научная статья по теме ISOLATION OF -GLUCOSIDASE-PRODUCING THERMOPHILIC BACILLI FROM HOT SPRINGS OF TURKEY Биология

Текст научной статьи на тему «ISOLATION OF -GLUCOSIDASE-PRODUCING THERMOPHILIC BACILLI FROM HOT SPRINGS OF TURKEY»

МИКРОБИОЛОГИЯ, 2009, том 78, № 1, с. 68-78

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

УДК 579.8.083.12(560)

ISOLATION OF a-GLUCOSIDASE-PRODUCING THERMOPHILIC BACILLI

FROM HOT SPRINGS OF TURKEY

© 2009 г. A. Coleri*, C. Cokmus*1, B. Ozcan**, N. Akkoc*, and M. Akcelik*

* Ankara University, Faculty of Science, Department of Biology 06100 Tandogan Ankara, Turkey **Mustafa Kemal University, Faculty of Sciences and Letters, Department of Biology, 31040 Hatay, Turkey

Поступила в редакцию 20.05.08 г.

Abstract - From 42 different hot springs in 6 provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the a-glucosidase production capacities by using pNPG as a substrate. a-Glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced a-glucosidase at significant levels comparable with that of the reference strains tested, thus five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g) were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes of a-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to a-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular a-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in a-glu-cosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable a-1,4-glucosidase-produc-ing bacilli that have biotechnological potential with a-glucosidases located at different cell positions were obtained.

Key words: isolation, thermophilic bacilli, 16S rDNA, a-glucosidase.

INTRODUCTION

a-Glucosidase (a-D-glucoside glucohydrolase, EC 3.2.1.20) occur in microrganisms as intracellular, extracellular, or cell-bound enzymes, and hydrolyze terminal, non-reducing a-1,4-linked-D-glucose residues in short-chain oligosaccharides including maltose, which are formed by other amylolytic enzymes, like a-amylase (EC 3.2.1.1) or glucoamylase (EC 3.2.1.3) [1]. They have a number of potential applications in fundamental research, industrial starch processes, synthesis of oligo-, di-, and trisaccha-rides, as an indicator for sterilization control, and in a-amylase assay in clinical laboratories. They are usually found in association with other amylolytic enzymes which accomplish complete degradation of starch, and are widely distrubuted throughout the three major kingdoms [2, 3].

The industrial demand for enzymes that can withstand harsh conditions has greatly increased over the past decade. Therefore, thermostable enzymes, which have been isolated mainly from thermophilic organisms, have found a number of commercial applications because of their overall inherent stability. Advances in this area have been possible with the isolation of a large number of thermo-

1 Corresponding author (e-mail: cokmus@science.ankara.edu.tr).

philic microorganisms from different ecological niches and subsequent production of valuable enzymes [4, 5].

Typically, enzyme production in the course of Bacillus fermentation processes occurs during a relatively short period of time, with very low-cost carbon and nitrogen sources. Members of the genus Bacillus have been used in multiple research works aimed on screening and purification of thermostable enzymes [6, 7]. a-Glucosidases from B. cal-dovelax DSM411 [8], B. flavocaldarius KP1228 (FERM-P9542) [9], B. thermoamyloliquefaciens KP1071 (FERM-P84776) [10], G. stearotherothermophilus ATCC 7953 [11], G. stearothermophilus ATCC 12016 [12], G. ther-modenitrificans HR010 [13], Geobacillus HTA-462 [14], and Bacillus sp. DG0303 [15] have been well characterized.

The aim of this study was to isolate and screen a-glu-cosidase-producing thermophilic microorganisms from Turkey hot springs, in order to determine their enzyme production capacities and to define the a-glucosidase production types in some of new isolates by measuring the change in the enzyme levels in intracellular and extracellular fractions.

MATERIALS AND METHODS

Bacterial Isolates and Standard Strains

One hundred and ninety-one samples of water, soil, and sediments were collected from 42 different hot springs and high-temperature well pipelines of geographically widespread locations in the provinces of Ankara, Aydin, Denizli, Izmir, Manisa and Nevsehir in Turkey. The water temperature and pH of these geothermal regions were between 60-90°C and 6.0-9.0, respectively. a-Glucosidase-producing strains: A333 and A343 were isolated from soil and sediment samples of Germencik (Aydin) and Salavatli (Aydin), respectively. F84a and F84b were both obtained from sediment samples of Kizil-cahamam (Ankara) high-temperature wells pipelines. E134 was isolated from a water sample of Kozakli (Nevsehir) hot spring. Geobacillus stearothermophilus ATCC 12980, G. stearothermophilus ATCC 43223, An-oxybacillus flavothermus DSM 2641, Anoxybacillus ke-stanbolensis NCIB 13971, Anoxybacillus gonensis NCIB 13933, Anoxybacillus ayderensis NCIB 13972 were kindly provided by Prof. Dr. Ali Osman Belduz (Karadeniz Technical University, Turkey) and G. stearothermophilus ATCC 7953 (DSM 5934), G. thermoglucosidasius DSM 2542, Bacillus amyloliquefaciens DSM 7 by DSMZ (the German Collection of Microorganisms and Cell Cultures).

Isolation of Thermophilic Amylolytic Bacilli

For enrichment, the water, sediment, and soil samples were incubated with shaking at 60°C and 250 rpm for 24 h in 5 ml of the G. thermoglucosidasius medium (MI) containing 1% soluble starch (Sigma S2004), 0.5% pepton, 0.3% yeast extract, 0.3% meat extract, 0.3% K2HPO4 and 0.1% KH2PO4 (pH 7.0) [16]. Pure cultures showing different colony morphology were isolated by streaking on plates of the same medium containing 3% agar. All the isolates were tested for their Gram reaction, cell and colony morphology, motility, sporulation, thermophilic growth, catalase and amylase activity [17]. Cell morphology, sporulation and motility were determined by phase-contrast microscopy of freshly prepared wet mounts [17, 18].

After cultivation of new isolates on MI agar plates at 60°C for 24 h, the plates were treated with iodine solution (0.2% I2 in 2% KI) in order to determine amylolytic activity. The thermophilic amylolytic isolates with starch digestion zones around their colonies were selected for further investigations on a-glucosidase production capacities [7, 16]. All the isolates were maintained on nutrient agar slants at 4°C and stored in nutrient broth containing gly-cerol at -80°C.

16S rDNA Amplification and Sequencing

Genomic DNA was extracted according to Marmur [19] from the cultures growing in nutrient broth for 18 h at 60°C. The gene encoding 16S rDNA was amplified by PCR with the 16S bacteria-specific 27Fa forward primer

(5'-AGAGTTTGATCCTGGCTCAG-3') and the 1492R reverse primer (5'-GGTTACCTTGTTACGACTT-3') using the method of Bond et al. [20]. The amplification products were purified from agarose gel using Qiagen Qi-aexII Gel Extraction kit. These products were cloned into PCR2.1 vector with TOPO TA Cloning Kit (Invitrogen) and sequenced by using M13 (-21) and M13R primers peculiar to PCR2.1 vector.

Phylogenetic Analysis

Homology search was carried out by using the basic BLASTN search program at the NCBI Web-site. 16S rDNA sequences were aligned by using Clustal W [21]. Tree distance matrix was calculated on the basis of the algorithm of Jukes and Cantor [22]. A phylogenetic tree was constructed by the neighbor-joining method and evaluated by bootstrap sampling (1000 replicates) using the MEGA 4 program [23].

Enzyme Assay

a-Glucosidase activity was determined spectrophoto-metrically by measuring the hydrolysis of para-nitrophe-nol a-D-glucopyranoside (pNPG, "Sigma" N1377) as substrate, by the modified methods [16, 24, 25]. The standard reaction mixture in a total volume of 4 ml contained: 33.3 mM potassium phosphate (pH 6.8), 2 mM pNPG and 500 |l of an appropriately diluted enzyme solution. The reaction was carried out at 37°C for 10 minutes and stopped by adding 1 M Na2CO3. The release of pNP (para-nitro-phenol) was measured by absorption at 400 nm, and one unit of enzyme was defined as the amount of enzyme needed for hydrolysis of 1 | mol pNPG per minute at 37°C, pH 6.8. The millimolar extinction coefficeint of pNP at 400 nm and pH 6.8 was measured as 18.5 L mM-1 cm-1, and was used to calculate the product yield. All the enzyme assays were performed at least three times.

Growth Conditions for a-Glucosidase Production

In order to determine intracellular or extracellular a-glucosidase production capacities of thermophilic, amy-lolytic isolates and enzyme-producing reference strains (G. stearothermophilus ATCC 12980, G. stearothermo-philus ATCC 43223, G. stearothermophilus ATCC 7953, and G. thermoglucosidasius DSM 2542), the organisms were cultured on MI agar plates at 60°C for 24 h. Actively growing cells were then resuspended in 0.85% NaCl to an absorbance value of 0.16-0.3 at 660 nm. For each of the isolates, 0.5 ml of the resulting suspension was inoculated into 3 test tubes of 5 ml enzyme prod

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