Abstract: Actinomycetes were isolated from the sediment samples collected from the shrimp pond and examined for their α-amylase activity. Among many strains, the strain AE-19 which was tentatively identified as Streptomyces aureofasciculus showed higher α-amylase activity and it was taken for further study. Impact of various physical and chemical factors such as pH, temperatures, sodium chloride concentrations, carbon and nitrogen compounds on the α-amylase activity of S. aureofasciculus was studied. It was found that the enzyme activity was maximum at pH 9, temperature 45°C, 0.05% NaCl concentration, carbon compound mannose and nitrogen compound L-histidine. These findings suggest that the strain can effectively be used in large scale production of α-amylase enzyme for commercial purposes, after testing and ascertaining the strain`s capability in large scale fermentations.
INTRODUCTION
Several enzymes are involved in hydrolysis of starch. Among them, α-amylase (endo-1,4, α-D-glucan glucohydrolase) which is an extracellular enzyme, randomly breaks 1,4 α-D-glucosidic linkage in the linear amylose chain (Patel et al., 2005). Though α-amylase can be obtained from various sources, this enzyme from microbial sources generally meets the industrial demands. The amylase family enzymes are of great significance due to their wide application potentials. Interestingly, the first enzyme produced industrially was an amylase from a fungal source in 1894, which was used as pharmaceutical aid for the treatment of digestive disorder (Crueger and Crueger, 1989). Amylases find potential applications in a number of industrial processes such as 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 pharmaceutical and fine chemical industries if enzymes with suitable properties could be prepared (Fogarty and Kelly, 1980). Considering its wide applications, a significant interest exists in α-amylase enzyme research especially for microbial resources. Though major portion of commercially important α-amylase is derived from bacterial sources such as Bacillus species, fungal α-amylase is preferred more in human consumption applications. From the available literature, it is found that there are studies on actinomycetes from different marine environs but there is not even a single work on the actinomycetes of shrimp ponds. Hence, the present investigation aims to study the α-amylase activity of actinomycetes isolated from the sediments of a shrimp pond under different experimental conditions with pH, temperatures, sodium chloride concentrations, carbon and nitrogen compounds and identify the potential α-amylase producing actinomycetes using chemotaxonomical and conventional methods of identification.
MATERIALS AND METHODS
Isolation of Actinomycetes
The sediment sample was collected from a shrimp pond (Lat. 11° 28’
53.7” N and Long. 79° 45’ 31.1” E), located opposite to the
Vellar estuary, southeast coast of India, by inserting a sterilized polyvinyl
corer (10 cm) into the sediments. The center portion of the 2 cm sediment sample
was taken out with the help of a sterile spatula. The collected sample was transferred
to a sterile polythene bag and taken immediately to the laboratory. After arrival
to the laboratory, the sample was air-dried aseptically for one week. The air-dried
sediment sample was incubated at 55°C for 5 min (Sivakumar et al.,
2005); then, 10-fold serial dilutions of the sediment samples were prepared,
using filtered and sterilized 50% seawater. One of the serially diluted samples
was plated in the Actinomycetes Isolation Agar medium (Hi-Media, Mumbai) in
duplicate petriplates after suitable dilution. To minimize the bacterial and
fungal contaminations, all agar plates were supplemented with 20 and 100 mg
L-1 of nystatin and cycloheximide respectively (Kathiresan et
al., 2005). The actinomycete colonies that appeared on the petriplates were
counted from 5th day onwards, upto 28th day. All the colonies that were growing
on the petriplates were separately streaked in petriplates, subcultured, ensured
for their axenicity and maintained in slants.
Assay of α-amylase Activity
α-amylase activity was assayed, following Summer's method. One ml of
buffered substrate prepared by dissolving 1 g starch in 100 mL of phosphate
buffer (pH-6.9) by boiling for 5 min at 80 to 85°C was added to 1 mL of
phosphate buffer in a series of tubes and equilibrated for 5 min at 28°C.
To all these tubes, 50 μL of tissue enzyme was added and allowed for reaction
for 10 min. The reaction was stopped by adding 2 mL of DNSA (3, 5-di-nitro-salicylic
acid). The contents were boiled exactly for 3 min. in water bath and cooled
for 20-25 min after which, the colour developed was read at 540 nm in a spectrophotometer.
α-amylase activity represented as total activity has been expressed as
the amount of micromoles of maltose liberated per minute of incubation per gram
of sample at 28°C.
Effects of pH, Temperature, Sodium Chloride, Carbon and Nitrogen Compounds
on α-Amylase Activity of the Actinomycete
Amylase activities of the actinomycete strain was measured at different
pH, temperature, sodium chloride concentration, carbon compounds and nitrogen
compounds. The experiments were conducted in 250 mL Erlenmeyer flasks containing
the sterilized Amylase Production Medium (APM) (Na2 HPO4
6 g, KH2 PO4 3 g, NH4Cl 1 g, NaCl 0.5 g, CaCl2
0.15 g, MgSO4, 7H2O 0.25 g, Casein hydrolysate 0.20 g,
Yeast extract 0.10 g, Starch 20 g, Water 1000 mL) broth at 15 lbs pressure for
15 min. After sterilization of the broth by autoclaving, the flask was cooled
and the strain was inoculated and incubated differently for different parameters
as described in the following paragraphs by taking one parameter at one time.
Appropriate controls were maintained in all the experiments.
Effect of pH
Effect of pH was studied by varying the pH from 4 to 9. After inoculation
of the strain, it was incubated for seven days at 45°C.
Effect of Temperature
After the inoculation of the strain in APM broth, it was incubated at various
temperatures viz. 35, 40, 45, 50, 55 and 60°C for seven days.
Effect of Sodium Chloride Concentrations
To study the tolerance towards sodium chloride, the APM broth prepared with
distilled water was added with sodium chloride of varying concentrations viz.,
0.05, 0.5, 1, 2, 3 and 4%. After the inoculation of the strain, it was incubated
at 45°C for seven days. Simultaneously, a control was maintained without
sodium chloride.
Effect of Various Carbon Compounds
The APM broth was used for studying the effect of various carbon compounds
such as arabinose, xylose, inositol, fructose, rhamnose, sucrose, raffinose
and mannose. The broth was distributed into different flasks and 1% of each
carbon source was then added before inoculation of the strain and incubated
for seven days at 45°C.
Effect of Various Nitrogen Compounds
The APM broth was used for studying the influence of different nitrogen
sources viz. L-asparagine, L-phenylalanine, L-histidine and L-hydroxyproline.
The broth was distributed into various flasks and 0.8 mL of each nitrogen compound
was then added and incubated for seven days at 45°C.
At the end of the incubation period, the α-amylase activity was determined by the procedure as described earlier.
Taxonomic Investigation
The genus level identification was made for the strain AE-19 using the cell
wall composition analysis and micromorphological studies (Lechevalier and Lechevalier,
1970). Characterization of the strain AE-19 was made by following the methods
described by Shirling and Gottlieb (1966) using standard yeast extract-malt
extract agar (ISP-2). The species level identification of the strain was based
on the keys of Nonomura (1974) and Bergey's Manual of Determinative Bacteriology
(Buchanan and Gibbons, 1974).
RESULTS AND DISCUSSION
Enzyme Assay
Among 20 strains of actinomycetes, only 8 strains showed α-amylase
activity. Out of these 8 strains, one strain AE-19 exhibited maximum enzymatic
activity (2.09 μg maltose mL-1 h-1) and it was selected
for further studies. Umamaheswary et al. (2005) isolated 40 strains of
Streptomyces from the fish, Mugil cephalus collected from the
Vellar estuary and out of them, only the strain LG-33 showed good L-glutaminase
activity. Sivakuamr et al. (2006) reported that strain (LG-10) of Streptomyces
sp. isolated from the fish, Chanos chanos showed good L-glutaminse
activity. Sahu et al. (2007) isolated 40 strains of Streptomyces
from different parts of three estuarine fishes viz., M. cephalus,
C. chanos and Etroplus suratensis for L-asparaginase and out of the
40 strains, only six strains showed significant L-asparaginase activity. From
the present findings and also from other studies, it could be inferred that
though the marine environs are harbouring a good number of actinomycete strains,
only a few of them are capable of to producing novel enzymes which have very
good industrial and commercial applications.
Effects of pH, Temperature, Sodium Chloride, Carbon Compounds and Nitrogen
Compounds on Amylase Activity
The selected isolate AE-19 was allowed to grow in different pH, temperatures,
salinities, carbon sources and nitrogen sources. pH of the medium played an
important role in amylase production. The enzymatic activity was optimum at
pH 9 (Fig. 1). Hamiton et al. (1999) observed the maximum
amylase activity at pH 6 while Patel et al. (2005) reported maximum amylase
activity at pH 7. Whereas, Sodhi et al. (2005) found that the amylase
activity was higher at pH 6.5 and temperature 60°C. But in the present study,
the maximum activity was recorded at pH 9 which indicates that the strain prefers
alkaline conditions for better enzyme production. The strain AE-19 showed maximum
amylase activity at the temperature of 45°C (Fig. 2) which
indicates that the enzyme is not thermostable. Stamford et al. (2001)
reported 70°C as optimum temperature for the production of amylase enzyme
by Nocardiopsis sp. Kundu (2006) reported the maximum amylase activity
at 50°C while Nipkow et al. (1989) observed the maximum enzyme activity
at 60°C. This reveals the fact that the optimum conditions for enzyme production
differ species to species.
Since the strain was isolated from the shrimp pond, it was felt that is necessary to understand the influence of different sodium chloride concentrations on the enzymatic activity of AE-19. Good activity was recorded at 0.05% sodium chloride concentration (Fig. 3). Saramma et al. (1994) reported that the amylase from Vibrio sp. required 1-2% sodium chloride concentration for the maximum enzyme activity. Likewise, Kundu (2006) also observed the maximum amylase activity at 1-2% sodium chloride concentration by the actinomycete strain. However, in the present study, the test strain was isolated from the shrimp pond. Thus the isolated strain could not be a true marine form thereby it might not be able to produce higher amount of the enzyme at higher sodium chloride concentrations. This could be ascribed to the fact that the enzyme production starts to decrease with the increasing NaCl concentration.
Among the different carbon sources used, the enzyme activity was maximum in rhamnose and mannose and minimum was recorded in sucrose (Fig. 4). Hamilton et al. (1999) tested the amylase activity in various carbon sources and found that the highest amylase activity was shown by the Bacillus sp. with lactose as carbon source and the enzyme was not produced when glucose and fructose were used as carbon sources.
Among the different nitrogen sources used, the enzyme activity was maximum in L-histidine and minimum in L-hydroxyproline (Fig. 5). But Patel et al. (2005) and Kundu (2006) observed maximum amylase activity in L-Phenylalanine as nitrogen source in Aspergillus oryzae, A. awamori and Streptomyces galilaeus.
Present investigation on the determination of optimal process parameters for the strain AE-19 for its better enzyme activity has yielded important results, indicating that the strain can be used for large scale industrial production of the enzyme. However, these parameters have to be tested in large scale cultures in automated incubators so as to confirm the optimum conditions for enzyme production before industrial application.
Taxonomic Investigation
The strain AE-19 possesses LL-Diaminopmelic (LL-DAP) and the test strain
contains glycine in its cell wall. Presence of LL-DAP along with glycine indicates
the cell wall chemotype-I. The strain with chemotype-I does not have any characteristic
pattern of sugars (Lechevalier and Lechevalier, 1970). The species belonging
to the wall type I are Streptomyces, Streptoverticillium, Chainia,
Actinopycnidium, Actinosporangium, Elyptrosporangium, Microellbosporia,
Sporichthya and Intrasporangium (Lechevalier and Lechevalier, 1970).
The micromorphological observations of the strain AE-19 reveal that it belongs
to the genus Streptomyces. The morphological, physiological and biochemical
characteristics of the amylase producing strain AE-19, tested in the present
study, are shown in Table 1.
| Fig. 1: | Effect of pH on α-amylase activity of the strain AE-19 |
| Fig. 2: | Effect of temperature on α-amylase activity of the strain AE-19 |
| Fig. 3: | Effect of sodium chloride concentration on α-amylase activity of the strain AE-19 |
These characteristics were compared with those of the Streptomyces species, given I the Key of Nonomura (1974) and the Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). The comparison showed that the present strain differed with the reverse side pigmentation and utilization of carbon source i.e., sucrose. The test strain coagulates the milk whereas the reference strain S. aureofasciculus is not coagulating the same. Except these, all the other characters are exactly similar to those of S. aureofasciculus. Hence, the strain AE-19 has been tentatively identified as S. aureofasciculus.
| Fig. 4: | Effect of carbon compounds on α-amylase activity of the strain AE-19 |
| Fig. 5: | Effect of nitrogen compounds on α-amylase activity of the strain AE-19 |
| Table 1: | Comparison between the strain AE-19 and Streptomyces aureofasciculus |
| +: Positive; -: Negative | |
CONCLUSION
From the present study, it is concluded that the sediments of the shrimp pond are good source materials for the isolation of potential actinomycetes. The present study has revealed that the tentatively identified species, S. aureofasciculus isolated from the sediments possesses good α-amylase activity. The study has also standardized the growth parameters of the actinomycetes for the maximum enzyme production, which can be effectively used in the large scale production of enzyme for commercial purposes.
ACKNOWLEDGMENTS
Authors thank Prof. T. Balasubramanian, Director, Center of Advanced Study in Marine Biology and the authorities of Annamalai University for providing with necessary facilities. One of the authors (M.K.S.) is thankful to the Ministry of Environment and Forests, Government of India for granting the fellowship.