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And Lipase-Producing Halococcus Agarilyticus GUGFAWS-3 from Marine Haliclona Sp

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And Lipase-Producing Halococcus Agarilyticus GUGFAWS-3 from Marine Haliclona Sp Annals of Microbiology (2018) 68:851–861 https://doi.org/10.1007/s13213-018-1391-6 ORIGINAL ARTICLE Isolation and culturing of protease- and lipase-producing Halococcus agarilyticus GUGFAWS-3 from marine Haliclona sp. inhabiting the rocky intertidal region of Anjuna in Goa, India Sanket Krishnanath Gaonkar1 & Irene Jeronimo Furtado1 Received: 23 March 2018 /Accepted: 24 October 2018 /Published online: 19 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018 Abstract Three extremely halophilic bionts GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 were isolated from a marine, white sponge, attached to rocks, in the intertidal region of Anjuna, Goa, India (15° 34′ 05″ N, 73° 44′ 17° 40′ E). Because the sponge had irregular tubes arranged in clusters, it was identified as Haliclona sp. All sponge bionts produced protease and lipase. GUGFAWS-1 and GUGFAWS-2 were euryhaline Eubacteria, growing from 0 to 30% NaCl concentration. The biont, GUGFAWS-3, was a haloarchaeon having glycerol diether moieties in its cells and showed R-O-R and long isoprenoid chains, in FTIR. The haloarchaeon, GUGFAWS-3, simultaneously produced extracellular 49.5 U mL−1 of protease and 3.67 U mL−1 of lipase, in the presence of 25% NaCl. It grew as dark orange-red colonies at 5–30% NaCl. Its growth was sensitive to bile salts and resistant to 700 U of penicillin. Cells were Gram-negative cocci, arranged in pairs, and 1-μm size in SEM micrograph. It possessed bacterioruberin with absorption at 387, 468, 492, and 523 nm. The 16S rRNA gene sequence of GUGFAWS-3 was 99.1% similar to Halococcus agarilyticus 62E (T) of the family Halococcaceae of the domain Archaea. This study is the first evidence of retrieval and culturing of Halococcus agarilyticus strain GUGFAWS-3 (MF425611) from marine Haliclona sp. with ability to simultaneously produce protease and lipase extremozymes of ecological and biotechnological significance. Keywords Marine Haliclona sp. Haloarchaea . Protease . Lipase . Extremozymes Introduction or fatty acid ester. Proteases (3.4.21-24) hydrolyze proteina- ceous material of plant and animal origin (Zhao et al. 2008). Globally, haloarchaea and extremely halophilic eubacteria are Extracellular lipases with optimum activity in the range of investigated for the production of commercially important 3–4 M NaCl have been isolated from Natronococcus sp. proteases and lipases, which function at 3–4 M NaCl and (Bhatnagar et al. 2005; Boutaiba et al. 2006), Haloarcula water activity of aw 0.75 (Grant 2004; Gupta et al. 2015). marismortui (Camacho et al. 2009), and from unidentified Lipases (3.1.1.3) and esterases (3.1.1.1) hydrolyze long- haloarchaeal strains (Ozcan et al. 2009). To this day, only a chain fatty acyl-glycerol and short-chain acyl-glycerol, respec- single lipase from Haloarcula sp. G41 has been purified and tively. These enzymes carry out amidation, transesterification, studied for biotechnological application by Xin and Hui-Ying and enantioselective reactions (Matsumoto et al. 2004). Their (2014). Similarly, proteolytic enzymes with optimal activity in production is induced by oil, fatty acid, fatty acid alcohol, and/ the range of 3–4.3 M NaCl have been reported by: Norberg and Hofsten (1969)inHalobacterium salinarium strain I, Izotova et al. (1983)inHalobacterium halobium,Kamekura and Seno (1993)inHaloferax mediterranei R4, Vidyasagar Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-018-1391-6) contains supplementary et al. (2006)inHalogeometricum borinquense strain TSS101, material, which is available to authorized users. Shi et al. (2006)inNatrinema sp. R6-5, Manikandan et al. (2009)inHaloferax lucentensis VKMM007, and Akolkar * Irene Jeronimo Furtado et al. (2009)inHalobacterium sp. SP1. [email protected] As intertidal reaches are exposed to tidal fluxes with fluc- tuating salinity and also to effluents, having animal and petro 1 Department of Microbiology, Goa University, Taleigao Plateau, Goa 403 206, India industries waste, we envisaged that sponges from these 852 Ann Microbiol (2018) 68:851–861 reaches would be a good source of extremely halophilic bionts were incubated at RT for 15 days, and checked for growth with polymer-degrading capabilities. Earlier, Velho-Pereira or inhibition of growth. and Furtado (2014) reported haloarchaeal bionts from marine The resistance of each isolate to antibiotics was evaluated sponges, inhabiting Mandapam, India. by spread plating, individual cultures, in their log phase, on During this study, we successfully isolated and character- modified Miller-Hinton agar with 25% NaCl (w/v), for lawn ized a biont GUGFAWS-3, as Haloccoccus sp., and the host growth. Antibiotic Discs (octa-disks; HiMedia Laboratories sponge as Haliclona sp. Interestingly, this biont simultaneous- Ltd., Mumbai, India) impregnated with appropriate antibiotic ly produces protease and lipase extremozymes, functional in such as bacitracin (5 μg), penicillin (10 U), ampicillin (5 μg), the presence of 25% NaCl, and is of great commercial and ciprofloxacin (5 μg), streptomycin (10 μg), vancomycin biotechnological significance. (30 μg), gentamycin (10 μg), and linezolid (30 μg) were placed on agar and monitored after 15 days for zone of inhi- bition of growth. Materials and methods Phenotypic characterization of microbial bionts Isolation of microbial bionts from the marine sponge The three distinct isolates GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 were individually subjected to Gram reaction. A piece of 2 × 2 cm2 was excised from a marine sponge, Phenotypic characteristics of cell morphology, catalase, nitrate growing attached to rocks in the intertidal region (located at reductase, and motility were examined as described by 15° 34′ 05″ N, 73° 44′ 17° 40′ E), in Anjuna, Goa, India. The Smibert and Krieg (1994). sponge sample was washed and macerated, using sterile motor Cellular morphology of each of the three halophilic isolates and pestle. The macerate was added to 100 mL of sterile NaCl GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 was deter- Tryptone Yeast Extract Medium (NTYE) (Braganza and mined, using a light microscope (Nikon H600L) and 100X oil Furtado 2009) consisting of 20 g MgSO , 5 g KCl, 0.2 g 4 immersion lens. Gram character was studied using the modi- CaCl , 3 g yeast extract, 5 g tryptone, and 250 g crude salt/ 2 fied gram staining method of Dussault (1955). The slide was NaCl per liter, pH 7 (adjusted with 1 N NaOH). Penicillin to air dried and observed under oil immersion lens. final concentration of 300 U/mL was also added. Flasks were Biochemical tests such as utilization of citrate and produc- incubated in a REMI BOD CIS-24 PLUS at 150 rpm, at 30 °C. tion of acid from D-glucose, sucrose, and lactose were done After 8 days, an aliquot of this growing culture broth was according to Norberg and Hofsten (1969). Stocks of D-glu- serially diluted and spread on to NTYE agar. Plates were in- cose, sucrose, and lactose were prepared separately and added cubated at room temperature (RT, 28 to 30 °C) for 15 days. to the basal medium to a final concentration of 0.5% (v/v). Three distinct colonies were picked, purified, and designated Each tube was inoculated with 2% (v/v) inoculum of each of as GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 denoting the three isolates separately in 20% NaCl with OD of 1 at GU (Goa University), G (Gaonkar), F (Furtado), A (Anjuna), A . All tubes were incubated at RT for 8 days. WS (white sponge), and 1, 2, and 3 as strain numbers. 600 Thin smear of cells of GUGFAWS-3 freshly grown in a synthetic medium with 20% NaCl (NSM) (Aguiar and Physiological characterization of microbial bionts Furtado 1996; Raghavan and Furtado 2004) was prepared on a clean glass coverslip (2 × 2 cm), air dried, and exposed Isolates GUGFAWS-1, GUGFAWS-2, and GUGFAWS-3 to 2.0% (v/v) glutaraldehyde in 20% NSM at RT overnight. were grown separately in (i) Tryptone Yeast extract (TYE) Coverslip was exposed to an increasing gradient of acetone- broth (Aguiar and Furtado 1996)consistingof(g/L)20 water of 30%, 50%, 70%, and 90% each for 10 min, respec- MgSO4, 5 KCl, 0.2 CaCl2, 3 yeast extract, and 5 tryptone, tively, and finally to 100% acetone. Coverslip was air dried pH 7 and (ii) TYE with salt concentration ranging from 5 to and mounted on to an SPI-Module sputter and observed under 30% at RT, pH 7, on a rotary shaker at 150 rpm. Growth was SEM (ZEISS E VO 18 special edition) for morphological measured at 600 nm using a Shimadzu UV–Vis details. spectrophotometer-1601 (Japan). The isolates were spot inoculated on the NTYE agar medi- Preparation of whole cells and chemotaxonomic um with pH varying from 5, 6, 7, 8, and 9 and incubated at RT analysis for 15 days. The temperature required for optimum growth of the isolates was determined, by inoculating culture on NTYE GUGFAWS-1, GUGFAWS-2, GUGFAWS-3 were individu- agar and incubating at RT, 37 °C and 45 °C. ally grown in NTYE at RT and 150 rpm. After 8 days, cells Individual cultures were spot inoculated onto were harvested at 2432g for 10 min at 4 °C using an MacConkey’s agar containing 25% NaCl (w/v) and plates Eppendorf 5417 R centrifuge. Cells of each culture were Ann Microbiol (2018) 68:851–861 853 resuspended separately to an absorbance 2 at 600 nm and Culturing of Halococcus sp. for production studied for their susceptibility to water (Fukushima et al. of extracellular protease and lipase 2007), pigment profile (Raghavan and Furtado 2005), and presence of glycerol diether moieties (GDEMs) (Ross et al. Isolate, GUGFAWS-3, pre-grown in the NSM liquid medi- 1981). Haloferax alexandrinus KF796625 was used as a stan- um with 0.2% glucose (NGSM), was inoculated into the dard haloarchaeon.
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