Selective Isolation and Molecular Identification of l-glutaminase producing Bacteria from Marine Sediments
Kiruthika J.1* and Saraswathy N.2
1. Government College of Technology, Coimbatore-641013, Tamil Nadu, INDIA
2. Kumaraguru College of Technology, Coimbatore-641049, Tamil Nadu, INDIA
*mailkiruthika@yahoo
Abstract
L-Glutaminase (L-glutamine amidohydrolase E. C.
3.5.1.2) is a therapeutic enzyme which catalyses the conversion of L-glutamine to L-glutamic acid and ammonia. It has gained importance due to its potential application as anti-cancer and flavor enhancing agent. With an aim to isolate a potential L-glutaminase producing strain, soil samples were collected from different river banks of Kerala and Tamil Nadu, India. Among the several strains, based on the enzyme activity, one of the potent isolate was selected and identified.
The morphological and biochemical characterization revealed that the potent isolate belongs to the genus Vibrio. Further, 16s rRNA gene (1.4 Kbp) of the potent isolate was amplified and analyzed which showed that it had about 99% similarity with Vibrio sp. IMSF-06 (NCBI accession no. GQ907023) and the next homologue was Vibrio azureus; HNS029 (NCBI accession no. JN128263). Thus, the strain was ultimately identified as Vibrio azureus strain JK 79 (GenBank Acession Number JQ820323). Keywords: L-glutaminase, Isolation, Identification, 16 S rDNA analysis, Vibrio azureus JK 79.
Introduction
L-glutaminase (L-glutamine amidohydrolase E.C. 3.5.1.2) catalyses the hydrolysis of L-glutamine to L-glutamic acid and ammonia. This is an essential enzyme for the synthesis of various nitrogenous metabolic intermediates. Glutaminase is synthesized by various bacteria, fungi, yeast, moulds and filamentous fungi.5,7This enzyme is involved in glutamine catabolism in micro-organisms. Mammalian cells also synthesis this enzyme which is involved in the generation of the energy using glutamine as the major respiratory fuel. Thus, many types of tumor cells as well as actively dividing normal cells exhibit high rates of glutamine utilization.6
Cancer cells, especially Lymphatic tumor cells cannot synthesize L-glutamine and hence require large
amount of L-glutamine for their rapid growth. Thus these cells depend on the exogenous supply of L-glutamine for their survival and rapid cell division. Hence, the use of amidases deprives the tumor cells from L-glutamine and causes selective death of L-glutamine dependant tumor cells. L-asparginase and L-glutaminase earned attention due to their anti-tumor properties.1
The terrestrial bacterial source of L-asparginase is currently used for the treatment of leukemia but this is known to cause a lot of side effects and hence there is a need for an alternative enzyme drug which is more compatible to human blood and induces less or no side effects in patients. The marine environment particularly sea water, which is saline in nature and chemically closer to human blood plasma, can provide microbial enzymes that are safe with no or less side effects when administered for human therapeutic application. Yet another important fact about marine microbial enzyme is that they show high level of salt tolerance. Hence there is an increasing interest in the marine micro-organism for therapeutic purposes.3,13 Another important application of L-glutaminase is in food industry as flavor enhancing agent. It increases the glutamic acid content of the fermented food there by imparting a unique flavor.15Since the sources for L-glutaminase are limited, the search for potential strains that hyper-produce enzyme with novel properties for their industrial production is being pursued all over the world.9 Screening of microbes for any bio-product involve either sample collection from the ecological
sites (soil and /or water), followed by screening of microbes from that sample or screening already available micro-organisms. The most efficient way of finding new microbial source of enzyme is to screen large number of microbes due to their diversity and versality.6Identification of bacteria by morphological and biochemical characterization is a difficult task as it is time consuming and also is accompanying many difficulties due to differences in physical and biochemical characteristics of the same strain grown in different habitats.    A combination approach of 16 S rRNA sequencing and biochemical characterization of organisms by Bergey’s Manual of Systematic Bacteriology is being currently used for the authentic identification of bacteria.6,8 In the present study we describe the selective isolation of a potent marine bacterial strain that hyper- produces the L-glutaminase enzyme. The molecular identification of the novel strain was performed by 16S rDNA sequence analysis.
Material and Methods
Sample Collection: Soil samples were collected from different river banks of Tamil Nadu (Chennai, Ramanathapuram, Parangipettai, Mangrove forest) and
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Kerala (Thrissur, Calicut, Alapuzha, Cochin, Chavakkadu) in sterile polythene bags and transported to
the laboratory. The samples were stored at 40C till further use. Enrichment and Screening for L-glutaminase Isolate: One gram of soil sample was suspended in 100 ml of sea water and mixed well by keeping it in incubator cum shaker (100 rpm, 370C) for 30 minutes for the complete mixing of the soil sediment. About 1.0 ml of the supernatant solution was serially diluted in sterile sea water and 0.1 ml of this was spread on minimal glutamine agar (MGA) medium. The constituents of MGA (grams/litre) include 0.5 KCl; 0.5 MgSO4; 1.0 KH2PO4; 0.1 FeSO4; 0.1 ZnSO4; 10 glutamine, 0.12 Phenol red and cycloheximide (20 µg/ml). L-glutamine acts as the sole carbon and nitrogen source, Phenol red acts as pH indicator and antibiotic cycloheximide retards the fungal growth. The plates were incubated at 370C for 48 hours.
Only the bacteria which synthesise L-glutaminse can grow in MGA media and the extracellular production of l-glutaminase was confirmed by the formation of pink color zone around the colonies. Single colonies were selected and maintained in glutamine agar medium. All the media in the present work was prepared with sea water which has 30 ppt (parts per thousand) of salinity. Seventeen morphologically different isolates were selected and L-glutaminase activity was determined.
Enzyme Assay: For the seventeen strains, L-glutaminase activity was measured by Imada et al4method. The different isolates were grown for 24 hrs at 370C and 180 rpm and harvested by centrif
ugation at 10000 rpm for 20 minutes and the supernatant was used for enzyme assay. An aliquot of 0.5 ml of the sample was mixed with 0.5 ml of 0.04M L-glutamine solution in the presence of 0.5 ml of distilled water and 0.5 ml of phosphate buffer (0.1 M , pH 8). The mixture was incubated at 370C for 30 mins and the reaction was arrested by the addition of 0.5 ml of 1.5 M trichloro acetic acid. To 0.1 ml of the mixture, 3.7 ml of distilled water and 0.2 ml of Nesslers reagent were added. The absorbance was measured at 450nm using a UV-Visible spectrophotometer. The liberated ammonia content was measured and one international unit of L-glutaminase was defined as the amount of enzyme that liberates one µ mole of ammonia under optimal conditions. The enzyme yield was expressed as Units/ml (U/ml).
Phenotypic and Biochemical Identification: The morphological analysis such as texture, color and margin of the potent strain Est1 was determined. The biochemical characterization tests were carried out according to Bergey’s manual of Systematic Bacteriology.
Molecular Characterization of the Isolate: The Genomic DNA was isolated from the culture Est1 using Chromous Genomic DNA isolation kit (RKT09) and the steps were followed according to the manufacture’s protocol. The
Primers 16s FP 5'-AGAGTRTGATCMTYGCTWAC-3' and 16s RP 5'-CGYTAMCTTWTTACGRCT-3' were used to amplify the 1.4 Kbp 16s rDNA gene. The PCR was carried out with initial denauration at 940C for 5 min, followed by 35 cycles of denaturation at 940C for 30 sec, annealing at 550C for 30 sec , extension at 720C for 2 min and final extension at 720C for 5 min using Thermal Cylcer ABI2720.
The amplified PCR product was analysed by agarose gel electrophoresis under standard conditions. The Chromus PCR purification Kit was used to recover the amplicon from the agarose gel. Purified PCR product was sequenced directly by ABI 3500 XL Genetic Analyzer using Big Dye Terminator version    3.1”Cycle sequencing kit. The nucleotide sequences of the 16s rDNA obtained were subjected to Blast analysis for gene homology search and was deposited in GenBank database.
Phylogenetic Analysis of the strain: The 16s rRNA sequence data was aligned with System Software aligner and analyzed to identify the bacterium and its closest neighbors. The high scoring sequences similar to 16s rDNA gene were identified and retrieved from GenBank database. The Phylogentic Tree Builder uses aligned sequences and a distance matrix was generated using the Jukes-Cantor corrected distance model. When generating the distance matrix, only alignment model positions was used, alignment inserts were ignored and the minimum comparable position is 200. The tree was created using Weighbor with alphabet size 4 and length size 1000.
Results and Discussion
Isolation of the Micro-Organism: Among the soil samples collected from the river banks of different regions in Kerala and Tamil Nadu, seventeen isolated were selected based on the morphological difference and appearance of pink colur zone around the colonies (Figure 1). Minimal Glutamine Agar (MGA) medium was used for the selective isolation of the L-glutaminase producing bacteria. Only the bacteria which produce L-glutaminase enzyme extracellularly, can grow in the MGA medium as L-glutamine is the only carbon and nitrogen source available in the media.
Further the MGA media was supplemented with 0.012% Phenol red (pH indicator) and this gives a yellow colour to the media. The bacteria utilize the L-glutamine by hydrolyzing it with L-glutaminase and this leads to the production of glutamic acid and ammonia. The accumulation of ammonia in the media changes its pH to alkaline and due to the presence of phenol red in the media, the bacterial colonies are surrounded by pink zone. All the seventeen isolates were designated based on the place where the soil samples were collected. The isolates include Ram 1, Ram 2, Est 1, Est2, Man, Thri 1, Thri 2, Thri 3, Cali 1, Cali 2, Cali 3, Ala 1, Ala 2, Ala 3, Koc 1, Chav 1 and  Che 1.
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Determination of L-glutaminase Activity: The L-glutaminase activity of all the 17 isolates was determined by Imada et al 4 method. One international unit of enzyme activity is defined as the amount of enzyme required to liberate one micromole of ammonia under standard conditions. The enzyme activity of the different isolates is summarized in figure 2. Among the 17 isolates (Ram 1, Ram 2, Est 1, Est2, Man, Thri 1, Thri 2, Thri 3, cali 1,cali 2,cali 3,ala 1,ala 2,ala 3,koc 1,chav 1 and che 1), the isolate Est1 isolated from the estuary region of Prangepettai (Lat. 110 42’ N; Long. 790 46’ E) east coast of India, showed the maximum enzyme activity ( Figure 2). Hence, the isolate was selected for further identification.
Biochemical and Molecular Characterization of Isolate: The colonies of the Est1 strain were found to be pale white in color with smooth surface. Gram staining was performed and the bacterium was found to be Gram negative and rod shaped in structure (Fig 4, Table1). All the other biochemical characterization tests were carried out (Table 4) along with cultural characteristics of the isolate (Table 2 and 3). From the results, it was concluded that the isolate Est1 belongs to genus Vibrio  and was designated as marine Vibrio azureus  strain JK-79. The isolate was further confirmed by growing it in Vibrio selective media TCBS Agar (Thiosulfate-citrate-bile salts-sucrose agar Fig. 3)
Figure 1: L-glutaminase activity of marine Vibrio azureus  strain
JK-79 on MGA medium
Figure 2: Enzyme activity of the seventeen bacterial isolates .
20406080100120140R A M  1
R A M  2
T H R I  1
T H R I  2
T H R I  3
M A N
E S T  1
E S T  2
C A L I  1
C A L I  2
C A L I  3
A L A  1
A L A  2
A L A  3
C H E N  1
K O C  1
C H A V  1
Y i e l d  U /m l
Bacterial Isolates
Enzyme Activity
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Table 1
Morphological Characteristics of marine Vibrio azureus  strain JK-79
S.N. Colony Morphology
Result 1 Colour Dull White 2 Shape Circular 3 Margin Entire 4 Texture Smooth 5 Elevation Raised 6 Optical Opaque 7 Pigmentation Non-Pigmented 8 Staining Gram Negative
9 Cell shape Rod 10 Motility Motile 11 Appearance Dull
12 Arrangement Singles  or Short Chain
13 Endospore Negative 14
Flagella
Positive
Table 2
Effect of pH on the growth of marine Vibrio azureus
strain JK-79
S.N. pH Result 1    5 + 2    6 + 3 7 + 4 8 + 5 9 + 6
10 -
Table 3
Effect of temperature on the growth of marine Vibrio
azureus strain JK-79
S.N. Temperature (ºC)
Result 1    4 - 2 10 - 3 25 + 4 28 + 5 30 + 6 35 + 7 37 + 8 40 + 9 45 + 10 50 + 11 55 - 12 60 -
Figure 3: Green Colonies of marine Vibrio azureus
strain JK-79 on TCBS Agar
Figure 4: Gram staining of marine Vibrio azureus
strain JK-79
Table 4
Biochemical Characteristics of marine Vibrio azureus
strain JK-79
S.N. Cultural characteristics Result
1 Indole test -
2 Methyl Red Test -
3 Voges Proskauer Test +
4 Gas Production from
Glucose
-
5 Citrate utilization +
6 Casein Hydrolysis +
7 Starch Hydrolysis +
8 Gelatin Hydrolysis +
9 Urease -
10 Nitrate reduction +
11 H2S Production -
12 Cytochrome Oxidase +
weigh翻译13 Catalase Test +
14 Oxidation/Fermentation +
15 Arginine Dihydrolase -
16 Lysine decarboxylase
-
17 Ornithine decarboxylase -
18 Phenylalanine
deamination
+
19 ONPG _
20 Mealonate utilization +
21 Esculin hydrolysis +
22 Arabinose _
23 Xylose _
24 Adonitol _
25 Rhamnose _
26 Cellobiose +
27 Melibiose _
28 Saccharose +
29 Raffinose _
30 Trehalose +
31 Glucose +
32 Lactose _
33 TCBS agar Green
Colonies
34 Mac Conkey Agar -
Molecular Characterization of the Marine V ibrio Azureus strain JK-79: The molecular identification of the isolate Vibrio JK 79 was evaluated by the isolation and sequencing of the 16 s rRNA of this strain. The gene sequence was about 1.4 Kbp and contains 1439 bp nucleotides which was submitted in GenBank (National centre for biological Information, USA, Accession Number
JQ820323). The blast analysis revealed that the isolate was found to be most similar (99%) to Vibrio sp. IMSF-06 (NCBI accession no. GQ907023) and the next homologue was Vibrio azureus; HNS029 (NCBI accession no.
JN128263). Phylogenetic tree was constructed by taking the sequences obtained in the blast search given in fig. 5 and from this it was observed that the isolate belongs to the Vibrio azureus family.
Figure 5: Phylogenetic tree based on 16S rRNA gene
sequence comparision showing the relationship between
members of genus Vibrio and marine Vibrio azureus
strain JK-79
C onclusion
The present study focuses on the selective isolation of the potent L-glutaminase producing marine bacteria. The isolates potential in L-glutaminase production was evaluated by Imada et al4method. Based on the enzyme activity, the potent isolate was selected and further biochemical and molecular identification (16s rDNA analysis) was carried out to identify the micro-organism.
The isolate was found to be Vibrio azureus (GenBank Accession Number JQ820323).This strain isolated from the marine sediments has the potential for L-glutaminase  synthesis and suggests that this can be used as a potent source of the enzyme. Further studies on the media optimization for the large scale production of the enzyme from this strain are in progress.
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(Received 08th March 2013, accepted 27th May 2013)
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