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Document heading

Statistical optimization of glutaminase production from Lactobacillus casei using response surface methodologySajitha Nagamony*, Lakshminarayanan Ramasamy1, Vasanthabharathi venkataraman2, S. Jayalakshmi Singaram3CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, Tamilnadu, India.

Asian Pacific Journal of Tropical Biomedicine (2012)1-6

Asian Pacific Journal of Tropical Biomedicine

journal homepage:www.elsevier.com/locate/apjtb

*Corresponding author: N.Sajitha *CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, Tamilnadu-, India.Email: [email protected]

1. Introduction Enzymes are essential to life because they speed up metabolic reactions to a very great extent, but do not undergo any change in themselves. Enzymes are chiral catalysts, producing mostly or only one of the possible stereo isomeric products. A chiral molecule is a type of molecule that lacks an internal plane of symmetry and has a non-super imposable mirror image. Examples can be found for many chiral compounds comprising alcohols and diols, amines, amides, 毩-amino acids, 毬-amino acids, amino alcohols, carboxylic acids and esters. The fermentation of wine, leavening of bread, curdling of milk into cheese, and brewing of beer are few examples of enzymatic reactions. The uses of enzymes in medical field include killing

disease-causing microorganisms, promoting wound healing and diagnosing certain diseases. In the industry they are used for degrading oil spills and wastes into harmless compounds, in cleaning stains, and in fermentation processes to make alcoholic beverages, acids, chemicals etc. L-Glutaminase (L-glutamine amidohydrolase EC 3.5.1.2) catalyzes the hydrolysis of L-glutamine to L-glutamic acid and ammonia . The use of enzyme as drug is a crucial facet of today’s pharmaceutical industry. L-glutaminase a potent anti-leukemic agent[1].The action of glutaminase plays a major role in the cellular nitrogen metabolism of both, prokaryotes and eukaryotes[2]. L-glutaminase can be derived from plant as well as animal sources but microbial enzymes are generally meeting the industrial demands. Bacterial glutaminases and glutaminase-asparaginases are structurally heterogeneous since the existence of tetramers, dimers or monomers with markedly different subunit masses have been described. Even though many bacteria has the ability to produce L-glutaminase enzyme, only few

ARTICLE INFO ABSTRACT

Article history:Received 15 April 2011Received in revised form 27 April 2011Accepted 28 June 2011Available online 28 June 2011

Keywords:IsolationScreeningIdentificationOptimization using RSMPartial purificationCharacterizationAntibacterial and Antioxidant.

Objective: To isolate and optimize the glutaminase producing bacteria using Response Surface Methodology. Methods: In the present study, the water, sediment samples and mangrove detritus were collected from the vellar estuary (Tamil Nadu, India) and the potential strains of glutamines production were screened in minimal medium plates. The optimization of glutaminase enzyme was done by response surface methodology. Result: Totally 25 strains were collected. Among them Lactobacillus casei in was found to be the potential strain. Hence this potential strain was used for further optimization using Response Surface Methodology, partial purification using ammonium sulphate precipitation, characterization, stability, antimicrobial and antioxidant application. The stability of glutaminase from Lactobacillus casei was studied and better stability was observed at pH 7 to 8. The thermal stability was observed at 30°C to 40°C. The SDS PAGE of glutaminase enzyme shows two different molecular weights. The molecular weight of partially purified glutaminase enzyme was 23kDa and 34kDa. The glutaminase enzyme shows moderate antibacterial activity. The glutaminase enzyme showed antioxidant activity. Conclusion: Thus the present study revealed that this Lactobacillus casei strain is highly suitable for industrial applications especially in medicinal and food industries.

Contents lists available at ScienceDirect

et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-62

bacteria produce more L-glutaminase in their minimum growth, which is selected for large-scale industries as high potential strains. The marine environment has vast bacterial diversity and high fluctuations in their salinities and temperatures become the source for the salt-tolerant; thermo-stable L-glutaminase[3].All living cells produce L-glutaminase but only certain microbial strains have the potential for industrial production of this enzyme[4].

2. MATERIALS AND METHODS

2. 1. Collection of samples

The surface water samples were collected using pre sterilized McCartney bottles allowing enough air space in the bottles to facilitate thorough mixing. Precautionary measures were taken to minimize the contamination. Sediment samples and mangrove detritus were collected using a sterile spatula. The central portion of the collected samples was aseptically transferred in to sterile polythene bags. Both the water and sediment samples were transferred to the laboratory in an ice box maintained at 4o C for further study. Most of the samples were processed with in 30min. of collection.

2. 2. Isolation of Glutaminase producing bacteria

For isolation of glutaminase producing organisms, water and sediments collected from the vellar estuary were used. 1g/ml of sample was suspended in 99ml sterile 50% aged sea water and was serially diluted in 9ml blank upto 106 and 0.1ml from each tube was spreaded on MRS agar plates and incubated at 37曟 for 48hrs. MRS agar contain 20 g of glucose, 10 g of peptone , 10 g of beef extract, 5 g of yeast extract, 5 g of sodium acetate, 2 g of potassium phosphate dibasic (K2HPO4), 1 g of Tween 80, and 2 ml of trace solution (5 g of CaCl2, 145 mg of boric acid, 125 mg of iron sulfate (FeSo4), 75 mg of calcium sulfate (CaSo4), 4.3 mg of manganese sulfate (MnSo4), 108 mg of zinc sulfate (ZnSo4), 125 mg of Na molybdate (Na2MoO4 � 2H2O), 7 g of nitrilacetate, 15gm Agar) per liter.

2. 3. Screening of glutaminase producing bacteria

The isolated strains were streaked in minimal media (KCl 0.5g, MgSo4.7H2O 0.5g, FeSo4.7H2O 0.1g, ZnSo4.7H2O 1.0g, KH2PO4 1.0g, L-Glutamine 0.5%, Phenol red 0.012g , Aged Sea Water 1000 ml, pH 7.2 + 7.4) plates to screen the potential glutaminase producing bacteria. The colour change of the medium from yellow to pink was considered as an indication for the L-glutaminase production. This colour change is due to change in the pH of the medium, as L-glutaminase causes the breakdown of amide bond in L-glutamine and librates ammonia. Hence extend of colour change was used to select the most potential strain.

2. 4. Identification of potential strain

The positive strains were isolated and were identified up to species level following Bergey’s manual of Determinative bacteriology[5]and the identified strain was stored in minimal media slant for further study.

2. 5. Estimation of crude extracellular glutaminase enzyme activity

The organism was cultured in minimal broth and was centrifuged; the cell free culture broth was used as crude enzyme. The activity of glutaminase was determined by estimating the amount of NH3 liberated from glutamine. The method of[6]was followed which is given below. Enzyme preparation of about 0.5ml was added to 0.5ml of 0.04M L-glutamine dissolved in 0.5ml of distilled water. To this 0.5ml of 0.1M phosphate buffer (pH-8) was added and incubated at 37°c for 30min. After incubation 0.5ml of 1.5M trichloroacetic acid was added to stop the enzyme reaction. Then 0.1ml of above mixture was taken and 3.7ml of distilled water was added. Then 0.2ml Nessler’s reagent was added to it. Absorbance was measured at 450nm using a UV spectrophotometer. One international unit of glutaminase is defined as, the amount of enzyme that liberates one micromole of ammonia under optimum conditions. The enzyme yield was expressed in units/ml/min.

2. 6. Optimization of process parameters

2. 6. 1. Identifying the significant variables using Plackett- Burman design The Plackett-Burman experimental design is a two factorial design, which identifies the critical physico-chemical parameters required for elevated glutaminase production by screening n variables in n + 1 experiments [7]. The variables chosen for the present study were Wheat bran (A), Coconut cake (B), Groundnut cake (C), Glucose (D), Urea (E), Yeast extract (F), Malt extract (G), Soyabean meal (H), Ammonium chloride (J), Ammonium nitrate (K), K2HPO4 (L), CaCl2 (M), MgCl2 (N) and Glutamine (O). The experimental design for the screening of the variables is denoted in Table 3. All the variables were denoted as numerical factors and investigated at two widely spaced intervals designated as -1 (low level) and +1 (high level). The effect of individual parameter on glutaminase production was calculated by the following equation:E = ( ∑ M+ - ∑ M- ) / N ( 1 ) Where E is the effect of parameter under study and M+ and M- are responses (glutaminase activities) of trials at which the parameter was at its higher and lower levels respectively and N is the total number of trials.

2. 6. 2. Response Surface Methodology Response surface methodology (RSM) was used to estimate

et al./Asian Pacific Journal of Tropical Biomedicine (2012)1-6 3

main effects on response, i.e. glutaminase yield (Table 4). Central composite design (Two level factorial: quarter fraction) consisting of seven main critical independent variables, (i) Wheat bran (0 - 4 %) (ii) Yeast extract (0 - 2 %) (iii) MgCl2 (0 to 4%) (iv) Glutamine (0 - 0.04 %) (v) pH (5 - 9) (vi) Temperature (25-450C) (vii) Incubation period (0 - 72 hours) and salinity (0 - 30 ppt) were chosen based on the initial screening. For each factor, a conventional level was

set to zero as a coded level. These eight factors, each with five coded levels consisting of 90 experimental runs and 2.82843 alpha values were used to analyze the experimental data to allow better estimate of the experimental error and to provide extra information about yields in the interior of the experimental region[8]. The experimental data were fitted according to Eq. (2) as a second-order polynomial regression equation including individual and cross effect of each

Table 3Observed response and predicted values of glutaminase enzyme

Wheat bran (%)Yeast

Extract (%)MgCl2 (%) Glutamine (%) pH

Temperature(�C)

Incubation Period (Hours)

Salinity (ppt) Glutaminase Production U/ml/min

Observed Predicted-1 -1 -1 1 1 -1 -1 -1 0 2.0441 -1 -1 -1 -1 1 -1 1 5 16.821

-1 -1 -1 -1 1 1 1 1 3 11.131-1 1 -1 1 -1 1 1 1 2 7.8461 -1 -1 -1 1 -1 -1 1 2 10.6731 -1 -1 1 1 1 1 -1 7 34.552

-1 1 1 1 1 1 -1 -1 3 11.8010 0 0 0 0 0 0 0 414 386.0860 0 0 0 0 0 0 0 414 386.0860 0 0 0 0 0 0 0 414 386.086

-1 1 1 -1 1 1 1 -1 0 0.103-1 1 1 -1 -1 1 1 1 0 11.3101 1 -1 1 -1 -1 -1 1 3 12.3270 0 0 0 0 0 0 0 414 386.0861 -1 1 -1 -1 1 1 1 46 55.732

-1 1 -1 -1 1 -1 -1 1 0 5.1191 1 1 1 1 1 1 1 170 187.0601 1 -1 -1 1 -1 1 -1 76 65.918

-1 -1 1 1 1 1 1 1 5 6.614-1 1 1 1 -1 -1 -1 -1 0 3.146-1 -1 -1 -1 -1 1 1 -1 3 0.1191 -1 1 -1 1 -1 1 1 80 81.3351 1 1 -1 1 1 -1 1 4 0.0081 1 1 -1 -1 -1 -1 1 0 3.420

-1 1 1 1 1 -1 -1 1 0 1.7600 0 0 0 0 0 0 0 414 386.0861 -1 1 1 1 1 -1 -1 8 5.5931 -1 1 -1 -1 -1 1 -1 70 62.699

-1 1 -1 -1 1. 1 -1 -1 2 20.1921 1 1 -1 -1 1 -1 -1 0 7.769

-1 -1 1 1 -1 -1 1 1 1 8.5431 -1 -1 1 1 -1 1 1 65 67.9911 1 -1 -1 1 1 1 1 30 39.3261 1 -1 1 -1 1 -1 -1 5 1.3871 1 1 1 -1 -1 1 1 230 211.738

-1 1 -1 1 1 1 1 -1 8 3.010-1 -1 -1 -1 -1 -1 1 1 0 2.8091 1 1 1 1 -1 1 -1 221 215.1521 -1 1 -1 1 1 1 -1 42 47.646

-1 1 1 -1 1 -1 1 1 5 10.293-1 -1 1 -1 1 1 -1 1 2 7.296-1 1 1 1 -1 1 -1 1 0 5.3871 -1 -1 1 -1 1 1 1 78 58.8891 1 -1 -1 -1 1 1 -1 11 19.065

-1 1 -1 -1 -1 1 -1 1 1 5.028


variable. Y = 毬0 + ∑ 毬i Xi + ∑ 毬ii Xi

2 + ∑ 毬ij Xi Xj (2) Where Y, 毬0, 毬i, 毬ii and 毬ij are the predicted response, a constant, a linear coefficient, a squared coefficient and an interaction coefficient representing Eq. (2) was used to build surfaces for variables. This model was likely to be useful as an approximation of the true response surface in a relatively

small region, and it is widely used in RSM for the following reasons: 1. the second-order model is very flexible. It can take on a wide variety of functional forms, so it will often work well as an approximation of the true response surface. 2. It is easy to estimate the parameters (the 毬’s) in the second-order model. The method of least squares can be used for this purpose. 3. There is considerable practical

Table 3Observed response and predicted values of glutaminase enzyme

Wheat bran (%) Yeast Extract (%) MgCl2 (%) Glutamine (%) pHTemperature

(˚C)Incubation

Period (Hours)Salinity (ppt)

Glutaminase Production U/ml/min

Observed Predicted0 0 0 0 0 0 0 0 414 386.086

-1 -1 1 -1 -1 1 -1 -1 0 8.785-1 -1 1 -1 1 -1 -1 -1 0 7.388-1 -1 1 1 -1 1 1 -1 0 5.8971 -1 -1 1 -1 -1 1 -1 32 45.6051 1 -1 -1 -1 -1 1 1 49 53.7550 0 0 0 0 0 0 0 414 386.0861 -1 1 1 -1 1 -1 1 2 18.680

-1 -1 -1 1 -1 -1 -1 1 0 1.1191 -1 1 1 -1 -1 -1 -1 6 5.6461 -1 -1 -1 1 1 -1 -1 6 12.484

-1 1 1 -1 -1 -1 1 -1 0 0.1571 1 -1 1 1 -1 -1 -1 6 19.4900 0 0 0 0 0 0 0 414 386.086

-1 -1 1 -1 -1 -1 -1 1 0 12.0251 -1 1 1 1 -1 -1 1 5 15.5321 1 -1 1 1 1 -1 1 5 12.8991 -1 -1 -1 -1 -1 -1 -1 2 2.287

-1 -1 -1 -1 1 -1 1 -1 9 10.972-1 1 -1 1 -1 -1 1 -1 0 0.937-1 -1 -1 1 -1 1 -1 -1 9 19.942-1 -1 -1 1 1 1 -1 1 9 22.2031 1 1 1 -1 1 1 -1 149 165.299

-1 1 -1 1 1 -1 1 1 3 12.9491 1 1 -1 1 -1 -1 -1 7 26.834

-1 1 -1 -1 -1 -1 -1 -1 0 5.005-1 -1 1 1 1 -1 1 -1 4 7.9560 0 0 0 -2.82843 0 0 0 90 101.1090 0 0 0 0 0 0 2.82843 285 271.6500 0 0 -2.82843 0 0 0 0 166 161.1020 0 0 0 0 0 0 0 414 525.6560 0 2.82843 0 0 0 0 0 288 287.082

0 0 0 0 0 0 0 -2.82843 276 261.4360 0 0 0 0 -2.82843 0 0 178 182.8710 0 -2.82843 0 0 0 0 0 265 238.0050 0 0 0 0 0 -2.82843 0 0 26.1662.82843 0 0 0 0 0 0 0 230 203.8790 0 0 0 0 2.82843 0 0 202 169.2150 0 0 2.82843 0 0 0 0 240 216.9850 0 0 0 0 0 0 0 414 525.656-2.82843 0 0 0 0 0 0 0 80 78.2070 0 0 0 0 0 2.82843 0 85 83.2520 2.82843 0 0 0 0 0 0 180 175.3320 -2.82843 0 0 0 0 0 0 160 136.7540 0 0 0 2.82843 0 0 0 160 120.978


experience indicating that second-order models work well for solving real response surface problems. Multiple regression analysis, response surface plots and statistical analyses were performed using Minitab 15 Statistical Software� (Minitab Inc., PA, and USA).

2. 6. 3. Statistical analysis Statistical analysis was done by ANOVA for analysis of the results. A probability level of p < 0.01 was considered as statistically significant.

2. 7. Production and Partial purification

The purification steps were performed at 4曟. The buffer used was 10 mM Tris-HCl (pH 8.0, buffer I). The culture was centrifuged at 3,000rpm for 1hr and supernatant was collected. The supernatant fluid containing the extra cellular enzyme was treated with solid ammonium sulphate saturation ranging from 60%, 70% and 80% with continuous overnight stirring and precipitated into the proteins. The precipitates collected by centrifugation at 3,000rpm for1hr were dissolved in 10mM Tris HCl buffer (pH 8.0). The enzyme solution was dialyzed against the same buffer for 24hrs with several changes to remove the salt and assay was done as per the[6]method. The dialyzed sample was further used for molecular weight determination using SDS-PAGE.

2. 8. Determination of molecular weight of glutaminase enzyme (SDS-PAGE) SDS-PAGE (Sodium-dodecylsulphate-polyacrylamide gel electrophoresis) The samples were solubilized in reducing sample buffer and equal amount of lyophilized Enzyme and was loaded into 12% SDS-Polyacrylamide gel and electrophoresis was carried out at constant current (30mA)[9].

2. 9. Antibacterial activity

2.9.1. Well diffusion method [10]

To test the antibacterial activity, the bacterial pathogens were swabbed on Muller Hinton agar plates. 0.1ml of the glutaminase enzyme was poured into the wells and the plates were incubated at 37曟 for 24hrs. The glutaminase enzyme inhibiting the growth of pathogens was assessed based on the zone of inhibition around the wells.

2. 10. Antioxidant activity

Total antioxidant activity of the enzyme was measured by the following methd[11]. 7.45ml sulphuric acid (0.6M solution),0.9942g sodium sulphate (28mM solution) and 1.2359g Ammonium molybdate (4mM) were mixed together in 250ml distilled water and labeled as Total Antioxidant Capacity (TAC) reagent. 100毺l of enzyme was dissolved in 1ml of TAC reagent. Blank was maintained with distilled water replacing the TAC reagent. Absorbance was measured at 695nm in a

spectrophotometer.

3. RESULT

3. 1. Isolation of glutaminase producing bacteria

The water, sediment with mangrove detritus was collected from vellar estuary. Here total heterotrophic bacteria were isolated from Zobell marine agar in water, sediment and mangrove detritus are 5.6伊103CFU/ml, 3.2伊104CFU/g and 2.5伊105 CFU/g repectively. The total number of Lactobacillus species were isolated from MRS media plates in water sample was 1.6伊102 CFU /ml, whereas that of sediment with detritus sample is 1.5伊105 CFU/g .Table 1Plackett–Burman experiment for screening of significant process variables affecting glutaminase productionA B C D E F G H J K L M N O Enzyme U/ml/min

Observed Predicted - - - - - - - - - - - - - - 111 111.3+ + + - - + + - + + - - - - 286 286.7+ + - - - - + - + - + + + + 284 284.3- + - + - + + + + - - + + - 273 273.1- + + + + - - + + - + + - - 294 294.1- - - + - + - + + + + - - + 294 294.9- - - - + - + - + + + + - - 114 113.5- - + - + - + + + + - - + + 116 115.7+ - + + - - - - + - + - + + 326 325.7- - + + - + + - - - - + - + 325 324.1- + - + + + + - - + + - + + 334 333.9+ + - - + + - + + - - - - + 350 348.9- + + - + + - - - - + - + - 168 168.1- + + - - - - + - + - + + + 136 136.3+ - + + + + - - + + - + + - 252 252.1+ - + - + + + + - - + + - + 330 330.9+ + + + - - + + - + + - - - 403 402.3+ + - + + - - - - + - + - + 395 395.3+ - - - - + - + - + + + + - 162 161.3+ - - + + - + + - - - - + - 303 303.5

3. 2. Identification and Screening of glutaminase producing bacteria

Figure 1. (a) - Plate showing the glutaminase activity on minimal agar medium. (b)- Control plate showing no glutaminase activity on minimal agar medium.

(a) (b)


Totally 25 Lactobacillus strains were isolated. The isolated Lactobacillus strains were screened using minimal agar medium for the production of glutaminase. The active glutaminase producing organism changed the colour of the medium from yellow to pink (fig 1). This colour change was due to change in the pH of the medium, as L-glutaminase caused the breakdown of amide bond in L-glutamine and librated ammonia. Totally 9 glutaminase producing Lactobacillus strains were selected and used for identification using Bergey’s manual. Among the 9 strains, Lactobacillus casei was found to be the potential strain which was used for further studies.

3. 3. Optimization

3. 3. 1. Screening of parameters using Plackett-Burman designTable 2Analysis of Variance for glutaminase Enzyme U/ml/min (Plackett-Burman design)

Source DF Seq SS Adj SS Adj MS F PMain Effects 14 168911 168911 12065.1 9729.90 0.000Residual Error 5 6 6 1.2Total 19 168917

Pareto Chart of the Standardized Effects(reponse is Enzyme U/ml,Alpha=0.05)

2.6

0 50 100 150 200 250Standardized Effect

DABNOFGKLMJHEC

Term

Fig. 2: Effects Pareto for glutaminase enzyme U/ml/min

The experiment was conducted in 20 runs to study the effect of the selected variables fig 2 represents the results of the screening experiments using Plackett-Burman design. Statistical analysis of the variance was performed and represented in Table 1, 2 .The model F value of 9729.9 implied that the model was significant. The values of Prob < 0.05 indicated model terms were significant. The magnitude of the effects indicated the level of the significance of the variable on glutaminase production. Among the variables screened Glucose, Wheat bran, yeast extract, Coconut cake, MgCl2, Glutamine and Yeast extract, were identified as most significant variables influencing glutaminase production. Every microorganism evidences its own idiosyncratic physicochemical and nutritional requirements for growth and enzyme secretion.

3. 3. 2. Response surface methodology

The use of statistical models to optimize culture medium components and conditions has increased in present-day biotechnology, due to its ready applicability and aptness. In the present study, the significant variables necessary for enhanced glutaminase production were selected using the Plackett-Burman design. A large variation in glutaminase production (403 to 465.43 U/ml/min) from that mandated by the Plackett-Burman design experiments suggested a need for further optimization. The Central composite design (Two level factorial: quarter fraction) was employed to study the interactions among the significant factors and also to determine their optimal levels. The central composite design plan exploited in the present study enabled us to study and explore the culture conditions that would support a ~14 % increase in glutaminase production. A high degree of similarity was observed between the predicted and experimental values that reflected the accuracy and applicability of RSM to optimize the process for glutaminase enzyme production. Totally eight variables (i.e) Wheat bran, yeast extract, MgCl2, Glutamine, pH, temperature, incubation period and salinity were taken for RSM which gave maximal yield in the Plackett-Burman experiments. The parameters of Eq. (2) were determined by multiple regression analysis by the application of RSM. The overall second-order polynomial regression equation showing the realistic relationship between glutaminase activity (Y) and eight test variables in coded units was represented by Eq. (3). Y = 455.871 - 69.785 + 22.216 + 6.820 + 8.676 + 9.879 + 3.512 - 2.414 + 19.343 + 1.806 - 48.077 - 46.202 - 32.889 - 42.077 - 51.827 - 43.702 - 62.139 - 32.389 + 8.297 + 10.734 + 8.484 + 0.141 - 4.859 + 19.891 + 2.078 + 8.547 + 10.297 + 1.516 - 2.484 + 8.266 - 1.422 + 8.047 + 0.328 - 2.109 + 10.391 - 0.609 - 1.234 + 0.453 + 8.141 + 1. 891 - 1.516 + 0.484 - 2.203 - 5.016 + 1.547 + 2.359. (3) Multiple regression model assumes a linear relationship between some variable Y (dependent variable) and n independent variables C1, C2, C3, . . . Cn. Based on the result obtained with the multiple regression analysis, it was observed that temperature, some interaction of squared and some of interaction coefficient had a negative impact on glutaminase production. The analysis of variance (ANOVA) by Fisher’s statistical test was conducted for the second-order response surface model and the result showed that the computed F value for linear regression was much greater than the tabulated (F) > P value. Therefore, the model terms Wheat bran, yeast extract, MgCl2, Glutamine, pH, temperature, incubation period and salinity were significant (Table. 2). The goodness-of-fit of the model was checked by decisive the coefficient of determination (R2) and adjusted R2. When R2 is large, then, the regression has accounted for a large proportion of the total variability in the observed value of Y which favors the regression equation model[8, 12]. The observed values of R2 explained that the fitted model could explain 97.49% of the total variations and hence vouched for


adequacy of the model (Table 3). The adjusted R2 correctd the R2 value for the sample size and for the number of terms in the model. The adjusted R2 value (94.92%) and Predication of R2 (93.65%) in the present study advocated for a high significance of the model.These results reinforced that the response equation provided a suitable model for the CCD experiment(Table 4).Table 4Analysis of variance for glutaminase enzyme U/ml/min (Central Composite Design)

Source DF Seq SS Adj SS Adj MS F PBlocks 1 280507 280507 280507 284.83 0.000Regression 44 1400140 1400140 31821 32.31 0.000Linear 8 88678 88678 11085 11.26 0.000Square 8 1232918 1232918 154115 156.49 0.000Interaction 28 78544 78544 2805 2.85 0.001Residual Error 44 43333 43333 985Lack-of-Fit 36 43333 43333 1204Pure Error 8 0 0 0Total 89 1723979

The interaction effects and optimal levels of the variables were determined by plotting the response counter plots. The response counter plots are represented in Fig. 3. The optimum conditions for glutaminase production were proposed to be Wheat bran (2.6285%), yeast extract (1.1428%), MgCl2 (0.6286%), Glutamine (0.24%), pH (7.06), temperature (34.140C), incubation period (45.26 hrs) and salinity (15 ppt). The maximum glutaminase activity of 465.4261 U/ml/min was predicted by the model. The suggested medium composition was repeated. The validation experiment showed that the experimentally determined production values were in close agreement with the statistically predicted ones, confirming the model’s authenticity. The Lactobacillus casei strain produced 465.426 U/ml/min glutaminase under optimized culture conditions.

3. 4. SDS-PAGE

The SDS PAGE of glutaminase enzyme shows two different molecular weights. The molecular weight of glutaminase enzyme was 23KDa and 34KDa (Fig. 4).

3. 5. Antibacterial activity

The glutaminase enzyme obtained from Lactobacillus casei was used for testing antibacterial activity. Five bacterial food pathogens were used, among this E. coli and Listeria monocytogenes showed the maximum zone of inhibition when compared to all other pathogens

3. 6. Antioxidant activity

Free radical scavenging properties of glutaminase enzyme was assessed using total antioxidant method. The total antioxidant capacity of glutaminase enzyme along with the standard ascorbic acid. The activity of enzyme increased

with increase in concentration of the sample.

97.4

66.2

31

20.1

14.4

KDa

34KDa

23KDa

1 2

Fig. 4: SDS-PAGE of partially Purified Glutaminase1 – Protein marker2 – Sample (Glutaminase enzyme)

4. DISCUSSION

In the present study, totally 25 lactobacillus strains were isolated from Vellar estuary. Among this 9 potential glutaminase producing strains were screened. Among the 9 strains, Lactobacillus casei was found to be the potential strain for maximum glutaminase enzyme production. In the present study, wheat bran served as the best cheaper source, yeast extract served as the best nitrogen source and MgCl2 served as a mineral source and glutamine served as a inducer for glutaminase production. But in controversy,[2]

did the screening of the substrates and moistening media and best results were obtained with wheat bran moistened with 7 ml of 10% NaCl solution and sesamum oil cake with 7 ml of sea water. The optimum conditions for glutaminase production were anticipated to be Wheat bran (2.6285%), yeast extract (1.1428%), MgCl2 (0.6286%), Glutamine (0.24%), pH (7.06), temperature (34.140C), incubation period (45.26 hrs) and salinity (15 ppt). But the semi quantitative assay of the crude L-glutaminase produced at pH 7, temperature 30曟 and salinity of 3.5 % showed optimal enzymatic activity were described by[13]. The effect of pH, temperature and salt on the glutaminase activity of L. reuteri KCTC3594 was studied by[14]. The glutaminase worked optimally at pH 7.5. The optimum temperature was 40°C. To determine the salt dependence of glutaminase activity, the activity was assayed in a reaction mixture of 0 - 20% (w/v) NaCl. It was shown that L. reuteri KCTC3594 was salt-tolerant as the relative activity displayed 110 % in the condition of 5% NaCl and remained over 50% in


the presence of 20% NaCl. The maximum glutaminase production in the present study 465.4261 U/ml/min was predicted by the Central Composite Design. A large variation in glutaminase production (263 to 465.43 U/ml/min) from that mandated experiments and the statistical optimization. Maximum glutaminase production up to 146.91 U/l and specific activity was 0.45 U/mg proteins when all the variables were kept at their central values [1]. The model was used for optimization by numerical optimization. The model predicted maximum glutaminase production up to 149.98 U/l and specific activity of 0.488 U/mg protein could be achieved using the medium (g/l) sucrose 17.8, yeast extract 4.8, glutamine 5.0 and sodium chloride 55.6. Thus, overall 1.03-fold increase in glutaminase production with a 1.1-fold increase in specific activity was being predicted after validation of RSM. The optimized enzyme production (320U/ml) was obtained at different glutamine concentrations and different media volumes, while the interaction of other selected parameters at various glutamine concentrations revealed variation of enzyme production ranging from 240 to 280 U/ml[15]. The molecular weight of the glutaminase enzyme obtained in this study is 23 and 34 KDa (fig 4). But[14]obtained a molecular weight 70 and 50 kDa glutaminase enzyme produced Lactobacillus reuteri. The molecular mass of the native glutaminase enzyme and found it to be 86 and 43 kDa respectively, using gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Intact glutaminase(48.3 kDa, 170毺g/ml),was digested by incubation with 300 毺g protein/ml of the cell free extract from Micrococcus luteus K-3 at 30曟 for 24hrs, the glutaminase fragment(38.5 kDa) , and the small fragment(8kDa) were produced[16]. In the present study the pathogens used for antibacterial activity of glutaminase enzyme were Salmonella typhi, E.coli, Vibrio parahemolyticus, Listeria monocytogenes and Yersinia. Moderate antibacterial activity was obtained in the range of 6 to 10 mm in diameter, the highest being E.coli and Listeria monocytogenes (10mm). However, other therapeutic enzymes such as Lysozyme shows a broad antimicrobial spectrum. According to[17], 3 bacterial species, Bacillus stearothermophilus, C. thermosaccharolyticum, and C. tyrobutyricum, were found to be completely inhibited by the Lysozyme enzyme. Two species, Campylobacter jejuni and proteolytic C. botulinum type B, were weakly inhibited. Antioxidants are absolutely vital for maintaining optimal cellular and systemic health and well being, natural product as antioxidant agents have received much attention. In the present study the glutaminase enzyme show high antioxidant activity when compared to the standard ascorbic acid. The total antioxidant capacity of glutaminase enzyme increases with increase in concentration of the sample, while other enzymes like L-Asparaginase was investigated for antioxidant activity using DPPH assay and it showed that Bacillus sp. R36 possessed low scavenging activity with high SC50 values of 325.4 毺g/ml compared to the scavenging

activity of the well-known antioxidant (ascorbic acid, a. a., SC50 8.7 毺g/ml)[18].

5. CONCLUSION

As there is a great industrial insist for the glutaminase existance, it provoked the present cram to look for a promising strain with the desired nature from the Vellar estuary. Totally 25 Lactobacillus strains were isolated from sediment, water and mangrove detritus. Among this Lactobacillus casei was found as potential strain. For the high production of glutaminase, the media was screened (Plackett Burman design) and optimized statistically (Central composite design - Two level factorial: quarter fraction) using Minitab software. Thus the present study revealed that Lactobacillus casei strain is highly suitable for industrial applications especially in medicinal and food industries.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

The authors thank for the guidance of Dr S.Jayalakshmi, also for providing the facilities to carryout my whole research work. I would like to thankful to my seniors and friends who are all support me for my work.

References

[1] Iyer P, Singhal RS. Production of glutaminase (E.C.3.2.1.5) from Zygosaccharomyces rouxii: Statistical optimization using response surface methodology. Bioresour. Technol 2008; 99 : 4300-4307.

[2] Kashyap P, Sabu A, Pandey A, Szakacs G, Soccol CR. Extracellular L-glutaminase production by Zygosaccharomyces rouxii under solid-state fermentation. Proc. Biochem 2002; 38: 307-312.

[3] Renu S, Chandrasekaran M. Extracellular L-glutaminase production by marine bacteria. Biotechnol. Lett 1992; 14: 471-474.

[4] Nathiya K, Sooraj SN, Angayarkanni J. Optimised production of L-glutaminase: A tumour inhibitor from Aspergillus flavus cultured on agroindustrial residues. Afr. J. Biotechnol. 2011; 10:13887-13894.

[5] Buchanan RE, Gibbons NE, Cowan ST, Holt TG, Liston J, Murry RGE, Niven CF, Ravin AW, Stainer RY. Bergey’s Manual of Determinative Bacteriology (Eds) Williams and Wilkins Co: Baltimore 1974.

[6] Imada A, Igarasi S, Nakahama K, Isono M. Asparaginase and Glutaminase Activities of Micro-organisms. J. Gen. Microbiol 1973; 76:85-99.

[7] Plackett RL, Burman JP. The design of optimum multifactorial


experiments.[8] Haaland PO . Experimental design in biotechnology. Marcel

Dekker, New York 1990.[9] Laemmli UK. Cleavage of structural proteins during the assembly

of the head of bacteriophage T4. Nature1970; 227:680-685. [10] Reinheimer JA, Demkow MR, Candioti MC. Inhibition of coliform

bacteria by lactic cultures. Aust. J. Dairy Technol 1990; 45: 5-9.[11] Mitsuda H, Yasumodo K, Iwami F. Antioxidant action of indole

compounds during the autoxidation of linoleic acid. Eiyo to Shokuryo1996; 19: 210-214.

[12] Mukherjee B, Khuri AI, Sinha BK, Ghosh M. Design issues for generalized linear models: a review. Stat Sci 2009; 21:376-399.

[13] Balagurunathan R, Radhakrishnan M, Somasundaram ST. Lglutaminase Producing Actinomycetes from Marine Sediments -Selective Isolation, Semi Quantitative Assay and Characterization of Potential Strain. Aust J Basic & Appl Sci 2010; 4(5): 698-705.

[14] Jeon JM, Lee HI, Han SH, Chang CS, Seong So J. Partial Purification and Characterization of Glutaminase from Lactobacillus reuteri KCTC3594. Appl. Biochem. Biotechnol 2009; 402-751.

[15] Sathish T, Prakasham RS. Enrichment of glutaminase production by Bacillus subtilis RSP-GLU in submerged cultivation based on neural network - genetic algorithm approach. J. Chem. Technol. Biotechnol 2010; 85: 50-58.

[16] Yoshimune K, Shirakihara Y, Shiratori A, Wakayama M, Chantawannakul P, Moriguchi M. Crystal structure of a major fragment of the salt-tolerant glutaminase from Micrococcus luteus. K-3. Biochem.Biophys.Res.Commun 2006; 346: 1118-1124.

[17] Hughey VL, Johnson EA .Antimicrobial Activity of Lysozyme against Bacteria Involved in Food Spoilage and Food-Borne Disease. Appl. Environ.Microbiol 1987; 2165-2170.

[18] Moharam ME, Amira M, Eldeen G, El-sayed ST. Production, immobilization and anti-tumor activity of L- asparaginase of Bacillus sp R36. J. Amer. Sci 2010; 6(8): 157-165.

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