Int J Pharm Pharm Sci, Vol 10, Issue 4, 122-127Original Article


ISOLATION, SCREENING AND DETERMINATION OF Α-AMYLASE ACTIVITY FROM MARINE STREPTOMYCES SPECIES

SATHYA RENGASAMY, USHADEVI THANGAPRAKASAM*

Centre for Research and Development, PRIST University, Tamil Nadu, India
Email: ushadevi29@gmail.com

Received: 26 Dec 2017 Revised and Accepted: 15 Mar 2018

ABSTRACT

Objective: This study was aimed to isolate potent amylase producing Streptomyces from the marine source.

Methods: Soil samples were collected from less explored mangrove regions of Muthupet, Tamilnadu. Isolation of Streptomyces was performed by serial dilution plate technique using starch casein agar (SCA) (pH 7.2 and temp 28 °C). Morphological and biochemical characteristics were studied using Bergey’s manual of systematic bacteriology. Preliminary screening and quantification of amylase activities were analysed in selected Streptomyces isolates by starch agar plate and dinitrosalicylic acid (DNS) method respectively.

Results: Totally 65 isolates were separated from the marine soil. Among them, 23 strains showed different morphological features. These strains were subjected to amylase activity. Eight Streptomyces isolates (S1-S8) exhibited positive for amylase activity. The zone of clearance was exhibited in the range of diameters between 4-20 mm. Fermentation was prompted with inorganic salt starch agar, international Streptomyces project (ISP-4) media at 28 °C and incubated in an orbital shaker at 250 rpm for 96 h (pH 7.5). The quantitative estimation of amylase activity was exhibited selected eight isolates in the range between 2.4±0.002-5.9±0.005 (U/ml). The Streptomyces species S4, S5 and S6 exhibited strong amylase activity in both qualitative and quantitative level.

Conclusion: This work motivating the amylase producing Streptomyces are originated in mangroves and it proved Streptomyces sp. S6 has a more efficient source of amylase production.

Keywords: Streptomyces, Isolation, Screening, Quantitative determination, Amylase

© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2018v10i4.24447

INTRODUCTION

Amylase (EC: 3.2.1.1) plays a wide range of biotechnological applications in food industry, fermentation, textile and paper industries and having above 25% demand on a global scale [1-2]. Amylase has been used successfully in starch saccharification, brewing and distilling industries [3]. Presently, amylase production has reached up to 65% of the world market and continuously increasing the usage [4]. α-amylases have been derived from various sources such as plants, animals and microorganisms. Microbial amylase meet industrial demand [5] as there is a possibility of increasing the levels of microbial enzyme synthesized by classical genetic techniques, continuous culture selection, induction and optimization of growth conditions for the enzyme of interest [6].

Streptomyces are an economically important group of organisms among actinobacteria family. They are responsible for the production of about half of the discovered metabolites, notably antibiotics, antitumor agent, an immunosuppressive agent, enzymes and enzymes inhibitors. This insight has been a driving strength towards other drug discovery platforms including high throughput combinatorial production and rational drug design [7]. Streptomyces are known to produce the array of antimicrobial and antioxidant compounds [8]. Thus, it is crucial that new group of Streptomyces from unexplored or underexploited marine environment be pursued as a resource of novel compound [9].

The occurrence of amylase from Streptomyces and the genus considered as an active source of amylases [10]. Notably, Streptomyces avermitilis, Streptomyces sp. SLBA-08, Streptomyces strain A3, Streptomyces rochei BTSS 1001 are used for the production of amylase. Microbial amylase successfully used in medicinal research [11,12]. The most significant bacterial amylases are Bacillus, Streptomyces, Micrococcus, Escherichia, Proteus and Serratia [13,14].

Microbial enzyme technology has in recent years, grown to a multi-million dollar industry, exploration of microbial strains for discovering enzyme with novel properties has come close to the screening programs that multinational firms have been undertaking for the discovery of newer enzymes. The marine Streptomyces species are capable of producing a range of bioactive compounds, as well as enzymes [15]. Streptomyces adopted an incredible quantity of valuable products and are expected to the sources of many commercially important metabolites, including novel enzymes. Development in biology has to a great extent to improve the potential to make libraries of enzyme variants, but a vital challenge is to develop good screening tools that can identify the best performing strain [16].

Multi prospective application and demand pave the mode for growing native amylase production and searching for the more efficient process. Microbial bioprocess can meet more easily the current market demand for industrial enzymes [17]. Specifically, Streptomyces have been used to synthesize amylases [18,19]. Hence, the present study made an effort to screen and determine the amylase activity from marine Streptomyces.

MATERIALS AND METHODS

Chemicals and kits

Chemicals used in this study of starch casein agar media (SCA), Inorganic salt starch agar media (ISP-4), Iodine and maltose were purchased from Hi-Media (Mumbai-India). Dinitrosalicylic acid (DNS) purchased from Sigma Aldrich, (Mumbai, India). All chemicals were grade and all working reagents were prepared with deionized water.

Isolation and selection of Streptomyces

Soil samples were collected from Muthupet Mangrove forest (Lat.10° 20’N and Long.79 °35’E) in Tamilnadu during May 2012 situated on the south-east coast of India. Isolation of Streptomyces was performed by serial dilution plate technique using starch casein agar (SCA) medium [20]. Based on the colony morphology as white coloured, powdery, dried, and rough, with irregular and regular margins were determined Streptomyces species.

Characterization of marine isolates

Marine isolates features were studied for morphological, physiological and biochemical characteristics according to the method [21]. Colony color, mycelium nature, texture, shape were complemented to Bergey’s manual of systematic bacteriology [22]. Microscopic characterization was experimented by coverslip culture technique [23]. The mycelium structure and spore arrangement were observed through high power oil immersion (100 X) objective by light microscope (Olympus, CH20i). Biochemical characterization of isolates was performed by standard methods [24].

Screening of amylase

Isolated 23 Streptomyces were grown on starch agar medium and incubated at 28 °C for 72 h. After incubation, 3 ml of 1% iodine was flooded with each plate, and the development of stainless zone around the colonies indicated amylase production [25].

Flask scale fermentation

The spores suspensions (~106 spore ml-1) of eight Streptomyces inoculums were aseptically transferred individually into 50 ml of medium. Streptomyces isolates were cultured in inorganic salt starch agar, international Streptomyces project (ISP-4) medium (50% sea water; pH 7.5). The cells were grown aerobically in 250 ml Erlenmeyer flasks containing 100 ml medium at 28 °C and incubated in an orbital shaker at 250 rpm for 96 h. After fermentation, the cells were centrifuged at 5000 × g for 15 min. The cell supernatant was separated and quantitatively studied amylase activity.

Determination of biomass

The cells were grown aerobically in 250 ml flask containing 100 ml medium at 28 °C with continuous shaking in an Orbital shaker (250 rpm). Streptomyces growth was monitored by measuring culture turbidity at OD 600 nm and estimated the biomass content. During the stationary growth phase, the biomass was collected by centrifugation at 15,000 × g for 10 min at 4 °C. The cell suspension was washed twice with distilled water and dried in hot air oven at 80 °C overnight. The cell dry weight was calculated 100 ml of culture broth ≥ 0.8 OD 600 nm corresponds to mg 100 ml-1 of cell dry biomass.

Enzyme assay

Amylase activity was estimated using the method of [26] with minor modifications. To prepare the reaction mixture 1 millilitre of supernatant was mixed with 1 ml of solubilized starch solution and then incubated at 60 °C for 10 min. The reaction was stopped by adding 2 ml of dinitrosalicylic acid (DNS) reagent. The mixture was cooled in an ice water bath for 10-15 min and then centrifuged 5000 × g for 5 min at 4 °C. The quantity of enzyme was measured at 540 nm using UV-Vis Spectrophotometer (Techcomp, 2310), with a blank sample as a reference. One unit of enzyme activity was defined as the amount of amylase needed to produce 1 µmol of maltose per min under the assay conditions. Amylase activity were performed in triplicates, values calculated by the mean values along with standard error mean.

RESULTS

Isolation and identification of Streptomyces

Totally, 65 isolates were selected from the marine sample. Most of the isolates appeared like Streptomyces. Morphologically versatile 23 isolates selected for amylase screening experiments. The isolates were grown at pH 7.2 and temperature 28 °C. Morphological characteristics of the Streptomyces isolates are given in table 1.

Table 1: Morphological characterization of Streptomyces isolates

Isolates Appearance Aerial/substrate mycelium Size (mm) Shape Surface Reverse pigment Light microscopy
S1 White brown color, powdery Ash with sandal white 6 Button Smooth Brown Filamentous
S2 Dark brown with violet Yellow with brown 8 Double ring Smooth Pink Filamentous
S3 Powdery green with white Ash with yellow 5 Round Rough Green Rectus with filamentous
S4 Violet with yellow White with sandal 9 Line with dots Powder Dark brown Recti flexible
S5 Sandal with white colonies, Grey with cream 8 Tiny round Smooth Light yellow Spiral with filamentous
S6 Golden yellow with sandal White with ash 4 Round Smooth Orange Filamentous
S7 Yellow with Brown color Brown with white 3 Feather with round Rough Pink with brown Filamentous with spiral
S8 White with pink Ash with white 5 Root with line Smooth Red with pink Filamentous

S1-S8: Streptomyces species code; mm-millimeter.

Fig. 1: Colony morphology of Streptomyces isolates on starch casein agar plates, representative isolates of amylase producers associated with mangrove soil; S-Streptomyces species Isolate code; S1, S2, S3, S4, S5, S6, S7 and S8

The isolates showed well developed aerial and substrate mycelia with sporulation on starch casein agar media. The colony morphology of Streptomyces isolates is given in fig. 1. Microscopic views of spore chains arrangements of Streptomyces appeared like filamentous (F) flexible-rectifexible (RF), and filamentous with spirals (FS) (table 1). Isolates emerged different shapes like round, tiny ring, and button shape colonies, and the texture appeared powdery, rough and smooth spores. Isolates synthesized numerous reverse color pigments like green, yellow, red, golden yellow, brown and violet color on starch casein agar plate (fig. 2).

Fig. 2: Reverse side pigmentation on Streptomyces isolates, the pigmented isolates suitable for the identification of Streptomyces on genus level. S1-S8: Streptomyces species code

Biochemical characteristics of the isolates are given in table 2. Isolates are aerobic, G+ve, mesophilic, non-motile and non-endospore forming characteristics. Morphological and biochemical characteristics of marine isolates were appeared similar to the genus Streptomyces. This study provides genus level identification of Streptomyces.

Table 2: Biochemical characterization of Streptomyces isolates

Experiments S1 S2 S3 S4 S5 S6 S7 S8
Gram’s staining + + + + + + + +
Endospore − − − − − − − −
Starch hydrolysis +++ +++ ++ ++ ++ +++ +++ +
Citrate + + − − + + + −
Catalase + − − + + + − +
Urease + + + + + + + −
Nitrate reduction + − − + + + − +
CHO Fermentation Glucose + − − − + − + −

+= Positive, − = Negative. S1-S8: Streptomyces species code.

Screening of amylase activity

Primary screening of amylase production are given in fig. 3. Among 23 isolates, 8 Streptomyces isolates exhibited positive amylase activity in the starch medium. These potent isolates were displayed various diameters zone, ranging between 4-20 mm. Streptomyces S4, S5 and S6 exhibited higher activity than the other isolates.

Each Streptomyces produced the desirable zone of clearance. Especially, S6 exhibited higher amylase activity.

Fig. 3: Screening of amylase producing Streptomyces isolates from mangrove soil. The development of clear zone around the colonies was indicates positive for amylase activity. S1-S8: Streptomyces species code

Determination of biomass

The time course profile of eight Streptomyces species was monitored as optical density vs. incubation time (fig. 4). The maximum biomass production was recorded only in Streptomyces sp. S6, it has reached initial stationary phase at 96 h, and the maximum cell dry weight of 0.53 mg 100 ml-1. The S4, S3 and S8 isolates were reached in the range between 0.41-0.53 mg 100 ml-1.

Quantification of amylase activity

Amylase production was determined roughly (without optimization), the potent isolates produced desirable quantity. The results of extracellular amylase activity by dinitrosalicylic acid (DNS) method are given in table 3. The crude fraction of enzyme absorption was measured at 540 nm, the measurement provides the validation of amylase production. The crude fraction of amylase activity was observed in the range of 2.4±0.002-5.9±0.005 (U/ml).

Fig. 4: Time course profile of amylase producing streptomyces isolates, the growth phase was determined with ISP-4 medium. The growth rate was observed regularly at 24 h time intervals and the concentration was measured at OD 600 nm during 24-168 h. The right portion of the curve was observed only in S4-S6, and high level of growth exhibited during 96 h

Table 3: Qualitative and quantitative determination of amylase activity by Streptomyces

Isolates Clear zone (mm) Cell dry biomass (mg 100 ml-1) Enzyme activity (U/ml)
S1 4.0±0.81 0.23±0.003 2.4±0.002
S2 9.0±0.26 0.35±0.004 2.6±0.005
S3 7.0±0.31 0.24±0.003 3.5±0.001
S4 15±0.54 0.41±0.001 4.6±0.002
S5 18±0.98 0.51±0.004 5.6±0.005
S6 20±0.12 0.53±0.001 5.9±0.009
S7 12±0.81 0.41±0.005 4.1±0.002
S8 9.0±0.54 0.32±0.001 3.5±0.003

S1-S8: Streptomyces species code; mm-millimetre; mg-milligram; U/ml-Unit/ millilitre, Amylase screening and activity was performed in triplicates and the mean values along with standard error mean (mean±SEM., n=3).

DISCUSSION

This study was aimed to isolate potent amylase producer from a marine source. Streptomyces produce the incredible source of intercellular and extracellular enzymes vital for industrial and economic use [27]. However, the distribution of Streptomyces in the marine is chiefly unexplored or underexploited habitats be pursued as sources of novel compounds for industrial usage [28]. Mangrove is a high moisture, high salinity and hypoxia to tolerant ecosystem [29] which breeds many kinds of novel microorganisms which are the main source of vital compounds [30]. Many researchers investigated the muthupet mangrove vegetation, isolation and characterization of bioactive compounds from Streptomyces [31-32]. A novel Streptomyces strain was isolated from mangroves soil at Tanjung Lumpur, Malaysia [33].

Morphological and biochemical features of isolated Streptomyces were similar to Bergey’s manual of systematic bacteriology [22]. Furthermore, they are ubiquitous in nature and showed a higher diversity in color of colonies, secreted pigments, etc., compared to other bacteria. Strains can be readily differentiated by variation in colors of their aerial and substrate mycelium and based on physiological and biochemical characteristics [34]. The similar microscopic characterization of Streptomyces spore morphology and hyphae features was studied [35].

More studies involving isolation and screening of amylase by starch agar plate. Amylolytic activity was identified by Bacillus subtilis B19 [36]. Amylase production on the agar plate was proved by Aspergillus versicolor and Penicillium sp [37]. Amylase production has been done by numerous strains notably S. gulbargensis, Streptomyces strain A3, S. avermitilis, S. rochei BTSS 1001 [38-39]. This work coincided with the similar determination of previous analysis of amylase on starch agar.

Amylase is one of the most significant industrial enzymes. Every year several tons of amylase is used in the various industrial applications in Iran [40]. High level of extracellular amylase production was achieved newly isolated alkali-thermotolerant strain Streptomyces gulbargensis DAS 131[38]. Aquatic actinomycetes were exhibited amylase activity 62.97 U/ml [41]. Box-Behnken design method highly improved amylase activity 145.32 U/ml (pH 6, temp 35 ° C) [42]. Aspergillus fumigatus NTCC1222 showed highest amylase activity (164.1 U/ml) was studied [43]. α-Amylase was derived from Bacillus sp. BCC 01-50 [44]. Recently highest amylase production was achieved through optimization by B. cereus. The production was carried out under submerged state fermentation. The maximum production of amylase was 281.1±0.65 U/ml in wheat bran supplemented medium [45].

Analysis of amylase activity in the crude extract, the results provide a significant quantity of amylase. Streptomyces S4, S5 and S6 were exhibited (4.9-5.6 U/ml) activity. Few reports coincide with the present amylase activity. Streptomyces cheonanensis VUK–A was roughly produced 4.3 U/ml after optimization, reached 11.2 U/ml [46]. Amylase activity was explored during the short period, significant quantity achieved by Streptomyces sp S4, S5 and S6. The isolates S4, S5 and S6 are considered an efficient for amylase production to the technological application.

CONCLUSION

In the present study, the marine Streptomyces isolates had the significant ability to produce industrial amylases. Streptomyces are considered as a worthy source for amylases, identified from mangroves. It is enormous importance as secrete extracellular amylases. The results are more virtual and low-cost production of amylase by inexpensive single media without optimization. Further study is in progress to scale up the amylase production using optimization. Therefore, the future work deals with the optimization and improvement of amylase will be explored.

AUTHORS CONTRIBUTIONS

Sathya Rengasamy and Ushadevi Thangaprakasam were designed and executed the experimental analysis and written the manuscript.

CONFLICT OF INTERESTS

No conflict of interest declared

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