, New Delhi for J. Agric. Technol. 1.
identification of fungi. Octávio LF, Daniel JR, Francislete RM, Carlos Bloch Jr, Carlos PS,
Maria FG (2000). Activity of wheat α-amylase inhibitors towards
burchid α-amylases and structural explanation of observed
specificities. Eur. J. Biochem. 267:2166-2173.
REFERENCES Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R (2000).
Advances in microbial amylases. Biotechnol. Appl. Biochem. 31:135-
AbouZeid AM (1997). Production, purification and characterization of an 152.
extracellular alpha-amylase enzyme isolated from Aspergillus flavus. Quang D, Nguyen Judiet M, Szabo R, Hoschke A (2000). Optimization
Microbios. 89(358):55-66. of composition of media for the production of Amylolytic enzymes by
Das S, Lyla PS, Khan SA (2008). Distribution and generic composition Thermomyces lanuginosus ATCC 34626. Food. Technol. Biotechnol.
of culturable marine actinomycetes from the sediments of Indian 38 (3):229-234.
continental slope of Bay of Bengal. Chin. J. Oceanol. Limnol. 26 Ravikumar S, Kathiresan K, Thadedus S, Ignatiammal M, Selvam MB,
(2):166-177. Shanthy S (2004). Nitrogen-fixing azatobacters from mangrove
Gupta A, Gautam N, Modi DR (2010). Optimization of a-amylase habitat and their utility as marine biofertilizers. J. Exp. Mar. Biol. Ecol.
production from free and immobilized cells of Aspergillus niger. J. 312:5-17.
Biotech. Pharm. Res. 1(1): 001-008. Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: Molecular
Gupta N, Das S, Basak UC (2007). Useful extracellular activity of Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol.
bacteria isolated from Bhitarkanika mangrove ecosystem of Orissa Biol. Evol. 24:1596-1599.
coast. Malaysian J. Microbiol. 3(2):15-18. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG
Gupta R, Gigras P, Mohapatra H, Goswami VK and Chauhan B (2003). (1997). The Clustal X Windows interface: flexible strategies for
Microbial ά-amylases: a biotechnological perspective. Process multiple sequence alignment aided by quality analysis tools. Nucleic
Biochem. 38:1599-1616. Acids Res. 25:4876-4882.
Hapwood DA, Bil MJ, Charter KF, Kieser T, Bruton CJ, Kieser HM, Yabuki M, Ono N, Hoshino K, Fukui S (1977). Rapid induction of
Lydiate DJ, Smith CP, Ward JM, Schrempf H (1985). Genetic amylase by non-growing mycelia of Aspergillus oryzae. Appl.
manipulation of Streptomyces: a laboratory manual. John Innes Environ. Microbiol., 34:1-6.
Founadtion, Norwich, UK, pp. 81.
Kathiresan K, Manivannan S (2006). α-Amylase production by
Penicillium fellutanum isolated from mangrove rhizosphere soil. Afr.
J. Biotechnol. 5 (10):829-832.
Khoo SL, Amirul AA, Kamaruzaman M, Nazalan M, Azizan MN (1994).
Purification and characterization of alpha-amylase from Aspergillus
flavus. Folia Microbiol (Praha). 39(5):392 - 398.
Lin LL, Hsu WH, Chu WS (1997). A gene encoding for α-amylase from
thermophilic Bacillus sp., strain TS-23 and its expression in
Escherichia coli. J. Appl. Microbiol. 82:325-334.