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-glucosidase and -amylase inhibitory activity

International Journal of Integrative Biology

Letter

A journal for biology beyond borders

ISSN 0973-8363

-glucosidase and -amylase inhibitory activity of Micromonospora sp. VITSDK3 (EU551238) K R Suthindhiran, M A Jayasri, K Kannabiran* School of Biotechnology, Chemical and Biomedical Engineering,VIT University, Vellore, India Submitted: 25 Feb. 2009; Accepted: 8 Jun. 2009

Abstract An Actinomycete strain designated as VITSDK3 was isolated from marine sediment sample collected at Marakkanam, the southern coast of India. The strain was characterized using polyphasic taxonomy and identified as Micromonospora sp. The strain is Gram-positive, non-motile and aerial mycelium is absent. The cell wall type is meso-DAP, with xylose and arabinose as characteristic cell wall sugars, MK-10(H6) and MK10(H4) as major menaquinones and contains branched iso and anteiso fatty acid profile. The 16S rRNA sequence analysis indicates that the strain is clustered within the genus Micromonospora. Similarly, based on comparative phenotypic and phylogenetic analysis the isolated strain is identified as a member of genus Micromonospora. The strain is able to grow between 6-22% NaCl concentrations. Maximal growth of the strain is seen on the ISP medium 9, supplemented with 50% sea water and 25% soil extract with a temperature of 28°C, pH of 7.4 and salt concentration of 10% (w/v). The media and cultural conditions for optimal growth have been optimized under shake-flask conditions by measuring the dry weight of the mycelium. The organic solvent extract of the strain is screened for the -glucosidase and -amylase inhibitory activity and shows concentration dependent activity. The extract shows 74.43% inhibition on -glucosidase and 74.32% inhibition on -amylase at 100g/ml. Our investigations indicate that the isolated strain is halophilic, which produces extra cellular metabolites found to be very effective in inhibiting the enzymes -glucosidase and -amylase. The results suggest that the strain produces the secondary metabolites which could be used for the treatment of glucose metabolism related disorders. Keywords: Marine actinomycetes; Micromonospora sp. VITSDK3; secondary metabolites; -glucosidase and -amylase inhibition.

INTRODUCTION Actinomycetes, usually isolated from terrestrial areas are known to be rich sources of novel and pharmaceutically important compounds. As the terrestrial actinomycetes have been extensively studied, the probability of discovering new drugs and compounds is decreasing. Paralellely, the ocean is a fruitful area that can be exploited for the actinomycetes which could contribute to the discovery of novel bioactive compounds (Lam 2006). However, only a small proportion of marine actinomycetes have been isolated and even fewer are successfully grown in the laboratory. Bioactive compounds from marine actinomycetes have been found to be an important *

Corresponding author: K. Kannabiran, Ph.D. Biomolecules and Genetics Division, School of Biotechnology, Chemical and Biomedical Engineering, VIT University,Vellore-632014, Tamil Nadu, India Email: [email protected]; [email protected]

International Journal of Integrative Biology ©OmicsVista Group, All rights reserved

source of drugs for potential targets (Fiedler et al., 2004). These bioactive compounds are secondary metabolites which enhance survival fitness under extreme conditions in the ocean and continue to serve as potential chemical weapons against specified targets. (Proksch et al., 2002). Thus, secondary metabolites from marine actinomycetes have become the subject of commercial interest. Hence, it is anticipated that isolation and characterization of marine actinomycetes could be useful in the discovery of novel bioactive compounds (Fenical 2006). Specific enzyme inhibitors are biochemical tools that have potential utility in the treatment of diseases. It has become evident that actinomycetes are the potential producers of various enzyme inhibitors (Bull et al., 2007). α-amylase and α-glucosidase inhibitors are drug targets for the treatment of diabetes, obesity and hyperlipaemia. Inhibitors inhibit the action of these enzymes results in reduction in starch hydrolysis which IJIB, 2009, Vol. 6, No. 3, 115


shows beneficial effects on glycemic index. These inhibitors can retard the liberation of glucose from dietary complex carbohydrates and delay glucose absorption, resulting in reduced postprandial plasma glucose levels and suppress postprandial hyperglycaemia. (El-Ashry et al., 2000; Franco et al., 2002; Jayasri et al., 2009). Screening of α-amylase and α-glucosidases inhibitors from marine sources has received much attention throughout the world. The marine actinomycetes especially have emerged as a promising new resource yielding unusual chemical structures with potent enzyme inhibitory activity (Imada 2006). But the studies on marine actinomycetes in Indian peninsula are scanty. Taking this into an account, the present study is aimed at isolating the bioactive actinomycetes resulted in isolation of a strain VITSDK3. The cytotoxicity of the crude extract of this strain is reported earlier (Suthindhiran et al., 2008). In this manuscript, we report the isolation, taxonomy and α-amylase and αglucosidase inhibitory activity of Micromonospora sp. VITSDK3.

MATERIALS AND METHODS Sampling, isolation and maintenance The strain VITSDK3 was isolated from the marine soil from Marakkanam [Latitude (N) 12o20′, Longitude (E) 79o95′)] costal region, the south east coast of India, collected at a depth of 400 cm. The soil samples were dried in laminar air flow for 8-12 h, kept at 42ºC for 1030 days in a sterile petri dish and the strain was isolated from these preheated samples. The strain was isolated from the starch casein agar supplemented with antibiotics, cycloheximide (25 g/ml) and nalidixic acid (25 g/ml) (Himedia, Mumbai, India). Plates were incubated at 28±2ºC for 7–18 days. All the media were prepared with varying salt concentrations (3, 5, 7, 9, 12, 15, 18, 21% (w/v)) to enhance the growth of halophilic actinomycetes. The isolated strain was sub-cultured and maintained in slant culture at 4ºC as well as at 20% (v/v) glycerol stock at -80ºC.

Optimization of nutritional and cultural conditions The culture and media conditions were optimized by inoculating the strain in different culture media (SCA, ISP medium 2, ISP 3, ISP 4, ISP 5, ISP 6, ISP 7, ISP 9, Potato agar, Czapek’s agar, Modified Bennett's agar, Sucrose/nitrate agar, Glucose/nitrate agar, Water agar, Glucose/peptone agar, Glycerol/calcium malate agar and Nutrient agar) and the growth was measured by incubating and measuring the dry weight of the mycelium. The effect of culture conditions like


incubation temperature (15, 27, 37, 50ºC), NaCl concentration (6, 12, 15, 18, 22 and 24%) and the pH (5.0, 6.0, 7.4 and 9.0) was also investigated. The carbon and nitrogen sources required were studied by inoculating the isolates into the ISP9 agar with different sugars (D-glucose, sucrose, starch, D-xylose, Dgalactose, maltose, L-arabinose, fructose, lactose, inositol and glycerol) and nitrogen (oraganic nitrogen sources like peptone, yeast extract, casein and inorganic sources like ammonium sulphate, ammonium nitrate and urea) sources. The concentrations for all the carbon sources and carbon utilization tests were done as described earlier (Shirling and Gotileb, 1966; Williams et al., 1989). Based on the above studies the growth medium and cultural conditions for VITSDK3 were optimized.

Taxonomy Methods of International Streptomyces Project (ISP) (Shirling and Gottlieb, 1966) were used to determine the morphology, culture media, physiological and biochemical characterization. The morphology of the spore bearing hyphae with the entire spore chain and the aerial mycelium was observed in light microscope and phase contrast microscope. The substrate mycelium and the matured single spores were further analyzed using scanning electron microscope (Hitachi, S-3400N). The cell wall fraction and sugar composition were analyzed using the method of Hasegawa et al. (1983); Lechevalier and Lechevalier (1980). The whole-cell sugar composition was determined as described by Becker et al. (1965) and Lechevalier and Lechevalier (1980). Identification of polar lipids was done using the method of Minnikin et al. (1984). Menaquinones were determined as described by Collins (1985). Fatty acids were analyzed by inoculating the strain in TSB agar plates [trypticase soy broth (BBL), 3%(w/v); Bacto agar (Difco), 1.5%(w/v)], incubated for 7 days at 28oC. The fatty acids were extracted, methylated and analyzed using the standard MIDI (Microbial Identification) system (Kampfer and Kroppenstedt, 1996). The DNA was isolated by HiPurA bacterial DNA isolation and purification kit (Himedia, India) and amplified by PCR using a master mix kit, Medoxmix (Medox, India) as per user manual. The primers and the PCR conditions were adapted from Rainey et al. (1996). The primers and the methodology for the sequencing were adapted from Mincer et al. (2002); Rainey et al. (1996) and Nathan et al. (2004). The sequencing was carried out for both the sense and antisense directions. The similarity and homology of the16S rRNA sequence was analyzed with similar existing sequences available in the databank - National Center for Biotechnology Information (NCBI) using BLAST search. The DNA sequences were aligned and phylogenetic tree was constructed by neighbor joining method using ClustalW IJIB, 2009, Vol. 6, No. 3, 116


software (Saitou and Nei, 1987). The phylogenetic tree based on Maximum-parsimony method was also carried constructed using MEGA version 3.1 (Kumar et al., 2001). A bootstrap analysis was performed for 1000 replicates. The DNA was isolated by the method of Marmur (1961) and the G+C content was determined using the thermal denaturation method of Marmur and Doty (1962).

Fermentation and extraction of secondary metabolites Well grown slant cultures of the isolates in ISP9 were inoculated into 50 ml medium in 250 ml Erlenmeyer flasks containing the ISP9 production medium with sea water: 25%, distilled water 75%, pH 7.2, and was incubated for 2 days in rotary shaker (200rpm) at 28ºC. 10% of these inoculums were transferred into 200 ml production medium in 1 l Erlenmeyer flasks. The inoculated cultures in the production medium were incubated for 72 h on a rotary shaker (200 rpm) at 28ºC. After fermentation the broth was centrifuged at 4000 rpm for 10 min at 10ºC and the filtrate was separated. The supernatant was extracted twice with ethyl acetate (400 ml) and washed with 500 ml water. The extract was then concentrated in rotary vacuum and lyophilized using a freeze drier (Thermo, USA) at 5ºC for 5 min.

glucosidase solution (1.0 U/ml) were incubated in 96 well plates at 25ºC for 10 min. After pre-incubation, 50 µl of 5 mM p-nitrophenyl-a-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) was added to each well at 5 sec intervals. The reaction mixtures were incubated at 25ºC for 5 min. After incubation, absorbance readings were recorded at 405 nm by micro-array reader and compared to the control which had 50 µl of buffer solution in place of the extract. The α-glucosidase inhibitory activity was expressed as inhibition % and was calculated as follows: % Inhibition 

[(Control405 - Extract 405 )] 100 Control540

RESULTS

Chemicals Porcine pancreatic -amylase (EC 3.2.1.1), αglucosidase from Baker’s yeast (EC 3.2.1.20), pnitrophenyl-a-D-glucopyranoside and dinitrosalicylic acid was purchased from Sigma (India).

Inhibition assay for -amylase activity 500 µl of extract and 500 µl of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) containing -amylase solution (0.5 mg/ml) were incubated for 10 min at 25ºC. After pre-incubation, 500 µl of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) was added to each tube at 5 sec intervals. The reaction mixtures were then incubated at 25ºC for 10min. The reaction was stopped with 1.0 ml of dinitrosalicylic acid color reagent. The test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted after adding 10 ml distilled water and absorbance was measured at 540 nm. % Inhibition 

[(A 540 Control - A 540 Extract)] 100 A 540 Control

Inhibition assay for -Glucosidase activity A volume of 50 µl of sample solution (extract) and 100 µl of 0.1 M phosphate buffer (pH 6.9) containing αInternational Journal of Integrative Biology ©OmicsVista Group, All rights reserved

Figure 1: Scanning electron micrograph of matured single spores. Photomicrograph was taken after 8 days incubation in ISP 9 medium. The size of the spore is 0.6–0.9 μm. Bar 1 μm.

Taxonomy The isolated strain VITSDK3 is a Gram-positive, nonacid fast, non-motile, aerobic actinomycete. The aerial mycelium is absent and produces well branched yellowish brown substrate mycelium on ISP9 medium. The mature single spores on the tips of substrate mycelium were observed (0.6-0.9 m) (Fig. 1). The diameter of the substrate mycelium was between 0.4 and 0.7 m. The colonies were abundant and yellowish to brown in color in ISP9 medium. However the colonies were raised and brown in color on sporulation. The spores were warty but not spiked. The colonies were white in color when cultivated in starch casein agar and ISP2 medium. The characteristics of the strain grown in different media are tabulated (Table 1 [Supplementary data] ). No diffusible pigments were observed. There is no melanin production and the strain hydrolyses starch. The strain utilizes D-glucose, sucrose, D-galactose, L-arabinose, fructose, lactose and

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L-rhamnose, but not D-cellobiose, D-xylose, trehalose and glycerol. Diaminopimelic acid (DAP) analysis shows the presence of meso-DAP in the cell wall peptidoglycan. Xylose and arabinose are present in whole cell hydrolysates. MK-10(H4) and MK-10(H6) are the predominant menaquinones and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides as typical polar lipids (phospholipids type II sensu). The fatty acid profile comprised of iso and anteiso branched (fatty acid type 3b). The isolate was sensitive to tetracycline, neomycin, gentamycin, rifampicin, lincomycin, but resistant to penicillin G. The G+C content of the isolated DNA was 69.6 mol%. The sequence similarity values of 16S rDNA (1141bp) of the strain VITSDK3 revealed that it forms a separate subclade in the phylogenetic tree from its close relatives and the other members of Micromonospora.. The nearest neighbor Micromonospora sp. 206804 shows maximum similarity of 94% with a significant bootstrap support (Fig. 2 [Supplementary data] ). Furthermore, the phylogenetic tree based on maximumparsimony method (Fig. 3 [Supplementary data] ) supports the placement of the strain as a new species within the genus Micromonospora. Based on the molecular taxonomy and phylogeny, it is clear that the strain VITSDK3 belongs to the genus Micromonospora and represents a distinct phyletic line, indicating that the isolate could be a novel species. The 16S rRNA sequence of Micromonospora sp. VITSDK3 has been deposited in GenBank (NCBI, USA) under the accession number EU551238. A neighbor-joining tree based on 16S rRNA gene partial sequence shows that the isolate occupied a distinct phylogenetic position within the radiation including representatives of the family Micromonospora.

The effect of ethyl acetate extract of VITSDK3 on αglucosidase and α-amylase inhibition is given in Fig. 4. The ethyl acetate extract shows a significant αglucosidase inhibitory activity under in vitro conditions. Concentration-dependent inhibition of the activity of αglucosidase was observed at 5, 25, 50, 75 and 100 µg/ml concentration. The highest concentration (100 g/ml) of the extract tested shows a maximum inhibition of 74.43% on the activity of α-glucosidase. The percent inhibition was varied from 74.43 to 49.1% from the highest (100µg/ml) to the lowest concentration (5 g/ml). α-amylase inhibitory effect of ethyl acetate extract of VITSDK3 is shown in Fig. 4. A maximum inhibition (74.32%) of α-amylase activity was observed at 100 g/ml of ethyl acetate concentration and the percentage of inhibition was varied from 74.32% to 48.12% from higher (100 g/ml) to lower (5 g/ml) concentration.

DISCUSSION Microorganisms are known to produce a large variety of enzyme inhibitors that provide protection against insects and microbial pathogens. In particular, marine actinomycetes have emerged as potential producers of enzyme inhibitors. Marakkanam, the sampling site is situated in the state of Tamil Nadu, the south east coast of India, perched at a height of 14 meters (45 feet) above mean sea level. It is a narrow sandy coastal belt exhibiting tidal flat and marsh zones. It has large areas of salt pans near wide backwater with salinity of 32-38 parts per thousand. In the course of systematic screening for bioactive marine actinomycetes, the strain VITSDK3 was isolated at a depth of 400 cm.

Growth optimization Abundant growth of the strain was seen with ISP2, ISP9 and starch casein agar medium. The strain also shows moderate growth using ISP1, ISP6 and ISP7 media. The strain shows maximal growth when cultivated at temperature 28ºC; pH 7.4, with sea water (25%) and soil extracts (25%). The strain requires 10% salt concentration for optimal growth and sporulation. The strain is able to grow between 6-22% NaCl concentration. No growth occurs at 0, 24 and 26% salt concentration. The strain is capable of growing at 6% salt concentration, but shows poor growth and sporulation. Among carbon sources, glucose, starch and arabinose grown strains show abundant growth. The growth is more when the strain is cultivated with organic nitrogen sources compared with inorganic nitrogen sources. Peptone and yeast extract enhance the growth with abundant colonies with white sporulation.

α-glucosidase and α-amylase inhibition International Journal of Integrative Biology ©OmicsVista Group, All rights reserved

Figure 4: -glucosidase and -amylase inhibitory activity of strain VITSDK3 ethyl acetate extract. The values are mean ± SD.

The cultural, morphological, biochemical and physiological characteristics reveals that the strain belongs to the genus Micromonospora. The characteristics of the strain VITSDK3 at the genus level were also confirmed by cell chemistry which includes phospholipids type II sensu (Lechevalier et al., 1977) and fatty acid type 3b (Kroppenstedt 1985). The 16S rRNA (1141 bp) sequencing of the strain VITSDK3 shows maximum similarity with Micromonospora sp. IJIB, 2009, Vol. 6, No. 3, 118


206804 (94%). The strain forms a separate subclade in the phylogenetic tree constructed by neighbor joining method as well as maximum-parsimony method. The results clearly indicate that the strain belongs to the genus Micromonospora and represents a new species. Due to the unavailability of phenotypic data of the strain Micromonospora sp. 206804 from culture bank, we are unable to perform the comparative phenotypic studies as well as the DNA relatedness studies with our strain. However phenotypic comparison was carried out using other closest phylogenetic neighbors based on 16S rRNA sequence similarities (Table 2 [Supplementary data] ). To enrich the growth and yield of secondary metabolite, the culture characteristics were optimized by a systematic study and the suitability of a number of carbon and nitrogen sources were also evaluated and correlated. Optimization was carried out in batch culture and for each parameter the growth of the strain was measured as dry weight of the mycelium. As the samples were collected from the ocean-near-salt pans, the salt concentration in the media was increased and the growth was evaluated. When sea water was periodically enhanced with salt and nutrients, halophilic bacteria show good growth. The occurrence of actinobacteria in highly saline environments has been reported earlier (Gottlieb 1973; Tresner et al., 1968). Halophilic micro-organisms can be conveniently grouped according to NaCl requirements for growth (Ventosa et al., 1998). Extreme halophiles are able to grow in saturated NaCl and are unable to grow in the presence of NaCl concentrations less than 12%. Larsen (1986) defined that moderate halophiles are organisms growing optimally between 5 and 20% NaCl concentrations. VITSDK3 can be defined as a moderate halophile. However, it does not grow without NaCl in the medium, and was phylogenetically distant and exhibits different physiological characteristics from other strains of Micromonospora. The drugs for lowering glucose are inhibitors of amylase and -glucosidase enzymes. These enzymes break down the carbohydrates into absorbable monosaccharides (Stuart 2004). Inhibitors of these enzymes delay the absorption of ingested carbohydrates and thereby reduce the increase in blood glucose level. Therefore, investigation on such agents from new, unexplored sources has become important. Also, there is a need to find new, safe and effective therapeutic agents for the treatment of many diseases and disorders associated with carbohydrate metabolism. We therefore investigated the inhibitory effect of extracts from marine derived halophilic actinomycetes strain VITSDK3 on -glucosidase and -amylase. The crude extracts obtained with different solvents like methanol, chloroform, hexane and water were screened

for enzyme inhibitory activity. But only the ethyl acetate (cell free extract) soluble fraction demonstrated comparatively higher enzyme inhibitory activity against the enzymes. The ethyl acetate extract of VITSDK3 shows significant inhibition of both -amylase and αglucosidase. This may have potential in managing type II diabetes related disorders and the purified inhibitor could be of use as an indicator for combinational therapy. There are reports on enzyme inhibitor producing marine actinomycetes (Sugino and Kakinuma, 1978; Sakuda et al., 1986; Imada and Simidu, 1988; Imada and Simidu, 1992; Aoyama et al., 1995; Imada and Okami, 1995; Imada 2005). However, studies on bioactive marine actinomycetes from Indian peninsula are scanty. Most of the studies conducted in Indian Peninsula have been restricted to isolation, identification and maintenance of actinobacteria and few studies focus on their antagonistic properties against different microbial pathogens. The vast diversity and relatively unexplored nature of these unique sources of chemical diversity suggest that the marine actinomycetes are important sources of novel bioactive compounds. It is suggested that frequent and systematic screening for actinomycetes in the Indian peninsula could provide novel species as well as novel bioactive compounds.

Acknowledgement KS is thankful to the management of VIT University for the award of TRA fellowship (VIT/AR(G)/2006/562). We also thank Mr. Elumalai, Technician, Pondicherry University for the help in SEM photograph.

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