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L-Glutamate Oxidase Enzyme from Hypocrea jecorina: Production and Biochemical Characterization.

Byline: Fatih Mehmet Eynur and Emine Karakus

Summary- L-Glutamate oxidase (LGOX; EC 1.4.3.11) is the important enzyme that catalyzes the deamination of L-glutamic acid to 2-oxo glutarate and ammonium ions. In this study the microbial production of L-glutamate oxidase enzyme from Hypocrea jecorina pure culture was carried out and the optimum conditions and calculation of some kinetic parameters of the produced enzyme were also determined by using Nesslerization reaction. The enzyme shows maximum activity at pH 8.5 and 40oC in TRIS buffer. The kinetic parameters of the enzyme KM and Vmax values were determined as 2.58 mM and 83.33 U/mg protein respectively. The inactivation rate constants and half life (t1/2) were determined from the slope of thermal stability plots of the enzyme at 60 70 and 80oC temperatures. The activation energy (Ea) was found to be 1.40 kcal mol-1

Key Words: Glutamate oxidase Hypocrea jecorina Enzyme production L-glutamate Nessler reagent.

Introduction

Enzymes are biocatalysts produced by living cells to bring about specific biochemical reactions generally forming parts of the metabolic processes of the cells. Enzymes are highly specific in their action on substrates and often many different enzymes are required to bring about by concerted action the sequence of metabolic reactions performed by the living cell. All enzymes which have been purified are protein in nature and may or may not possess a nonprotein prosthetic group. The practical application and industrial use of enzymes to accomplish certain reactions apart from the cell dates back many centuries and was practiced long before the nature or function of enzymes was understood [1].

Microbial enzymes are numerous used in food pharmaceutical textile paper leather and other industries. L-amino acid oxidases (LAOX) catalyse the stereospecific oxidative deamination of amino acid substrates to the corresponding a-keto acids along with the production of ammonia and hydrogen peroxide via an imino acid intermediate [2].

L-Glutamate oxidase (LGOX; EC 1.4.3.11) belongs to the family of oxidoreductases specifically those acting on the CH-NH2 group of donors with oxygen as acceptor. The enzyme specifically catalyzes the oxidative deamination of L-glutamate in the presence of water and oxygen with the formation of 2-ketoglutarate ammonia and hydrogen peroxide [3-5].

L-Glutamate oxidase is the principle component in the L-glutamate determination. L- glutamate is the major excitatory neurotransmitter in the central nervous system of mammals. It has important role in Parkinson's disease epilepsy schizophrenia drug addiction and many brain diseases. Based on this information it is obviously very important to develop specific analytical methods for measuring this amino acid preferably in simple and reliable way [6-13].The hydrogen peroxide formed after L-glutamate oxidase reaction can be easily detected by several methods used glutamate oxidase such as biosensor and the chromogenic peroxidase reaction [4 14-19]. The enzyme is also important in fermentation industry food and clinical chemistry for fermentation process measurement evaluation of food quality and diagnosis of several kinds of diseases [20].

Although several microbial fermentation studies for LGOX production from different sources have been carried out [3 4 20 21] there was no paper related to LGOX production from Hypocrea jecorina. Because of the production of L-glutamate oxidase enzyme from Hypocrea jecorina has not been observed in the literature until now this study is original. In our study we represent microbial production and some biochemical characterization of L-glutamate oxidase enzyme from Hypocrea jecorina. The optimum parameters of the produced enzyme such as optimum pH optimum temperature buffer concentration thermal stability activation energy half time and storage stability of enzyme were also determined.

Results and Discussion

In this study we produced L-glutamate oxidase enzyme from Hypocrea jecorina pure culture and determined its biochemical characteristic such as optimum working conditions some kinetic parameters. Because L-glutamate oxidase (LGOX ) is an enzyme that specifically catalyzes the oxidative deamination of L-glutamate in the presence of water and oxygen with the formation of a-ketoglutarate ammonia and hydrogen peroxide [3 4] we determined the enzyme activity by measuring ammonium ions released as a result of glutamate oxidase enzymatic reaction. The results of some quantitative parameters of produced L-glutamate oxidase from H. Jecorina present in Table-1.

Table-1: Production of L-glutamate oxidase from H.

Jecorina.###

###NH4+ amount

###liberated from

###L-glutamate###Protein

###Specific activity

###oxidase###amount

###L-glutamate

###activity###(mg/mL)

###(U/mg protein)

###oxidase###s

Optimization of the Culture condition for L- glutamate Oxidase Production

In order to determine the effect of substrate (L-glutamate ) concentration on L-glutamate oxidase enzyme production from Hypocrea jecorina pure culture the enzyme production was carried out by adding L-glutamate in different concentrations ranging from 1 to 4%. Activity determinations were carried out on samples taken at the end of the incubation period. The obtained results were presented in Fig. 1.

As seen in Fig. 1 when added L-glutamate amount is incresed L-glutamate oxidase enzyme activity decreases. But the specific activity of the enzyme rapidly increases. Because Hypocrea jecorina uses L-glutamate as nitrogen source and only ammonium ions produced as a result of L- glutamate oxidase enzymatic reaction involved in the medium we didn't add any other nitrogen source. Therefore wen can determine correctly enzyme activity. In this way microorganism was directed to glutamate and used it as both carbon and nitrogen source and the production of glutaminase enzyme were carried out. It was found 4% of L-glutamate concentration for optimum L-glutamate oxidase production from H. jecorina.

Effect of buffer concentration

It was shown that our produced LGOX enzyme didn't show any response with phosphate buffer. Because of this we used TRIS-HCl buffer in all of the experiments. The effect of buffer concentration on our produced LGOX enzyme was examined by measuring the enzyme activity at each buffer concentration (Fig. 2). The optimum buffer concentration was determined as 0.05M TRIS-HCl at pH 8.5.

Effect of pH and temprature

Fig. 3 shows the pH-activity profile of produced LGOX from H. Jecorina. It can be seen in Fig. 3 there is not any LGOX activity until pH 6.0. The enzyme showed maximum activity between 6.0 and 9.0. After pH 8.5 the activity of L-glutamate oxidase enzyme the activity decreased very rapidly. The optimum pH of the enzyme is 8.5. The enzyme was more stable in alkaline pH than in acidic pH. It was reported that the optimum pH of LGOX from different microorganism species was also alkaline pH [22 23].

Enzymes have a maximum activity at a pH called optimum pH. The enzyme activity decreases below and above on their optimum pH. Because enzymes are not resistant to strong acids and bases the changes in media pH affect the ionic structure of enzyme and substrate. Electrostatic bonds formed between the positive and negative groups on enzyme affect by pH. The activities of enzymes lose extreme pH values [24].

It was observed that L-glutamate oxidase activity increased up to the optimum temperature. The maximum catalytic activity was obtained at 40 oC. L-glutamate oxidase enzyme activity produced from Streptomyces sp. 18G was also highest at 37 oC [24]. This result show that we can work the enzyme until 40 oC. This situation is very important in terms of its analytical and industrial uses.

Although the enzyme-catalysed reactions rates increase with temperature rise enzymes lose activity at high temperatures due to their protein structures. High temperatures can affect the condition of being dissociated functional groups which play a role in the enzymatic reaction the affinity of enzyme to activators and inhibitors resolution of oxygen which can be substrate in the reaction. The temperature being the enzymatic reaction rate reaches maximum is called the optimum temperature. The vast majority of enzymes show maximum activity between 30 and 40 C. Enzymatic denaturation generally begins above 45 C. But the enzymes showed the activity at high temperatures are also avaible [25].

Thermal stability of L-glutamate oxidase from H. Jecorina

Thermal stability experiments were carried out with L-glutamate oxidase enzyme from H. Jecorina. The enzyme was incubated in the absence of substrate at various temperatures. As it was shown in Fig. 5 the enzyme retained 75 % of its activity during a 45 min of incubation period at all temperatures. L-glutamate oxidase enzyme were lost about 5% of its original activity at 25oC for 15 min. After 50oC activity loss of the enzyme is bigger. It can be said that the enzyme can be used in bioanalytical and biosensor applications.

Thermal inactivation kinetix properties fort he enzyme were also determined. For the first phase of thermal inactivation for the thermal inactivation rate constants k were calculated from the slope of the curve for 60 70 80oC separately. From a plot of ln k- 1/T (Fig. 6) Ea (activation energy) was calculated from the slope of the straight line and found to be 1.40 kcal mol-1.

Inactivation rate constant and the half-life (t1/2) of L-glutamate oxidase from H. Jecorina were calculated and represented in Table-2. The higher the inactivation rate constant is the less thermostable enzyme [26]. From this reason as seen in the Table- 2 the enzyme is less stable at higher temperature. The half-life (t1/2) is another important parameter for the characterization and stability of L-glutamate oxidase enzyme. It is seen in Table-2 that the increasing in the temperature causes the decrease in t1/2 values.

Table-2: Inactivation rate constant and half-lives (t1/2)

Enzyme Kinetic Studies

Km and Vmax values for the enzyme at optimal pH and temperature were determined by Lineweaver-Burk plot [27]. The double reciprocal plot was linear (correlation coefficient of 0.7939) over L-glutamate concentration range (10-100 mM). Km and the Vmax values were determined as 2.58 mM and 83.33 U/mg proteine for L-glutamate oxidase from H. jecorina respectively

Storage Stability of L-glutamate oxidase

In order to determine the storage stability of L-glutamate oxidase the enzyme extracts produced from H. jecorina were kept in small erlenmayer flasks at 4oC for 90 days. The total activity measurements were carried out taking samples in order to determine the stability during 90 days. Results are shown in Fig. 8. As shown Fig. 8 enzyme activity decreased of 66% until 60 days. Any decrease in L-glutamate oxidase activity didn't observe after 60 days until 90 days. This indicates that L-glutamate oxidase can be used other bioanalytical studies such as biosensor construction glutamate assay studies etc.

Experimental

Materials

Hypocrea jecorina microorganism used in this study was obtained from Institute of Biochemical Technology and Microbiology Vienna Technical University. L-monosodium glutamate glucose potato dextrose agar (PDA) bovine serum albumin (BSA) Coomassie Brilliant Blue G-250 ethanol o- phosphoric acid trichloroacetic acid (TCA) KH2PO4 MgSO4.7H2O KCl NaCl NaOH NaH2PO4 Na2HPO4 KI HgCl2 were purchased from Sigma Chemical (St. Louis Mo. USA). All chemicals used in this study were of analytical grade and were used without further purification. The absorbance measurements were carried out with Agilent UV-Vis spectrophotometer. Sartorius-PB11 model pH-meter was used for pH measurements. The incubation sterilization heating cooling and storing procedures were made with VWR-incubating mini shaker Clifton brand water bath Certoclav brand autoclave Memmert brand oven Arcelik brand refrigerator respectively.

GFL brand water destilation device was used to obtain destilled water.

Fermentation Medium and Culture Conditions for Microorganism

Potato dextrose agar (PDA) was prepared to sustain growth and vitality of Hypocrea jecorina pure culture. 39 g potato dextrose agar (PDA) was dissolved in water and the final solution volume was completed to 1 liter with distilled water. This PDA solution was sterilized by autoclaving for 20 minutes at 121oC. After sterilization it was kept at 45 C in water bath and the solution was apportion into petri dishes and allowed to cool. The solidified media was stored refrigerator at +4oC until used. For preparing PDA slant agar medium 15 mL of the solution prepared was apportion into each tube and closed lids of the tubes and sterilized by autoclaving for 20 minutes at 121oC. After sterilization process the tubes were allowed to cool positioning of flat. The solidified media was stored at +4 oC until used.

The optimum temperature for maximum L- glutamate oxidase activity was determined different tempratures ranging from 30 to 70oC with heat- controlled circulating water bath. The specific activity of L-glutamate oxidase at a specific temperature was determined spectrophotometrically by addition of the produced enzyme extract to the mixture. The optimum temperature value obtained from these assays was used in all the other experiments.

Effect of Buffer ConcentrationTo examine the effect of optimum buffer concentration for L-glutamate oxidase the enzyme activity were measured at eight different buffer concentrations varying from 001 to 008 M.

Thermal Stability of L-glutamate oxidase by H. Jecorina

The glutamate oxidase enzyme solution was incubated for various time intervals (1545 min) at the specified temperature (2590oC) in a prewarmed incubator and rapidly cooled. After 5 ml of the sample portions were withdrawn at various time intervals during 45 min the activity the enzyme activity was measured at room temperature. The stability of the enzyme was expressed as remaining activity.

The data obtained from the thermal stability profile were used to determine thermal inactivation of the enzyme. For the first phase of thermal inactivation for the thermal inactivation rate constants k were calculated from the slope of the curve for 60 70 80oC separately. The temperature dependence of k was evaluated by using the Arrhenius equation (Ln k = C- Ea/RT). Where C is a constant of integration Ea is activation energy (cal mol-1) R (1.99 cal mol-1) is the universal gas constant and T (K) is the absolute temperature in Kelvin.

Kinetic Constants of L-glutamate oxidase

Km and Vmax values for the our produced L- glutamate oxidase from H. Jecorina were determined by measuring activities in the presence of various substrate concentrations between 10 and 100 mM at pH 8.5. The Km and Vmax values of the enzyme

were calculated by using the Lineweaver- Burk double reciprocal plot [22] in which the reciprocals of the initial velocities of L- glutamate oxidase activity were plotted against the reciprocals of the concentration of L-glutamate used.

Conclusion

In this study L-glutamate oxidase enzyme was produced from Hypocria jecorina pure culture. After production the kinetic and some biochemical characteristics were alo determined. Because LGOX enzyme has very important role for L-glutamate assay it can be thought that LGOX enzyme produced from microbial sources are important in respect to their primer role for glutamate assay and their biosensor application. We planned to investigate usage of their biosensor contruction.

Acknowledgement

We gratefully acknowledge for the financial support of Yildiz Technical University Science Research Projects Foundation (Project number: 2011- 01-02-KAP07) for the financial support of this work.

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Date:Aug 31, 2014
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