Isolation and Characterization of L-Glutaminase producing Bacteria

Being a significant protein L-glutaminases discovers potential applications in various divisions running from nourishment industry to restorative and cure. It is generally disseminated in microbes, actinomycetes, yeast and organisms. Glutaminase is the principal enzyme that changes glutamine to glutamate. The samples were gathered from soil of Taxila, Wah Cantt and Quetta, Pakistan for the isolation of glutaminase producing bacteria. After primary screening, subordinate screening was done which includes multiple testification such as purification, observation of morphological characters and biochemical testing of bacterial strains along with 16S rRNA sequence homology testing. Five bacterial strains were selected showing glutaminase positive test in screening, enzyme production via fermentation and enzymatic and protein assays. Taxonomical characterization of the isolates identified them as Bacillus subtilis U1, Achromobacter xylosoxidans G1, Bacillus subtilis Q2, Stenotrophomonas maltophilia U3 and Alcaligenes faecalis S3. The optimization of different effectors such as incubation time, inducers, carbon source, pH, and nitrogen source were also put under consideration. There was slight difference among incubation of bacterial culture, overall, 36 hours of incubation time was the best for glutaminase production by all the strains. Optimal pH was around 9 in Achromobacter xylosoxidans G1 and Alcaligenes faecalis S3, pH 6 in Bacillus subtilis U1, pH 8 in Stenotrophomonas maltophilia U3, pH 6-8 in Bacillus subtilis Q2. Best glutaminase production was obtained at 37°C by Bacillus subtilis U1and Bacillus subtilis Q2, 30°C for Achromobacter xylosoxidans G1, Stenotrophomonas maltophilia U3 and 25°C by Alcaligenes faecalis S3. The carbon sources put fluctuated effects on activity of enzyme in such a way that glucose was the best carbon source for Bacillus subtilis U1and Bacillus subtilis Q2, Sorbitol for Achromobacter xylosoxidans G1 and Alcaligenes faecalis S3 while xylose was the best for Stenotrophomonas maltophilia U3. Yeast extract and Trypton were among good nitrogen sources for Achromobacter xylosoxidans G1 and of Bacillus subtilis U1 respectively. Glutamine was the best inducer for Bacillus subtilis Q2, Alcaligenes faecalis S3 and Stenotrophomonas maltophilia U3, while lysine for Achromobacter xylosoxidans G1 and glycine act as good inducer in case of Bacillus subtilis U1. After implementation of optimal conditions microbial L-glutaminase production can be achieved and the bacterial isolates have a great potential for production of glutaminase enzyme and their applications.

inducers, carbon source, pH, and nitrogen source were also put under consideration. There was slight difference 33 among incubation of bacterial culture, overall, 36 hours of incubation time was the best for glutaminase production 34 by all the strains. Optimal pH was around 9 in Achromobacter xylosoxidans G1 and Alcaligenes faecalis S3, pH 6 in 35 Bacillus subtilis U1, pH 8 in Stenotrophomonas maltophilia U3, pH 6-8 in Bacillus subtilis Q2. Best glutaminase 36 production was obtained at 37°C by Bacillus subtilis U1and Bacillus subtilis Q2, 30°C for Achromobacter 37 xylosoxidans G1, Stenotrophomonas maltophilia U3 and 25°C by Alcaligenes faecalis S3. The carbon sources put 38 fluctuated effects on activity of enzyme in such a way that glucose was the best carbon source for Bacillus subtilis 39 U1and Bacillus subtilis Q2, Sorbitol for Achromobacter xylosoxidans G1 and Alcaligenes faecalis S3 while xylose 40 was the best for Stenotrophomonas maltophilia U3. Yeast extract and Trypton were among good nitrogen sources 41 for Achromobacter xylosoxidans G1 and of Bacillus subtilis U1 respectively. Glutamine was the best inducer for 3 Introduction 48 Being the most important entity in multiple biological and non-biological processes, enzymes 49 play crucial role in maintaining and sustaining industrial, commercial and economical products. 50 They include all biochemical response and accelerate the rate of response without being 51 expressing themselves in the last product [1]. 52 L-Glutaminase is amidohydrolase enzyme belong to hydrolytic class which catalyzes the 53 conversion of amino acid L-glutamine into L-glutamic acid, in the presence of water and releases 54 ammonia. This enzyme plays an important role in nitrogen metabolism at cellular level. This 55 enzyme is present in both microorganisms such as bacteria fungi and yeast as well as in macro-56 Pakistan, kept in aseptic conditions in the lab and stored at -4°C. One bacteria strain was also 78 isolated from old hydrolyzed glutamine sample.   161 Optimization of multiple conditions leading to production of glutaminase was done by estimating 162 optimal conditions such as effect of incubation time, pH, carbon source, nitrogen source and 163

Optimization of culture conditions for L-Glutaminase production
inducers. 164 165 Glutaminase production by bacterial isolates was done at 30°C in fermentation media and 166 activity was measured after specific intervals (0, 1, 2, 3, 4 and 5 days). 167 168 The condition of pH was optimized for the production of glutaminase by the selected bacterial 169 isolate by running fermentation at different pH (6-9). 170

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Selected bacterial strains were optimized for the effect of temperature at 25°C, 30°C and 37°C. 172 173 All the strains were checked for their enzyme producing ability at different carbon source 174 addition such as glucose, sucrose, lactose, maltose, xylose and sorbitol (1 %) in the production 175 media. 176

Effect of carbon source
Effect of nitrogen source 177 The effect of 0.1% nitrogen source was measured. The potent nitrogen sources tested were 178 Trypton, Yeast extract, Ammonium chloride and Sodium nitrate. 179 180 The glutaminase producing strains were checked for measurement of effects of different inducers 181 like Glycine, Glutamine and Lysine on glutaminase production and glutaminase activity was 182 measured after every 24 hrs of incubation. 183

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The present study showed the isolation and characterization of bacterial isolates with the 185 capability of producing L-glutaminase enzyme. 186

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The primary screening is basically the rapid plate assay technique which utilize phenol red as pH 188 indicator. Soil samples were inoculated in the media and colonies displaying pink color 189 formation around the colonies were picked and purified and further again tested for enzyme 190 production on individual strains. Twenty bacterial colonies gave positive result in purified form 191 changing the color of media from yellow to pink (Fig 1). Five isolated Q2, U3, U1, S3 and G1 192 were selected for further analysis. After the plate rapid assay, the strains were also tested in 193 liquid media exhibiting same results. 194  195 The secondary screening was done in media by point inoculation of bacterial strain in agar media 196 and glutaminase production was indicated as color change in agar plates. The zone of hydrolysis 197 was measured in millimeter, where glutaminase enzyme hydrolyze the substrate glutamine in a 198 medium. The results showed that all the bacterial strain produce glutaminase giving zone of 199 hydrolysis of 14-32 mm (Fig 2, Table 1). Further all the selected bacterial isolates were grown in 200 liquid media (Fig 3)

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and U1 was having large sized colonies. As the shape of colonies is concerned, colonies of G1 226 strains were rhizoid in shape while circular shape exhibited by S3 and Q2 strains and U1 and U3 227 showed filamentous shape. All the strains were having dome shaped colonies except U1 which 228 exhibited flat elevation. Colonies of all the strains were opaque and moist and showed no 229 pigmentation (Table 2). 230 231 The microscopic observations such as shape, size and arrangement of cells was of different 232 phases and features and the gram staining of strains revealed that all the strains were gram 233 negative except U1 strains. The shape of the strain G1 was cocci while all others were rods 234 (Table 3). 235 Biochemical testing 241 The biochemical tests were random except that of the strain U3 which was negative for all of the 242 test and oxidase was positive for all of the test except U3. Strains G1, S3 and G2 were catalase 243 positive and citrate utilization positive while Mannitol salt agar test was positive for G1, G2 and 244 U1. Indole production was positive for G1 and Q2 and glucose fermentation was positive for S3, 245 G2 and U1 while only strain G1 and S3 were urease positive (Table 4). 246

Microscopic characters of the isolates
Molecular characterization 249 The agarose gel electrophoresis showed amplified sequences of 16S rRNA gene in bacterial 250 isolates. The amplified product was of 1500 bp ( Figure 5). The sequences were blast in NCBI 251 database and on the basis of the homology to the sequences (> 90%) with other bacterial strains 252 in the database the bacterial isolates were identified as Bacillus subtilis U1, Achromobacter 253 xylosoxidans G1, Bacillus subtilis Q2, Stenotrophomonas maltophilia U3 (Table 5).    285 Results showed that Glutaminase production by Achromobacter xylosoxidans G1, Alcaligenes 286 faecalis S3 on production media at 37ºC showed that optimum pH is 9. The strains Bacillus 287 subtilis U1, showed maximum activity at pH 6, Stenotrophomonas maltophilia U3 shown best 288 activity at pH 8 while Bacillus subtilis Q2 also showed glutamine activity between the pH range 289 of 6-8 maximally (Fig 8). Achromobacter xylosoxidans G1 worked best giving activity of 57.369 290 U/ml/min at pH 9 at 3 rd day of incubation, the activity 29.126 U/ml/min of Bacillus subtilis Q2 291 was highest at pH 6 and 8 on 2 nd day, U3 showed maximum activity at pH 8 at third day that was 292 46.34 U/ml/min, Bacillus subtilis U1 showed highest activity of 37.069 U/ml/min at pH 6 while 293 S3 showed highest activity of 57.36 U/ml/min at pH 9. 294 Achromobacter xylosoxidans G1 and Stenotrophomonas maltophilia U3 showed maximum 299

Effect of pH
Glutaminase activity at 30°C at 3 rd day (44.1 IU/ml/min and 41.9 IU/ml/min respectively). While 300 Alcaligenes Faecalis S3 best activity (39.71 IU/ml/min) was observed at 25°C at 3 rd day of 301 incubation (Fig 9). 302 IU/ml/min respectively (Fig 10). 313 314 The production of L-glutaminase by strain Achromobacter xylosoxidans G1 was at its higher 315 peak when yeast extract was used as nitrogen source showing 22.947 IU/ml/min activity (Figure  316 11). The poorest nitrogen source was sodium nitrate and ammonium sulphate at day 1 and day 2 317 respectively. In the case of Bacillus subtilis U1 the Trypton was the best nitrogen donor showed 318 maximum activity up to 61.7828 U/ml/min (Fig 11). 319

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The effect of inducers on all the strains were observed for 4 days. The peak achieved at 3 rd day 321 with strain U1 (Fig 12). 322 The inducers such as Glycine, Glutamine and Lysine influenced the glutaminase activity in a 323 strain G1. The interesting feature was that all the inducer showed parabolic effect on enzyme's 324 activity such that increasing at starting point and showed highest activity at 3rd to 5th day and 325 ultimately decreased at 6th day. The glutamine tends to effect greatly on all of strain like its 326 activities in U3, S3 and Q2 were 41.92, 37.069 and 38.6 U/ml/min at day 2 nd , 4 th and 3 rd day 327 except G1 where lysine enhanced the activity by 46.33 IU/ml/min of enzyme and Bacillus 328 subtilis U1 showed maximum activity with glycine (41.48 IU/ml/min) (Fig 12). 329

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Since, the disclosure of L-glutaminase for its properties like anti cancerous, different microbial 331 sources is the focal point of enthusiasm for the isolation of the protein. L-glutaminase action is 332 studied in living organisms, plant tissues and microorganisms including microscopic organisms, 333 and actinomycetes. Microbial L-glutaminase (L-glutamine amido hydrolase EC 3.5.1.2) has 334 gotten more prominent consideration for its potential biotechnological applications and 335 effectiveness in wide range. 336 In the present study we have isolated 4 bacterial strains Bacillus subtilis U1, Stenotrophomonas 337 maltophilia U3, Bacillus subtilis Q2 from different soil samples and Achromobater xylosoxidane 338 G1 that was isolated from the old L-Glutamine sample. In general, glutaminases from E. coli, 339 The sequencing of L-Glutamisae producing strains of U1 showed 98.32% homology with 360 Bacillus subtilis strain B92, S3 has shown 97.87% homology with Alcaligenes faecalis strain 361