Novel Actinomycetales bacterium-PV7 isolated from Kanyakumari Marine Sediments: A Prospective Source for Industrial and Pharmaceutical Enzyme Production

 

K.V Bhaskara Rao¹*, Veena S², Pooja S², Shriya Y², Shivram R²

¹Professor, Department of Biomedical Sciences, School of Bio Sciences and Technology, VIT University, Vellore, TN - 632 014, India

²Molecular and Microbiology Research Laboratory, Department of Biomedical Sciences, School of Bio Sciences and Technology, VIT University, Vellore - 632 014, Tamil Nadu, India

 

ABSTRACT:

Research for the selection of different enzyme producing microorganisms are increased around the world. Marine actinomycetes are one of the most investigated group since they are considered as a potential source of biotechnologically interesting products. The aim of the present study was to isolate commercially useful enzymes producing marine actinobacteria from Kanyakumari marine sediments. A total of 10 actinobacterial colonies were isolated which were designated as PV-1 to PV-10. In primary screening PV1, PV5, PV7 and PV8 showed good activity for cellulase and lipase enzyme production with a zone of hydrolysis of 10 mm and above. The isolate PV7 showed maximum activity with a zone of hydrolysis of 20 mm and 19 mm for cellulase and lipase enzyme respectively. Among all isolates PV7 and PV10 showed higher protease enzyme production with a zone of hydrolysis 19 mm and 14 mm respectively. In secondary screening PV7 showed maximum activity of 18.65 U/ml for cellulase followed by 15.32 U/ml of protease and 11.34 U/ml of lipase activities. The potential isolate PV7 was identified as Actinomycetales bacterium-PV7 using molecular methods by 16S rRNA sequencing. This study concludes that marine actinobacterial can be considered as source for the production of industrial and pharmaceutical enzymes.

 

 

 

 

 

INTRODUCTION:

Marine actinobacteria are gram positive filamentous bacteria with very high (68-78%)  G+C contents¹. Actinobacteria are present in various marine environments and ecological habitats². Marine actinobacteria to manage with the marine environmental stress and for their survival have developed a complex stress managing ability. This stress management is being unraveled for multiple purposes³.

 

Many reports are there on the bioactive potential of many actinobacterial origin metabolites⁴. These metabolites have been employed in assorted applications in environmental, industrial sectors and biomedical field⁵. Marine organisms, the prime reservoir of biological diversity are recognized as a rich source of novel bioactive compounds. Many natural products (more than 1000) available in India are produced from marine microbes, out of which 60-70% of secondary metabolites available are produced from marine actinobacterial⁶. Hence marine actinobacteriaare known richest and potential source of secondary metabolities and bioactive compound respectively. Marine actinobacteria isolated from deep sea sediments and salt pans soils are the major economically and biotechnologically important marine microorganism. Many enzymes that are used in different industries and pharmaceutical industries are produced from animals, plants and microorganisms. At present animal and plant based enzymes are that are widely used in different industrial sectors viz., leather, pharmaceutical, food and textile industries has been replaced by enzymes that are of microbial origi ⁷. Microbes due to their speedy growth rate and easy to handle have received the top priority in the field of enzyme production to meet the present market demand ⁸.

 

Marine actinobacteria due their harsh environmental habitat are capable of producing more stable enzymes at very high temperatures and more alkaline condition. Though production of many antibiotics from marine actinobacteria has been reported by many researchers, the enzyme production ability of marine actinobacteria is very less explored. Hence, this study was centered on the isolation of marine actinobacteria from Kanyakumari marine sediments and to check their ability to synthesise industrially and pharmaceutically important enzymes which can be a smart phenomenon to achieve future demands.

 

MATERIALS AND METHODS:

Chemical and Media:

All the medias and chemicals used in this study were purchased from Hi-media Laboratory Pvt. Ltd., Mumbai and Sis-co Research Laboratories (SRL) Pvt. Ltd, Mumbai, India.

 

Sample Collection:

Marine sediments were collected from the Kanyakumari beach (8.0866ºN, 77.5544ºE), Tamil Nadu, India. Samples were collected using sterile spatula at a depth of 15-20cm from the top layer of soil and then all the samples were aseptically transferred into sterile bag and transported to Biomolecules laboratory, VIT University. The collected sediments were then stored at 4ºC for further studies.

 

Isolation of Marine Actinobacteria:

Collected samples were serially diluted in 50% sea water and 1mL of appropriate dilutions were inoculated by following spread plate method on Starch casein agar (SCA), Actinomycetes isolation agar (AIA), Kuster’s agar (KA) and Ken knight and Munaiersagar (KMA) plates. All the media were supplemented with potassium dichromate (100µg/ml) and nalidixic acid (100µg/ml) to avoid all kinds of fungal and bacterial growth respectively. Plates were then incubated at 28⁰C for up to 30 days to check the growth of marine actinobacteria. Leathery and powdery colonies were isolated, purified and stored at 4ºC for further use⁹. The diversity of isolated marine actinobacterial was determined up to genus level by using morphological and cultural characteristics as described in International Streptomyces project (ISP) manual¹⁰.

 

Primary Screening for Enzyme Production:

Primary screening of all the actinobacterial isolates was carried out to check their ability for the production of different enzymes like cellulase, lipase and protease was done using the previously adopted methods by ¹¹. All the isolated cultures were inoculated on agar media with their respective substrates and incubated at 28⁰C for 5 days. For production of cellulase, lipase and protease enzyme carboxymethyl cellulose (CMC), tributyrin and casein were used as substrates respectively. After incubation period respective indicator was flooded in all plates and zone of clearance around the streaked culture were documented as positive results for enzyme activity.

 

Production Medium Preparation:

All the positive actinobacterial isolates (1mL) were inoculated into 50mL of production medium consisting of Soluble starch: 25g, Glucose: 10 g, Yeast extract: 2 g, CaCO_3: 3 g and 1mL of trace elements in 1000mL distilled water) separately. These Erlyen Mayer flasks were incubated at room temperature in arotaryshaker at 120rpm for 7 days. After the incubation period the contents of the flask were centre if used for 10 min. at 10000 rpm and the clear supernatant was collected to carry out the enzyme activities¹².

 

Secondary screening for enzyme production:

Cellulase activity:

Cellulase activity of the positive isolates was determined by the glucose equivalents amount released in the reaction mixture. The reaction mixture (10mL) was prepared using cell free supernatant (0.5mL), CMC (0.5%) which is completely dissolved in 0.1 M of phosphate buffer of pH 7 and finally made-up to 10mL using sterile distilled water. Reaction mixture was then incubated at 37ºC for 15 min. After the incubation period stopping reagent 3,5-dinitrosalicylic acid was added reaction followed by boiling of the same for 10-15min. At 620 nm the amount of glucose released was then measured using a UV-Vis spectrophotometer (Systronics-2202) and the final quantity was determined. One unit of cellulase activity was defined as glucose quantity in microgram that is liberated per min per ml of enzyme¹³.

 

Lipase activity:

One milliliter of cell free supernatant was added to the substrate solution consisting of 10mL of homogenized olive oil (10%) in gum arabic (10%), 2mL of CaCl₂ (0.6%) and 5mL of phosphate buffer (0.2 mol/L) with pH 7.2. The enzyme substrate mixture was then incubated at 35ºC for 1h in an orbital shaker at 120 rpm. To stop the reaction after incubation period 20 ml of ethanol: acetone (1:1) was added. Finally, using phenophtaline as an indicator liberated fatty acid was titrated with 0.1mol/L of NaOH. One lipase unit was determined by the amount of the enzyme that released 1µmol fatty acid per min under standard condition¹⁴.

 

Protease Activity:

The protease activity of the positive isolates was determined by using a reaction mixture consisting of cell free supernatant (0.1mL) and 0.9 ml of casein (2%) in 0.1 M of NaOHKH₂PO₄ buffer (pH 7)¹⁵.  The reaction mixture was then incubated at 37º C for 30 min. After incubation period trichloroacetic acid (1.5mL) was added to stop the reaction. After 15 min., the reaction mixture was then centrifuged at 10000 rpm for 10 min. Finally the protein concentration was determined using Lowry’s method¹⁶. One unit of protease activity is determined by µg of tyrosine liberated per ml of enzyme.

 

Identification of potential Actinobacterial isolate

The potential actinobacterialisolates showing higher enzyme production activity was done by using the key of Nonomuraand also by following the methods of Shirling and Gottlieb ^{10, 17}.  Species level identification was done by using cell wall analysis and by micro-morphological studies¹⁸. Finally the complete genus level identification was done by using molecular characterization based on 16S rRNA sequencing. Using the Insta Gene TM Matrix Genomic DNA isolation kit the bacterial genomic DNA was isolated. The fragments were amplified bi-directionally using the forward Genomic DNA isolation Kit. Amplification of the fragments was done bidirectionally using the forward (5′-AGAGTTTGATCMTGGCTCAG-3′) and reverse (5′- TACGGYTACCTTGTTACGACTT -3′) primers. The complied sequence was then analyzed by ABI 3730xl capillary DNA sequence (ABI PRISM 310 GA, Tokyo, Japan). Finally the sequence was observed for similarity using NCBI BLAST similarity tool.

 

Statistical Analysis:

Using two way ANOVA values were analyzed between groups of same or different dpi.  All values are shown as mean ± Standard deviation (SD). The significance of the differences between the mean values was determined by using student’s t test. For all the statistical analysis done *P values <0.05 were considered significant.

 

RESULTS AND DISCUSSION:

Isolation of Marine Actinobacteria:

Marine actinobacteria were isolated on Starch casein agar, Actinomycetes isolation agar, Kuster’ sagar and Ken knight and Munaiersagar (KMA) media. From the collected sample a total of 10 actinobacterial colonies were isolated. Maximum number of actinobacterial colonies were isolated on starch casein agar (5), followed by actinomycetes isolation agar (3) and Kuster’s agar (2). In Ken night agar plates, noactinobacterial growth was observed. The isolates were identified up to genus level by using culture characteristic as mentioned in ISP manual. Diversity of actinobacteria was observed after conventional identification of all isolated cultures. Among these isolates, the majority were belonging to the genus Streptomyces sp (60%), followed by Actinomycetes sp (30%), Nocardia sp (4%) and Micromonospora sp (6%). Gulve and Deshmukh reported isolation of 90 marine actinobacterial in starch nitrate agar, starch casein agar and actinomycetes isolation agar from the Konkan coast of Maharashtra and Konkan lines lies between Mumbai and Goa¹⁹. Similarly, in another study isolation of 100 actinobacterial colonies was reported using two different Media from the samples collected from shrimp farms of Tiab Port²⁰. When compared with the previous reports in our study less no of marine actinobacterial colonies were isolated. This is may be due to the sampling time, number and frequency, in this study samples were collected and processed one time only.

 

Primary Screening for Enzyme Production:

So far there are no reports available on the extracellular enzyme production from marine actinobacteria of Kanyakumari marine sediments. Out of the 10 isolates, 4 isolates were found to show very good activity of cellulase and lipase, 2 isolates demonstrated higher protease enzyme production (Figure 1). Five isolates PV1, PV5, PV7 and PV8 showed zone of clearance of 13mm, 16mm, 20mm and 17mm respectively in cellulase activity. Similarly, In lipase activity all 4 isolates PV1, PV5, PV7 and PV8 came up with a zone of clearance of 12mm, 15mm, 19mm and 17mm respectively. In screening for protease production only 2 isolates PV7 and PV10 showed good activity with a zone of clearance of 19mm and 14mm respectively.  Remaining isolates showed very less or no activity in primary screening. In a previous report Sathya and Ushadevi (2014) reported protease and lipase enzyme production using marine Streptomyces sp isolated from mangrove sediments²¹. In their report for protease and lipase zone of clearance was observed in the range of 4-27mm and 4-14mm respectively. In another study cellulolytic activity of three different strains Streptomyces sp NIOT-VKKMA02, Streptomyces sp NIOT-VKKMA026, Streptomyces sp NIOT-VKKMA022 was reported with a zone of clearance of 2.8mm, 2.1mm and 1.7mm respectively²². Hence, the results obtained in the present study are in accordance with the available reports and also showed good results in production of all three enzymes with higher zone of clearance.

 

Figure 1.Primary screening of isolated marine actinobacteria for enzyme production (A) Cellulase production, (B) Lipase production, and (C) Protease production.

 

 

Secondary screening of positive isolates for enzyme production:

Enzyme activity of active isolates was done by using cell free extract collected after fermentation. All positive isolates selected from primary screening showed good enzyme activity. Among the four isolates (PV1, PV5, PV7 and PV8), higher cellulase and lipase enzyme activity was shown by PV7 with 18.65 U/ml and 11.34 U/ml respectively. In quantitative assay of PV7 for proteolytic activity the isolates exhibited 15.32 U/ml of protease activity (Figure 2) and remaining isolates showed 6 U/ml or less activity for all the three enzymes. The potential isolates PV7 exhibited better activity in the production of two industrial importance extracellular enzymes and 1 extracellular enzyme of pharmaceutical importance. Likewise, in a previous report on quantitative evaluation of enzyme production from marine actinobacteria, cellulose and protease activity of three different strains of Streptomyces sp isolated from Andaman and Nicobar marine sediments was reported in the range of 5-7U/ml and 3-11U/ml respectively²².In another study a maximum of 8.65U/ml of cellulolytic activity was reported using marine actinobacteria isolated from different sites in Chennai, Kerala and Pooombukar in south India²³. Likewise, 200U/ml lipase enzyme activity was reported using actinobacteria isolated from Sangumugham Vishinjam and Veli Coast of Thiruvananthapuram²⁴. Similarly Benattouche and Abbounl (2011) reported a specific lipase activity of 41. 6 U/ml from Pseudomonas aeruginosa isolated from water samples of Algeria¹⁴. Hence, present study showed better results with higher production of the three enzymes when compared with previous reports. Our study is the first report to the best of our knowledge on the isolation of cellulase, lipase and protease producing marine actinobacteria from Kanyakumari sediment sample.

 

Figure 2. Secondary screening of positive isolates for enzyme production (A) Cellulase activity, (B) Lipase activity, and (C) Protease activity. 

 

 

Identification of Potential Enzyme Producing Isolate:

Potential isolate PV6 was identified as Actinomycetales bacterium-PV7 using 16S rRNA sequencing. Identification up to genus level by cell wall composition analysis, micro morphological studies and key of Nonomura also showed similar results (Table 1 and Table 2). The spore chain morphology and spore surface analysis of isolate PV7 showed substrate mycelium that were extensively branched mycelium which carried rectiflexcible and very smooth surface (Figure 3). Blast search for potential isolate PV7 showed only 85% similarity with Actinomycetales bacterium. The potential isolate Actinomycetales bacterium-PV7 occupy a unique phylogentic position and also it forms a separate subclade from the other members of Actinomycetales bacterium, which was confirmed using 16SrRNA sequencing (Figure 4). It confirms that the potential isolated Actinomycetales bacterium-PV7 is a novel species. Chemotaxonomical morphology and 16S rRNA gene sequence data suggests that Actinomycetales bacterium-PV7represent a novel species when compared with particular type strains of species presented in Nonomura key.

 

Table 1.Characteristic features of potential isolate PV7

 

Table 2.Cell wall amino acids and whole cell sugars of potential isolate PV7

Strain Name	LL-DAP	Meso-DAP	Glycine	Whole cell sugar	Walltype
PV7	+	-	+	-	I

 

Figure 3: Scanning electron microscopic (SEM) image of potential isolate PV7

 

 

Figure 4: 16S rRNA sequencing phylogenetic tree view for Actinomycetales bacterium PV7

 

 

CONCLUSIONS:

Actinomycetales bacterium-PV7 isolated from Kanyakumari marine sediments was found to be a promising source for the production of industrial and pharmaceutically interesting enzymes like lipase, cellulase and protease. The potential strain Actinomycetales bacterium-PV7 can be used as an alternative source for large scale production of these enzymes. Isolated strain is a novel species with only 85% similarity with Actinomycetales bacterium. Extraction and characterization of enzymes from Actinomycetales bacterium-PV7 and optimization to increase the production will be focused in future.

 

ACKNOWLEDGEMENTS:

Authors are thankful to the management of VIT University for providing necessary facilities to carry out this study and also DST-FIST for providing SEM facilities.

 

REFERENCES:

1.     Hoare DS, Work E. The stereoisomers of α, ε-diaminopimelic acid 2.The stereoisomer's of alpha epsilon-diaminopimelic acid. II. Their distribution in the bacterial order acinomycetales and in certain Eubacteriales. Biochemical Journal. 65(3); 1957: 441–447.

2.     Good fellow M, Williams ST. Ecology of actinomycetes. Annual Review of Microbiology. 37(1); 1983: 189–216.

3.     Sigrid H, Espen F, Kjell DJ, Elena I, Trond EE, Sergey BZ. Characterization of Streptomyces spp. isolated from the Sea surface microlayer in the Trondheim fjord, Norway. Marine Drugs. 6(4); 2008: 620–635.

4.     Berdy J. Bioactive microbial metabolites. Journal of Antibiotics. 58(4); 2005: 1–26.

5.     Arai T. Actinomycetes, the boundary Microorganisms. University of California; 1967. P. 123-152.

6.     Suthindhiran K, Kannabiran K. Diversity and exploration of bioactive actinomycetes in the Bay of Bengal of the puducherry cost of India. Indian Journal of Microbiology. 50(1); 2010: 76–82.

7.     Stamford TL, Stamford NP, Coelho LC, Araujo JM. Production and characterization of a thermostable glucoamylase from Streptosporangium endophyte of maize leaves. Bioresource Technology. 83(2); 2002: 105–109.

8.     Kumar CG, Takagi H. Microbial alkaline protease; from a bio industrial view point. Biotechnology Advances Journal. 17(7); 1999: 561–594.

9.     Karthik L, Gavurav K, Bhaskara Rao KV. Diversity of marine actinomycetes from nicobar marine sediments and its antifungal activity. Pharmacology. 2(1); 2010: 221-227.

10.  Shirling EB, Gottlieb D. Methods for characterization of streptomyces species. International Journal of Systemic Bacteriology. 16(3); 1966: 313-340

11.  Leon J, Liza L, Soto I, Cuadra D, Patino L, Zerpa R. Study of marine actinomycetes isolated from the central coast of Peru and their antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. Revista Peruana de Medicina Experimental by Salud Publica. 28(2); 2011: 237-46.

12.  Veena S, Swetha D, Karthik L, Bhaskara Rao KV. Assessment of anti-typhoid and antioxidant activity of marine actinobacteria isolated from Chennai marine sediments. Der Pharma Lettre. 8(3); 2016: 166-72.

13.  Bernfield P. Amylases, α and β. Academic Press. New York USA 1955. P. 149-158.

14.  Benattouche Z, Abbouni B. Isolation Optimization and Purification of Lipase Production by Pseudomonas Aeruginosa. Journal of Biotechnology and Biomaterials. 1(7); 2011: 1-4.

15.  Kathiresan K, Manivannan S. Production of alkaline protease by Streptomyces sp. isolated from coastal mangrove sediment. Research Journal of Environmental Sciences. 1(4); 2007: 173-178.

16.  Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein estimation with the Folin-phenol reagent. The Journal of Biological Chemistry. 193(2); 195: 1265–275.

17.  Nonomura H. Key for classification and identification of 458 species of the streptomycetes included in ISP. Journal of Fermentation Technology. 58(3); 1974: 78-92.

18.  Lechevalier MP, Lechevalier H. Chemical composition as a criterion in the classification of aerobic actinomycetes. International Journal of Systemic Bacteriology. 20(4); 1970: 435-43.

19.  Gulve RM, Deshmukh AM. Enzymatic activity of Actinomycetes isolated from marine sediments. Recent Research in Science and Technology. 3(5); 2011: 80-83.

20.  Gozari M, Mortazavi MS, Bahador N, Tamadoni J, Rabbaniha SM. Isolation and screening of antibacterial and enzyme producing marine actinobaceria to approach probiotics against some pathogenic vibrios in shrimp Litopenaeus vannamei. Iranian Journal of Fish Science. 15(2); 2016: 630-644.

21.  Sathya R, Ushadevi T. Industrially important enzymes producing streptomyces species from mangrove sediments. International Journal of Pharmacy and Pharmaceutical Science. 6(10); 2014: 233-237.

22.  Balakrishnan M, Lawrance AR, Nambali VV, Ramalingam K. Novel marine actinobacteria from emerald Andaman and Nicobar Islands: a prospective source for industrial and pharmaceutical byproducts. BMC Microbiology. 13(145); 2013: 2-17.

23.  SelvamK, Vishnupriya B. Screening and Quantification of Marine Actinomycetes Producing Industrial Enzymes Amylase, Cellulase and Lipase from South Coast of India. International Journal of Pharma and Bio Sciences. 2(11); 2011: 1481-1487

24.  Lekshmi M, Ayona J, Navami SS. Isolation and screening of actinomycetes from marine samples for enzymeproduction. International Journal of Scientific Engineering and Research. 5(12); 2014: 199-204.

 

 

 

Accepted on 06.04.2017           © RJPT All right reserved