Indian Journal of Geo-Marine Sciences Vol. 44(4), April, 2015, pp.

Production of alpha by salt – Tolerant Actinomycete Streptomyces sp. – SBU3 isolated from marine sponge

S. Krishnakumar1* V. Dooslin Mercy Bai2 & J. Premkumar1

1* Faculty of Bio and Chemical Engineering, Department of Biomedical Engineering, Sathyabama University, Chennai – 600 119, Tamil Nadu, India. 2Department of Biomedical Engineering, Rajiv Gandhi College of Engineering and Technology., Puducherry - 607402,India.

*[E. mail: [email protected], [email protected]]

Received; revised

Marine environment harbors a number of macro and microorganisms that have specific metabolic activities. Marine actinomycete Streptomyces sp. – SBU3 was isolated from marine sponge Ircinia sp. from Cape Comorin coast (Lat.8o 21’ N and Long 77o 30’E), India for amylase activity. The effects of salinity, temperature, pH, carbon sources, nitrogen sources and incubation period were tested for alpha amylase production by the isolate. Highest activity (200 IU/ml) was obtained when the medium supplemented with 1% Nacl (w/v), glucose as a carbon source at 1 % (w/v) and casamino acid as a nitrogen source 1 % (w/v). Optimal alpha amylase production by Streptomyces sp. – SBU3 was observed in pH 7.5 at 30oC during 96h of incubation period. So for no sponge associated actinomycetes have been explored for media optimization for their alpha amylase production.

[Key words: Alpha amylase, marine sponge, salt tolerant actinomycete, Streptomyces sp.]

actinomycetes because these bacteria produce resistant spores that are known to be transported Introduction from land into sea where they can remain dormant for many years8. But the recent studies have The world’s oceans represent a resource shown that actinomycetes can be isolated from the of huge dimension for natural products however, coastal environments and there are evidences that marine microbes until last few decade was actinomycetes can be recovered even from marine relatively neglected area of marine natural sediments9, 10. Some bacteria permanently reside products1. Microbes from extreme environments in the sponge mesophyle and close interaction have attracted considerable attention in recent between the host and associated bacteria11, 12. years. Majority of the studies were on The studies on halophilic sponge associated extremophilic organisms. However, extremophilic actinomycetes for amylase enzyme are bacteria and actinomycetes are relatively less comparatively still rare. Hence the attempts were explored group2. It is widely accepted that made for the production of alpha amylase enzyme halophilic actinomycetes will provide a valuable by actinomycetes isolated from marine sponge resource for novel products of industrial interest, from Cape Comorin coast, India. including and antimicrobial agents 3, 4. Actinomycetes, Streptomyces spp. were Amylase enzymes are of great importance in isolated from the sponge of marine habitat. biotechnological applications ranging from food, Among the isolates, one strain produced fermentation, textile and paper industries5. extracellular alpha amylase which was confirmed Amylase can be derived from plants, animals and by starch and identified as microbes. But the majority of the enzymes used in Streptomyces sp. – SBU3. Under natural habitat the industry are of microbial origin because the organism produce low concentration of alpha microbial enzymes are relatively more stable6. amylase enzyme13. Hence an attempt has been Alpha amylase enzyme has been derived from taken to increase the productivity by optimizing several fungi, yeast, bacteria and actinomycetes7. the cultural conditions to meet the industrial The distribution of actinomycetes in the demands. marine environments remained largely Materials and Methods undescribed for many years. Most of the workers Marine sponge, Ircinia sp. (Class: suspected the indigenous nature of marine Demospongiae, Order: Dictyoceratida, Family: INDIAN J. MAR. SCI., VOL. 44, NO. 4 APRIL 2015

Spongiidae) was collected by SCUBA diving 250ml Erlenmeyer flask. The growth parameters from Cape Comorin coast (Lat.8o 21’ N and Long such as salinity, temperature, pH, carbon sources, 77o 30’E), India. Marine sponge sample was nitrogen sources and incubation period collected from 2 to 3 m depth for the enumeration influencing the production of amylase were of actinomycetes. Sponge sample was gently optimized independently. The experiments were rinsed with sea water and kept in ice chest box conducted in 200 ml Erlenmeyer flask containing and brought to the laboratory. Sponge sample was 100 ml of amylase production media. After chopped and homogenized with sterile aged sea sterilization 10ml of spore suspensions were water for the isolation of actinomycetes. inoculated, incubated at various experimental The actinomycetes, was isolated from conditions and optimized for enzyme production sponge Ircinia sp. by spread plate method using in different parameters such as salinity (0, 1, 2, 3, Starch Casein Agar (SCA) Hi-media, Mumbai, 4, 5% w/v), temperature (20, 25, 30, 35, 40 oC), India in aged seawater and distilled water (1:1) pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) carbon supplemented with antibiotics (Nalidixic acid sources (glucose, sucrose, maltose, fructose and 20µg ml-1; Nystatin 25µg ml-1; Cycloheximide lactose) at 1%(w/v), nitrogen sources (peptone, 100µg ml-1) to minimize the gram negative yeast extract, beef extract, casein and casamino bacteria, fungal and yeast contaminants acid) at 1%(w/v) and incubation period (24 – 120 respectively14 . Inoculated plates were incubated h) at 24h intervals. After required incubation at 28±2oC for 15 days. After incubation period the period, the media were centrifuged (REMI powdery colony was selected and streaked on TABLE TOP) at 5000 rpm for 20 min to harvest ISP2 (International Streptomyces Project) the crude enzyme. The culture supernatant was medium to get an auxenic culture and are stored assayed in triplicates for amylase activity. in the same medium for further amylase screening The amylase enzyme assay (AEA) was study. carried out using culture supernatant obtained Amylase producing Streptomyces (APS) after centrifugation. Assay was made by using was screened on Starch Agar (SA) plates the reaction mixture (5ml) consisted of 1ml of containing soluble starch 2 g, peptone 5 g, yeast crude enzyme solution and 4ml of soluble starch extract 1.5 g, Nacl 5 g, agar 18 g in aged seawater in phosphate buffer of pH 7.0. Reaction mixture and distilled water (1:1) at the pH of 7.0±0.2. The was incubated at 28oC for 30 min. Reaction was culture was incubated for 96 h at 28±2oC and the ceased by placing the reaction mixture in boiling amylase production was detected after flooding water bath21. The level of liberated glucose was the plates with iodine solution15. Hydrolysis was estimated by dinitrosalicylic acid method22 and indicated by clear zone around the colony was expressed as one International Unit (IU) of confirmed as amylase producing strain. The amylase activity, which is the amount of enzyme potential candidate strain was chosen and is required to liberate 1µ mol of glucose in one subjected to analyze cell wall composition and minute under experimental condition. micro-morphological characteristics16. Strain characterization and carbon assimilation was Results adopted by the methods followed by Shirling and The effect of various growth parameters Gottileb17. Genus level identification was adopted of actinomycete on amylase production was based on the keys of Nonamura18 and Bergey’s examined on amylase production medium. In the manual of determinative bacteriology19. present investigation, out of 20 actinomycetes The inoculum was prepared by isolate screened for amylase enzyme activity, one inoculating loop full of powdery spore was isolate exhibited promising activity. Isolate was inoculated in Modified Nutrient Broth (MNB) identified as Streptomyces sp - SBU3 based on the medium containing glucose 5g, peptone 5g, beef morphological, biochemical and carbon extract 3g, sodium chloride 5g, aged sea water assimilation studies is represented in Table 1. The 500ml and distilled water 500ml was used. potential isolate have profusely filamentous Inoculated flask was incubated in orbit shaker structure. Aerial mycelium was grey colour with incubator (NEOLAB-756610) at 28±2oC for 3 15 to 17 terminal smooth globose spores. LL- days20. diaminopimelic (LL-DAP) was present in the cell The amylase enzyme production medium wall along with glycine. This indicates chemo- consisted of soluble starch 5 g, glucose 2g, yeast type-1 cell wall, which is the characteristic of extract 1.5g, KH2PO4 10g, (NH4)2SO4 10g, Streptomyces species. Potential strain could MgSO4 0.3g, Cacl2 0.5g, FeSO4 0.012g, MnSO4 utilize glucose, arabinose, sucrose, mannitol, 0.005g, ZnSO4 0.005g, CoCl 0.007g in aged fructose, rhamnose and raffinose as a carbon seawater and distilled water (1:1) was prepared in source along with acid production. Eventhough 2 KRISHNAKUMAR et al.: ALPHA AMYLASE BY SALT – TOLERANT ACTINOMYCETE STREPTOMYCES SP.-SBU3 xylose, inositol and cellulose were not utilized by amylase activity of 140 IU/ml was recorded under the potential strain. Reverse side colour and experimental condition. melanin pigment was negative for the isolate Discussion Streptomyces sp.-SBU3. Environmental culture condition and amount of carbon and nitrogen sources in

Table 1- Morphological, biochemical and carbon assimilation characteristics of Streptomyces sp.- SBU3 Characteristics Test results 215 True mycelium Present 195 Facultative anaerobe + 175 Acid fastness - Spores in aerial mycelium + 155 Spores in substrate mycelium - 135 Sporangium on aerial mycelium - 115 No. of spores on aerial 15 – 17 mycelium 95 Shape of spores Globose 75 Spore motility + Amylase (IU/ml) activity Amylase 0 1 2 3 4 5 Cell wall type 1 Salinity (%) DAP isomer LL-DAP Spore chain morphology Recti-flexibles Fig.1- Effect of salinity on amylase production by Spore surface Smooth Streptomyces sp.-SBU3 Aerial mycelium colour Grey Reverse side colour - 105 Melanin pigment - Growth at 450C + Halophilic + Alkalophilic - 95 Carbon utilization tests Glucose + Arabinose + 85

Sucrose + (IU/ml) Xylose -

Inositol - 75 activity Amylase Mannitol + 20 25Temperature 30 (oC) 35 40 Fructose + Fig. 2- Effect of temperature on amylase production by Rhamnose + Streptomyces sp.-SBU3 Raffinose + Cellulose - 100 DAP – Diaminopimelic acid ; + : denotes positive; - : denotes negative 90

The amylase production medium was 80 supplemented with 1% NaCl (w/v) have induced maximum amylase yield (200 IU/ml). However 70 the NaCl concentration > 2% (w/v) gradually decreased and finally retarded the amylase 60 enzyme activity (Fig.1). Optimum temperature and pH influenced amylase activity is depicted (IU/ml) activity Amylase 50 5 5.5 6 6.5 7 7.5 8 Fig 2 & 3. Maximum amylase activity were pH perceived at 30oC (100 IU/ml) and 7.5 (95 IU/ml) Fig. 3- Effect of pH on amylase production by Streptomyces respectively. Among the carbon source studied, sp.-SBU3 glucose exhibited maximum amylase activity (150 IU/ml) followed by sucrose (145 IU/ml) when compared with other carbon source tested (Fig. 4). Lactose contributed least activity (125 IU/ml) under experimental condition. Among the nitrogen sources explored, casamino acid exhibited maximum amylase activity of 165 IU/ml followed by casein 154 IU/ml (Fig.5). Different incubation period on amylase activity is presented in Fig.6. When the isolate Streptomyces sp. - SBU3 was incubated at 96h, the maximum 3 INDIAN J. MAR. SCI., VOL. 44, NO. 4 APRIL 2015

concentration plays a vital role in regulating the metabolic activity of the organism. Thus NaCl concentration was one of the important factors 160 controlling the extracellular enzymatic activity. 150 Optimal NaCl concentration required for amylase production was determined by inoculating 140 Streptomyces sp - SBU3 on amylase production 130 medium containing different salinity (NaCl). After 96h incubation, the amylase activity showed 120 maximum of 200 IU/ml at 1% (w/v) salinity. The 110 very less enzymatic activity in the production Glucose Sucrose Maltose Fructose Lactose medium without NaCl confirmed Streptomyces sp Amylase activity (IU/ml) activity Amylase Carbon sources (1%) - SBU3 ionic strength required for its growth. Fig. 4- Effect of various carbon sources at 1 % (w/v) on Influence of temperature on amylase amylase production by Streptomyces sp.-SBU3 production is related to the growth of microbes. A brief lag period was established by the Streptomyces sp - SBU3 for enzymatic production at 20 and 25oC. Enzyme activity was observed to be higher at 30oC and it reached a maximum of 100 IU/ml which was correlated with earlier findings showed maximum activity of extracellular alpha amylase by Streptomyces albidoflavus24. Many previous researchers proved temperature optimum for amylase is found to be in the range of 25 to 30oC for the mesophilic bacteria25. The effect of different pH on amylase enzyme production was determined by inoculating Streptomyces sp - SBU3 on amylase Fig. 5 - Effect of various nitrogen sources at 1 % (w/v) on production medium. Initial pH affects the growth amylase production by Streptomyces sp.-SBU3 of microorganism also affecting the product stability in the medium. The pH of the growth medium plays an important role by causing certain morphological changes in the microorganism, enzyme and extracellular metabolites secretion26. Thus pH effect was studied from 5.0 to 8.0 pH range and amongst the pH used pH 7.5 exhibited maximum amylase activity of 95 IU/ml. Production of alpha amylase is very sensitive to initial pH of the fermentation medium27. These findings of Streptomyces sp - SBU3 used in the present study corroborated with the findings28, reporting that pH 7.5 – 8.0 is best for the production of alpha Fig. 6- Effect of Incubation period (h) on amylase amylase by Bacillus subtilis which is also a gram production by Streptomyces sp - SBU3 positive bacterium. culture media are important for the growth and The effect of carbon sources at 1 % (w/v) production of extracellular amylase in bacteria, level was determined on amylase production. optimization of culture conditions is important for Among the varied carbon sources tested, glucose maximum enzyme production by microbial exerted maximum amylase activity of 150 IU/ml. strains23. Large scale production of industrial Monosaccharide glucose exergonic substrate was enzymes usually involves a wide number of much favoring the production capability of searches for optimatization of production media. enzymes by Streptomyces sp - SBU3. This was achieved by a systematic study on the Interestingly controversial results were observed suitability of various nutritional requirements to in our present study showing glucose positive the growing microbes. Streptomyces sp - SBU3 is effect in enzyme production. However many halotolerant in nature and it requires higher ionic researchers proved its catalytic repression in strength for its growth. In this sense the NaCl enzyme production. Glucose has been known to 4 KRISHNAKUMAR et al.: ALPHA AMYLASE BY SALT – TOLERANT ACTINOMYCETE STREPTOMYCES SP.-SBU3 induce only a minimum level of amylase activity Engineering, Puducherry, India for providing all and support good growth of microbes29. the needed facilities for the study. Kathiresan and Manivannan30 insisted that, References glucose in production of amylase in controversial 1 Rateb, M. E.,Ebel, R., Secondary metabolites of fungi from to microbes. But the present study clearly marine habitates. Nat. Prod. Rep., 28(2) (2011) 290-344. 2 Jensen,P.R., Mafnas,C., Biogeography of the marine indicated that, glucose induced amylase actinomycete Salinispora . Environ. Microbiol., 8 (2006) production by Streptomyces sp - SBU3 when 1881-1888. compared to other carbon sources tested. 3 Mitsuiki, S., Sakai, M., Moriyama, Y., Goto, M., However there is no significant changes among Furukawa, K., Purification and some properties of the carbon sources used in our study. Nitrogen keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. TOA-1. Biosci. Biotechnol. 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