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

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Asian Pacific Journal of Tropical Biomedicine

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Document heading

Statistical optimization of glutaminase production from Lactobacillus casei using response surface methodology
Sajitha Nagamony*, Lakshminarayanan Ramasamy1, Vasanthabharathi venkataraman2, S. Jayalakshmi Singaram3
CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, Tamilnadu, India.

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

ABSTRACT 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 30C to 40C. 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.

2011

Keywords: Isolation Screening Identification Optimization using RSM Partial purification Characterization Antibacterial and Antioxidant.

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, -amino acids, -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. T he uses of enzymes in medical field include killing
*Corresponding author: N.Sajitha * CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, Tamilnadu-, India. Email: sajimicro@gmail.com

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 todays 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

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
T he 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.

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

2. 4. Identification of potential strain

The positive strains were isolated and were identified up to species level following Bergeys 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 37c 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 Nesslers 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. 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 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. 6. Optimization of process parameters 2. 6. 1. Identifying the significant variables using PlackettBurman design T he P lackett- B urman experimental design is a two factorial design, which identifies the critical physicochemical 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

2. 3. Screening of glutaminase producing bacteria

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

The isolated strains were streaked in minimal media (KCl

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.

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

Table 3 Observed response and predicted values of glutaminase enzyme Wheat bran (%) Yeast MgCl2 (%) Extract (%) Glutamine (%)

3
Glutaminase Production U/ml/min
5 0

pH -1 -1
1 1 1 1 1

Temperature Incubation Period Salinity (ppt) (C) (Hours)

-1 -1 -1
1 1

-1 -1 -1 -1 -1
0 0 0 1 1

-1
0 0 0

-1 -1 -1 -1 -1 -1
0 0 0 1

-1 -1 -1
0 1 1 1

-1
1 1 1

-1 -1 -1 -1
0 0 0 1 1 1

-1
1 1 1

Observed
3

Predicted
16.821 11.131 7.846 10.673 34.552 11.801 386.086 0.103 2.044

-1
0 1 1

0 0

0 0

-1 -1
0 0 0

7 3

0 0

414 414 0 0

414

386.086 11.310 55.732 5.119

-1 -1
1 0 1

1 0

-1
0

-1 -1 -1 -1 -1 -1 -1 -1 -1
0 1 1 1 1 1 0 1

-1 -1 -1
1 1 1 0

-1
0

-1
0

-1
1 1 0

386.086 12.327

-1
1

-1
1 1 1

-1 -1 -1
1 1 1 1 1 1

-1 -1 -1 -1 -1 -1
0 1 1 1 1

-1
1 1 1

-1 -1 -1
1 1 1

-1 -1 -1
1 1 1 1

-1 -1
1 1

-1 -1 -1
1 1 1 1 1

414 46 0

386.086 187.060 65.918 3.146 6.614 0.119

170 76 0 5

-1 -1 -1 -1 -1 -1 -1
1 1 1 1 0 1 1

80 4 0 0

81.335 0.008 3.420 1.760

-1
0 1 1

-1 -1
1 1 1 1

-1 -1 -1 -1
1 1 1 1 1 1

1 1

-1
0 1 1

-1
1

-1 -1
1

-1 -1 -1 -1 -1
1 1 1 1 1

-1 -1 -1
1

-1
1 1 1

-1 1. -1 -1
1

-1
1

-1 -1 -1
1 1 1 1 1

-1 -1 -1 -1 -1 -1
1 1 1 1 1 1 1 1 1

-1 -1 -1 -1
1 1 1

414 70 2 0 8

386.086 62.699 20.192 8.543 7.769 5.593

65 5

-1
1 1 1

-1 -1 -1 -1
1 1 1 1 1

30

67.991 39.326 1.387 3.010 211.738 215.152 47.646 10.293 7.296 5.387 58.889 5.028 2.809

230 0 8

-1 -1 -1 -1
1 1 1 1 1

-1 -1 -1 -1
1 1

-1 -1 -1
1

-1

-1 -1 -1

-1 -1

-1 -1 -1 -1

-1 -1
1 1

221 42 2 0 5

-1

-1
1

11 1

78

19.065

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

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

Table 3 Observed response and predicted values of glutaminase enzyme Wheat bran (%) Yeast Extract (%) MgCl2 (%) Glutamine (%)

pH -1 -1 -1 -1 -1 -1 -1 -1
1 1 0 1 0

Temperature
(C)

Incubation Period (Hours)

Salinity Glutaminase Production U/

(ppt)

-1 -1 -1
1 1 0 1

-1 -1 -1 -1
1 0

1 1 1

-1 -1 -1
0 1 1 1 1

-1 -1 -1
0 1

-1 -1
1 1 1

-1 -1
0

-1 -1 -1 -1
0 1 1 1

Observed
414 0 0 32 0

-1
1 1 1

-1 -1
1 1 1 0

-1 -1 -1 -1
1 0 1

-1 -1 -1
0 1 1 1 1

-1 -1
1

-1 -1 -1 -1 -1 -1 -1 -1 -1
1 1 1 1 0 1

-1 -1 -1 -1 -1 -1 -1 -1 -1
1 1 0 1

414 2 0

49

-1 -1 -1 -1 -1 -1
1 1 1 1 1 1

-1 -1 -1
1 1 1 1 1 1 1

-1 -1 -1 -1 -1 -1 -2.82843
0 0 0 0 1 1 1 1 1 1 1

-1 -1 -1 -1
0 1 1 1

6 6 0

414 0 5 5 2 9 0 9 9

-1 -1 -1 -1 -1 -1 -1
0 0 0 0 0 0 0 0 0 0 2.82843 0 0 0 1 1

-1 -1 -1 -1 -1 -1 -1 -1
0 0 2.82843 0 0 0 1 1 1

-1 -1
1

-1 -1 -1 -1 -1 -1 -1 -1
2.82843 0 0 0 288 0 1 1

-1
0 0 0 0

-1 -1
0 0 0 1

-1 -1 -1 -1
0 0 0 0 0

-1 -1
0 1 0 0

149 3 7 0

-2.82843
0 0 0 0 0 2.82843 0 0 0 0 0 0 0 0 0

0 0

285

90

166

414

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

-2.82843

-2.82843

0 0 0 0

0 0 0 0 0

0 2.82843 0 0

0 0 0 0 0

-2.82843

-2.82843 276
265 0 178

0 0 2.82843

0 0 0 0 0

0 0 0

230 240 80

202 414 85

0 0 0 0

-2.82843

2.82843 0

-2.82843

2.82843

180 160

160

ml/min Predicted 386.086 8.785 7.388 5.897 45.605 53.755 386.086 18.680 1.119 5.646 12.484 0.157 19.490 386.086 12.025 15.532 12.899 2.287 10.972 0.937 19.942 22.203 165.299 12.949 26.834 5.005 7.956 101.109 271.650 161.102 525.656 287.082 261.436 182.871 238.005 26.166 203.879 169.215 216.985 525.656 78.207 83.252 175.332 136.754 120.978

variable. 2 Y = 0 + i Xi + ii Xi + ij Xi Xj (2) Where Y, 0, i, ii and 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 s) in the second-order model. The method of least squares can be used for this purpose. 3. There is considerable practical

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

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 M initab 15 S tatistical 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. 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. T he 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.

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 5.6103CFU/ml, 3.2104CFU/g and 2.5 105 CFU/g repectively. The total number of Lactobacillus sp ecies were isolated from MRS media plates in water sample was 1.6102 CFU /ml, whereas that of sediment with detritus sample is 1.5105 CFU/g .
Table 1 PlackettBurman experiment for screening of significant process

variables affecting glutaminase production

A B C D E F G H J

2. 8. Determination of molecular weight of glutaminase enzyme (SDS-PAGE) 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 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 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. 100l 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 SDS-PAGE (Sodium-dodecylsulphate-polyacrylamide gel

- - - + + + + - - + - + - + + + - - - + - - - - - + + - + + - - + + - + - + + + - - + + - + + + - + + + - + + + + + + + + +

- - + - - + + - + + + - - +
+ + + + + +

- + + + +

method [10]

- + - + + - - - - + - - + + -

- - - - - - - - + + - - - + - + - + + + + + + + - - + + - + + - + + - - + + + + - - + + - + + + + - + + + + - - + + - - + - + - + + + - - - - + - + + - - + + - + + - + + - - - - + - - - - + - + - + - + - + + + - - + + - + + + + - - + + - + + + - + + - - - - - + - + - + - + - + + + + + + - - - - + +

K L M N O Enzyme U/ml/min Observed Predicted

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

3. 2. Identification and Screening of glutaminase producing bacteria

Figure 1. (a) - Plate showing the glutaminase activity on minimal agar

(a)

(b)

medium. (b)- Control plate showing no glutaminase activity on minimal agar medium.

Totally 25 Lactobacillus strains were isolated. The isolated Lactobacillus strains were screened using minimal agar medium for the production of glutaminase. T he active glutaminase producing organism changed the colour of the medium from yellow to pink ( fig 1 ) . T his 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 Bergeys manual. Among the 9 strains, Lactobacillus casei was found to be the potential strain which was used for further studies.

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

3. 3. Optimization 3. 3. 1. Screening of parameters using Plackett-Burman design

Table 2 Analysis of Variance for glutaminase Enzyme U/ml/min (PlackettBurman design) Source Main Effects Residual Error Total
2.6

DF
14 19 5

Seq SS Adj SS
168911 168917 6 168911 6

Adj MS
12065.1 1.2

9729.90

0.000

D A B N O F G K L M J H E C

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

50

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

Standardized Effect

100

150

200

250

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 P lackett- B urman 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. E very 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. T otally eight variables ( i.e ) W heat 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 Fishers statistical test was conducted for the secondorder 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 2 R 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

Term

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

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 4 Analysis of variance for glutaminase enzyme U/ml/min (Central Composite Design) Source DF Blocks 1 Regression 44 Linear 8 Square 8 Interaction 28 Residual Error 44 Lack-of-Fit 36 Pure Error 8 Total 89 Seq SS
280507 88678

with increase in concentration of the sample.

KDa
97.4 1 2

66.2

Adj SS
280507 88678

Adj MS
280507 31821 11085 2805 1204 0 985

1400140 1232918 78544 43333 43333 1723979 0

1400140 1232918 78544 43333 43333 0

284.83 32.31 2.85 156.49 11.26

0.000 0.000 0.001 0.000 0.000 31 20.1

34KDa 23KDa

154115

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 models authenticity. The Lactobacillus casei strain produced 465.426 U/ml/min glutaminase under optimized culture conditions.

Fig. 4: SDS-PAGE of partially Purified Glutaminase 1 Protein marker 2 Sample (Glutaminase enzyme)

14.4

4. DISCUSSION
I n 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 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 KCTC 3594 was studied by [14]. T he glutaminase worked optimally at p H 7.5. The optimum temperature was 40C. 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

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

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] . T he 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 (320 U/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 k D a 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, 170g/ml),was digested by incubation with 300 g protein/ml of the cell free extract from Micrococcus luteus K-3 at 30 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 g/ml compared to the scavenging

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

SC50 8.7 g/ml)[18].

activity of the well-known antioxidant (ascorbic acid, a. a.,

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. T otally 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.

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