Abstract

Extracellular enzymes produced by microorganisms have always been a fascinating area of research because of their wider applications in industry and ease in production by microorganisms. Actinomycetes, especially marine actinomycetes are considered as treasure house for various biologically active primary and secondary metabolites, having diverse applications such as antibiotics, drugs, enzymes etc. The present study is aimed at isolating such marine actinomycetes from the intertidal zones in Chennai coast and to characterize their potential to produce proteolytic enzymes. A total of 35 morphologically different marine actinomycetes were isolated and screened for their potentials to produce extracellular proteolytic enzymes. Among them, 13 strains (or species) have produced gelatinolytic activity while 11 have exhibited proteolytic activity in skimmed milk. Among the proteolytic actinomycetes, 5 strains have produced proteolytic activities in both skimmed milk and gelatin substrates, indicating their wide spectrum of proteolytic activity. When the culture filtrates of these 5 strains were tested for their proteolytic potential, Streptomyces AMETH1009 strain has produced maximum zone of proteolysis in both the substrates.

Keywords

Intertidal zones, marine actinomycetes, protease, extracellular, Streptomyces

Introduction

Proteases are the important industrial enzymes for their degradative and synthetic functions. Protease enzymes are physiologically and biochemically necessary to catalyse the biological processes. Hence, proteases occur ubiquitously in a wide diversity of sources such as in plants, animals and in microorganisms. Microbes are considered as an attractive source of commercial proteases productions because of the limited space requirement for their growth and ready susceptibility to genetic manipulation (Rao et al., 1998).

Proteases are one among the three largest groups of industrial enzymes which has been used in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes (Anwar and Saleemuddin, 1998; Gupta et al., 2002).

Oceans occupy more than 70% of our Earth’s surface. The numerous marine microorganisms present in the oceanic environment contain biochemical secrets which can provide new insights and understanding of enzyme physiology. Microbial enzymes are relatively more stable and active than the corresponding enzymes derived from plants or animals (Bull et al., 2000; Lam, 2006; Zhang and Kim, 2010) and therefore, marine microorganisms become potential resource for  the production of new enzymes.

Actinomycetes are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of about half of the discovered bioactive secondary metabolites, notably antibiotics, antitumor agents, immunosuppressive agents and enzymes (Lam, 2006). As marine environmental conditions are extremely different from terrestrial ones, it is surmised that marine actinomycetes have different characteristics from those of terrestrial counterparts and, therefore, might produce different types of bioactive compounds. The intertidal zone is the area where land and sea meet. This habitat is covered with water at high tide, and exposed to air at low tide. Several studies have concentrated on marine actinomycetes and their proteases. A black pigmented, proteolytic actinomycete strain Streptomyces carpaticus NBRC15390T isolated from a marine sediment sample collected near Kakinada of Andhra Pradesh, produced proteolytic activity on casein-skim milk agar (Haritha et al., 2012). In this context, the present study is aimed at isolating such marine actinomycetes from the intertidal zones of Chennai coast for their potentials to produce proteolytic enzymes.

Materials and Methods
Collection of marine sediment samples and isolation of marine actinomycetes

Marine sediment (soil) samples were collected from six intertidal zones of Chennai beaches such as Kanathur, Muttukkadu, Kovalam, Mamallapuram, Thiruvanmiyur and Devaneri, along East Coast of  the Bay of Bengal.

The sediments samples were collected in clean sterile bags by using sterile collecting spatula and brought to the laboratory. The samples were air dried at room temperature for 2 days, then the samples were subjected to pretreatment technique, by heating the samples at 70˚C for 12 hours. Ten grams of soil sample from each location was suspended in 95 ml sterile distilled water and kept shaken at 150xg at 35ºC in an Environmental Shaker. Then, this suspension was serially diluted up to 10^-2. One ml of the diluted sample was taken from 10^-2dilution and the samples were pour plated on Starch Casein Agar (SCA) medium and plates were incubated at 37°C for 5 days. Colonies with the characteristics of actinomycetes were subcultured in SCA medium and used for further experiments (Sangeetha, 2013).

Screening of protease producing actinomycetes - live cultures

Pure cultures of isolated actinomycete were screened for protease production using direct culture assay with two different substrates viz., gelatin and skimmed milk. Sterile water agar medium with 0.5% of the substrate alone was added and the medium was poured into the sterile Petri plates and allowed to solidify. After solidification, the culture was streaked on the medium using sterile inoculation loop. After 3 days of incubation period, the enzyme activity was visualized by observing clear zone. The plates were flooded with saturated ammonium sulphate solution to visualize the zone of proteolysis. The diameter of zones formed was measured for all the positive strains.

Screening of protease producing actinomycetes - culture filtrates

Selected potential strains of isolated marine actinomycetes were subjected for the screening of protease production. Initially, protease activity was tested using two different substrates viz., gelatin and skimmed milk. Sterile water agar medium with 0.5% of the substrate supplemented with 50μg/ml of chlorophenicol antibiotic was added to avoid the bacterial contamination. The medium was poured into the sterile Petri plates and allowed to solidify. After solidification, 8 mm diameter well were made using cork borer. Two ml of each broth culture (from the Starch Casein broth) was centrifuged for 15 min at 10,000xg. From this, 100μl of culture filtrate of marine actinomycetes grown for 7 days in Starch Casein Broth (SCB), was added into the respective well. After 24 hours of incubation, the enzyme activity was observed as a clear zone. For visualization of protease activity, the plates were flooded with saturated ammonium sulphate solution prepared in 1N HCl. The diameter of zones formed was measured for all the positive strains.

Screening of protease producing actinomycetes - culture filtrates

Classical approaches such as morphological, physiological and biochemical characterization, as described by Nonomura (1974) and Buchanan and Gibbons (1974), were used for identification. Characteristics features such as aerial mass colour, melanoid pigments, reverse side pigments, soluble pigments, spore chain morphology and spore surface were considered for identification.

Results and Discussion

Actinomycetes are virtually unlimited sources of novel compounds with many therapeutic applications and hold a prominent position due to their diversity and proven ability to produce novel bioactive compounds (Subramani and Aalbersberg, 2012). Globally, researchers have isolated marine actinomycetes from diverse marine samples such as subtidal sediments, surface sea water and deep sea sediments. One hundred and two actinomycetes were isolated from the subtidal sediments of Bismarck Sea and the Solomon Sea off the coast of Papua, New Guinea (Magarvey et al., 2004). Marine actinomycetes have already been explored for proteolytic enzymes. In India, totally 191 different marine actinomycetes were isolated from 256 different marine samples collected from the Bay of Bengal and its associated Pulicat lake and Pichavaram mangrove. Among them, 157 strains are reported to produce caseinase, 113 strains produce gelatinase and 108 strains produce both the protease enzymes (Ramesh et al., 2009). Ramesh and Mathivanan (2009) have isolated 208 strains of marine actinomycetes from a total of 288 marine samples and screened for industrial enzymes. Among 208 isolates, 183, 157, 116, 72 and 68 isolates produced lipase, caseinase, gelatinase, cellulase and amylase, respectively. Likewise, in the present study, a total of 35 actinomycetes strains were isolated from six different locations of the intertidal zones of the Bay of Bengal. Thirty five marine actinomycete strains with different morphological features were selected and named as AMET 1001 to AMET 1035. Pure cultures of marine actinomycetes were maintained in SCA (Fig. 1). There has been no remarkable difference observed in actinomycetes population among the locations. All the locations had actinomycetes in the range of 3.78-4.45 log cfu. The population of marine actinomycetes and number of strains isolated do not show much variation with reference to their locations.

Fig 1: Pure cultures of selected marine actinomycetes

In this study, all the 35 isolates were retained for the screening of proteolytic enzymes using qualitative plate assay with two substrates. Among the 35 isolates, 13 actinomycetes have produced gelatinolytic activity in the qualitative assay while 11 isolates exhibited proteolytic activity in skimmed milk. Among these proteolytic actinomycetes, 5 strains have produced proteolytic activities in both the substrates indicating their wide spectrum of proteolytic activity (Fig. 2a; Table 1).

Fig 2: Proteolytic activity of selected actinomycetes in Qualitative Plate Assay (a: Whole culture, b: Culture filtrate)

Fig 3: Colony morphology of AMETH1009 in Starch Casein Agar Medium

When the culture filtrates of these 5 strains were tested for their proteolytic potential, the strain AMETH1009 had produced more zone of proteolysis in both the substrates i.e., 1.50 cm in gelatinplates and 1.1 cm in skimmed milk plates (Fig. 2b). The strain AMETH1009 was subjected to morphological, microscopical and biochemical characterization. The selected strain showed a white coloured aerial mycelium, with pale pink coloured reverse pigmentation and brown coloured soluble pigmentation (Fig. 3). Microscopic studies have indicated that the strain has smooth spore surface, spherical shaped and long chained spores, typical characteristics of the genus Streptomyces (Tresner et al., 1961). (Fig. 4).

Table 1: Screening of marine actinomycetes for protease activity-Whole Culture Assay Values of zone of proteolysis indicating positive enzyme activity; ‘–’ indicates no enzyme activity

Fig 4: Microscopic characterization of aerial and substrate mycelium of AMETH1009. Note the morphology (a) Spore Chain -Aerial mycelium and (b) Substrate mycelium.

Conclusion

Marine actinomycetes are one of the important biological resources for many of the useful bioproducts, including industrial enzymes. Marine actinomycetes have been isolated from many of the ecosystems of marine environment. In the present study, bioprospecting of marine actinomycetes from intertidal coastal sediments was approached and found Streptomyces AMETH1009 strain having good potential in producing proteolytic enzymes. The study has increased the scope of utilizing marine actinomycetes for the production of industrial enzymes. Further studies are needed to characterize the proteolytic enzymes from this marine actinomycete.

Acknowledgement

The authors sincerely acknowledge the support and facilities rendered by the Management of AMET University for the completion of this work. Authors MV and SV thank the Management and Authorities for Full Time Research Fellowship.

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