Immobilization of glutaminase enzyme from Hypocria jecorina on polyacrylic acid: preparation and biochemical characterization

Abstract

L-glutaminase enzyme produced from Hypocrea jecorina pure culture and polyacrylic acid (PAA) in the presence ^Cu+ ions were composed the ternary complex at pH 7. The properties of free and immobilized enzyme were defined. The effect of various factors such as pH, temperature, heat, and storage stability on immobilized enzyme were investigated. The properties of immobilized enzyme were investigated and compared to those of free enzyme. Optimum pH and temperature of both enzyme were determined to be 8.0 and 50°C, respectively. Kinetic parameters of the immobilized enzyme (Km and Vmax values) were also determined as 0.38 mM of the Km and 10.9 U/L of the Vmax. No drastic change was observed in the Km and Vmax values. Thermal and storage stability experiments were carried out. The thermal stability studies indicated that the immobilization process tends to stabilize the enzyme.

Keywords::

• enzyme production
• L-Glutaminase enzyme
• immobilization
• microorganism
• polyacrylic acid
• ternary metal complexes

Introduction

Proteins (or enzymes) and polyelectrolytes (PE) in the presence of transient metal (M) ions (Fe³⁺, Ba²⁺, Cu²⁺, etc.) compose the water-soluble and water-insoluble ternary polycomplexes (Karahan et al. 2010, Dincer et al. 1998, El-Madani et al. 1998, Filenko et al. 2001, Shoukry et al. 2002). Such polyelectrolyte (PE)-metal (M) -protein polycomplexes (PMC) have been obtained by simple mixing of solutions of proteins, metal ions, and PE in water at different pH values. This method consists of the use of small concentrations of metal additives, which promote PE binding to protein molecule without causing any unexpected changes in the chemical structure of components. These studies revealed that in the presence of metal ions many nitrogen- and carboxyl-containing PE [poly(acrylic acid) (PAA), polyvinylpyridines, poly-N-vinylimidazole, polyconidine, poly(N-isopropylacrylamide) (poly(NIPAAm)) and their copolymers irrespective of their molecular mass] can form ternary PMC with different proteins. These complexes can be both soluble and insoluble in water. The contacts between proteins and PE are achieved via formation of a chelate unit in which the metal ions are located at the interface. The solubility of the polycomplexes depends on the nature of proteins and correlates with their isoelectric points (pI). In these systems, the metal ions promoted two effects: (1) the binding of PE to the protein molecules and (2) intermolecular aggregation of polycomplex particles. The solubility, composition, and stability of these polycomplexes depend on pH, metal/PE, and protein/PE ratios. Some of these polycomplexes reveal strong immunogenecity and provide a high level of immunological protection (Karahan et al. 2010).

The specificity of enzymes promises great improvements in various applications. Enzymes have large number of practical applications such as biomaterials (Kobayashi and Ikada 1991, Okada and Ikada 1992) and biosensors (Dindar et al. 2011, Yılmaz and Karakuş 2011, Karakuş et al. 2013), through immobilization on a variety of supports. Beside their specificity, the short catalytic life times of enzymes presently limit their usefulness. Immobilization of enzymes has been taken to improve catalytic stability of enzymes and can expand the application of the neutral catalysts. There are many materials, including synthetic organic polymers, biopolymers, hydrogels, inorganic supports, and smart polymers, to be used to immobilize to enzyme, and good activity retention, and enhanced thermostability are often observed (Zhang et al. 2009).

There are many methods, such as adsorption, entrapment, covalent binding, etc., for enzyme immobilization (Karakuş and Pekyardımcı 2009, Karakuş and Pekyardımcı 2012, Nakajima et al. 1989). When the support contains the relevant functional groups, covalent immobilization of enzymes becomes feasible. A range of functional groups which can be used in the covalent immobilization of enzymes include amino, hydroxyl, carboxyl, and phenolic groups. The physical structure and chemical composition of support can also influence the microenvironment of the immobilized species and consequently their biological properties (Chen et al. 2000).

L-Glutaminase is widely distributed in animal tissues, plants, and microorganisms including bacteria, fungi, and yeast. L-glutaminase (L-glutamine amidohydrolase E.C 3.5.1.2) is a hydrolytic enzyme, that hydrolises L-glutamine to L-glutamic acid and ammonium ions (Bülbül 2012). In food industry, L-glutaminase is used as a flavor enhancing agent by increasing glutamic acid content in food due to its catalysed hydrolysis reaction of L-glutamine to L-glutamic acid and ammonium (El-Sayed 2009). It also used in enzyme therapy for cancer especially for acute lymphocytic leukemia (Roberts et al. 1970, Pal and Maity 1992). L-glutaminase production by various bacterial genera under submerged culture conditions was extensively studied (Wade et al. 1971, Imada et al. 1973, Saxena and Sinha 1981, Yano et al. 1988, Shindia et al. 2007).

In this work, polyacrylic acid (PAA) was selected immobilization matrix for the immobilization of our produced glutaminase enzyme from Hypocrea jecorina in the presence of Cu²⁺ ions in aqueous solution. The immobilized enzyme was characterized by determining kinetic constants, optimum pH and temperature, thermal and storage stability. The properties of the PAA-glutaminase enzyme from Hypocrea jecorina-Cu ²⁺ complex were compared to those of free enzyme.

Materials and methods

Chemicals

Benzoilperoxide, 1-ethyl-3-(3-dimetilaminopropyl) carbodiimide hydrochloride (EDC), ethyl acetate, L-glutamine, 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, NaH₂PO_4, Na₂HPO₄, KI, and HgCl₂ were purchased from Sigma Chemical (St. Louis, Mo. USA). PAA was purchased from Aldrich. CuSO₄ 5H₂O were purchased from Merck (Darmstadt, Germany) and dissolved in ultrapure water. All experiments were made at + 4°C in cold room in order to avoid losing enzyme activity. 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, Arçelik brand refrigerator, respectively. Ultrapure water was obtained from Millipore MilliQ Gradient System.

Microorganism

In this study, H. jecorina microorganism obtained from Institute of Biochemical Technology and Microbiology, Vienna Technical University was used.

Cultivation

To sustain growth and vitality of H. jecorina microorganism, potato dextrose agar (PDA) was prepared. For this purpose, 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 min at 121°C. 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 + 4°C 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 min at 121°C. After sterilization process, the tubes were allowed to cool positioning of flat. The solidified media was stored at + 4°C until used.

H. jecorina was planted into each petri dish containing solidified culture media with sterile loop and incubated for 7 days in an incubator set at 30°C. After incubation period of 7 days the petri dishes contained produced H. jecorina were covered with parafilm and stored at 4°C until used. This procedure was repeated when needed fresh H. jecorina. To maintain vitality of the mold strains stored at 4°C, it was transferred to fresh mediums once a month.

To produce crude L-glutaminase enzyme extract from Hypocria jecorina, we used the our-modified fermenting liquid media method proposed by El-Sayed's method (El-Sayed 2009). The medium contains these components (in grams per liter): glucose as carbon source 1.0, KH₂PO₄ 0.1, MgSO₄.7H₂O 0.05, KCl 0.05, NaCl 5.0, L-glutamine as nitrogen source 4.0. After sterilization of 100 mL of the solution containing these liquid media components at 121°C for 20 min, the solution was incubated in water bath at 30°C for 30 min. 2 mL of Hypocria jecorina pure culture was added into this sterilized solution medium and incubated at 30°C and 150 rpm in shaking incubator for 30 min. After incubation of 30 min, we measured L-glutaminase activity. Experiments and enzymatic assay was performed in triplicates for each sample.

To maximize the yield of L-glutaminase from Hypocrea jecorina, several parameters such as effect of substrate and salt concentrations were investigated. To determine optimum substrate concentration for maximum L-glutaminase activity, it was prepared culture medium contained L-glutamine concentration ranging from 1% to 5% w/v separately. The effect of different doses of NaCl (0%–15% w/v) to the production medium to maximize enzyme activity was also studied. The effects of substrate and salt concentration on the enzyme production were evaluated by measuring L-glutaminase enzyme activity. The optimized parameter was incorporated at its optimized level in the all experiments. All the experiments were conducted in triplicate. After incubation of each fermentation sample, the crude enzyme was prepared.

Immobilization procedure

Preparation of polyacrylic acid (PAA)-Cu ²⁺-glutaminase enzyme ternary complex

First, polyacrylic acid (PAA) was synthesized and fractionated as explained in the literature (Miller 1978). Polymer was prepared by radical polymerization of acrylic acid in toluene with benzoilperoxide as initiator. PAA was fractionated from 3% to 4% solution in methanol by fractional precipitation with ethyl acetate. The weight-average molecular weight (Mw) of the fraction used in this study was 100 kDa. Polymer solution used in the study were prepared within ultrapure water in a cold room (5°C) by stirring over 12 h.

In order to produce PAA–Cu ²⁺ complex, CuS0₄ 5H₂0 solutions was added and the mole ratio of Cu ²⁺ ions to the PAA was kept constant at Cu ²⁺/PAA = 0.4 in ultrapure water. The solution was stirred at 20°C for 24 h. Then, pH was adjusted to pH 7 by adding 1 N NaOH solution.

To immobilize the enzyme or produce PAA-Cu ²⁺– glutaminase enzyme ternary complex, first, 1.2 mg/1 mL our prepared PAA-Cu ²⁺ complex was incubed 4.8 mg 1-ethyl-3-(3-dimetilaminopropyl) carbodiimide hydrochloride (EDC) to active carboxyl group on the surface of the polymer among 3 h at pH 5.5. After our produced glutaminase enzyme from Hypocria jecorina (1.2 Unit/1 mL) at pH 7 was added to this solution and mixed 12 h. The pH of the final solution was also adjusted to 7 (Scheme 1). The Cu ²⁺ ions/glutaminase enzyme and Cu ²⁺ ions/acrylic acid ratios were calculated using the equation;

Scheme 1. The hypothetical structure of ternary PAA-Cu²⁺–glutaminase enzyme from Hypocrea jecorina complex.

where C_CuSO4 is CuSO₄ concentration, C_PAA is PAA concentration (% 0,1), M_AA is molecular weight of the monomer of PAA polymer, and M_CuSO4 is molecular weight of CuSO_4.

Determination of glutaminase enzyme activity

To determine free and immobilized glutaminase enzyme activity, we used the method based on measurement of formed ammonium ions after the enzymatic reaction of the enzyme (Imada et al. 1973). L-glutaminase enzyme catalysis L-glutamine (L-Gln) to L-glutamic acid (L-Glu) and ammonium ions and the latter can react with Nessler's reagent to produce an orange product.

For this purpose, 0.5 ml of 0.1 M phosphate buffer (pH 8.0), 0.5 ml of distilled water and 0.5 ml of produced and immobilized L-glutaminase enzyme solution were added into 0.5 ml of 0.04 M L-glutamine solution and incubated at 37°C for 30 min for enzymatic reaction to occur. After incubation, 0.5 ml of 1.5 M trichloroacetic acid (TCA) was added to stop the reaction. 0.1 mL of final solution was added into 3.7 mL of distilled water and Nessler reagent, respectively. After the solution was incubated for 10 min for interacting with Nessler reagent and ammonium ions formed after L-glutaminase enzymatic reaction, the solution's absorbance was measured at 450 nm.

To determine the activity value of free and immobilized L-glutaminase enzyme, the amount of the NH₄⁺ ions formed as a result of enzymatic reaction must be determined by drawing ammonium standard graph. The ammonium ions were determined from standard curve obtained with ammonium chloride as standard. For this purpose, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL of standard ammonium chloride solutions were prepared. After adding Nessler reagent into each tube, the tubes were incubated 10 min at room temperature. The absorbance of each tube was separately measured against the blank containing 3.8 mL distilled water and 0.2 mL of Nessler reagent at 450 nm. The ammonium standard curve was obtained by plotting absorbance values against ammonium chloride concentrations.

One unit of glutaminase activity was defined as the amount of enzyme that liberated 1 μmol of ammonium ions produced per minute at room temperature. Specific activity is expressed as units per miligram of protein.

Protein determination

Protein concentrations were determined by the method of Bradford by using bovine serum albumin as standard (Bradford 1976). The amount of bound protein was calculated from the difference between the amount of protein introduced into the reaction mixture and the amount of protein after immobilization.

Effect of pH and temprature on L-glutaminase activity

The optimum pH values of our produced free and immobilized L-glutaminase enzyme from H. jecorina were determined for the pH range between 5.0 and 9.0 by using 0.1 M phosphate and TRIS buffers. After pH 9.0, we used 0.1 M TRIS buffer. The optimum pH values determined were used in all the other experiments.

Optimum temperatures to maximum free and immobilized L-glutaminase activity under mentioned assay conditions were determined between 30 and 70°C temperatures by a circulating water bath. The relative activities of both L-glutaminase at a specific temperature were determined spectrophotometrically by addition of the produced enzyme extract to the mixture. The optimum temperature values obtained from these assays were used in all the other experiments.

Thermal stability of L-glutaminase by H. jecorina

The thermal stability of free and immobilized L-glutaminase were investigated at four different temperatures between 40 and 70°C for varying periods of time in a temperature-controlled water bath. Enzyme solution was placed in a prewarmed incubator at the specified temperature and 0.5 ml of the sample portions were withdrawn at various time intervals during 45 min and residual activity assayed. The stability of the enzyme was expressed as remaining activity.

Kinetic constants of L-glutaminase

The kinetic parameters of our produced free and immobilized L-glutaminase from H. Jecorina, Km and Vmax values, were determined by measuring activities in the presence of various substrate concentrations between 10 and 100 mM at pH 8.0. The Km and Vmax values of the enzyme were calculated by using the Lineweaver–Burk double reciprocal plot, in which the reciprocals of the initial velocities of L-glutaminase activity were plotted against the reciprocals of the concentration of L-glutamine used (Lineweaver and Burk 1934).

Results and discussion

Glutaminase enzyme (EC 3.5.1.2) has received significant attention in the food industry owing to its potential as a flavor modulating agent, as it increases the glutamic acid content of the food imparting savory flavor. This unique taste called umami, elicited by meat, fish, and vegetable stocks, has been widely discussed in the literature and confirmed as the fifth basic taste beside sweet, acid, salty, and bitter (Weingand-Ziadé et al. 2003). L-glutaminase enzyme was widely found in microorganisms such as bacteria and fungi. L-glutaminase production by various bacterial genera under submerged culture conditions was extensively studied (Wade et al. 1971, Imada et al. 1973, Chantawannakul et al. 2003). There was no study previously related to glutaminase production from H. jecorina in the literature

In this study, first, we produced L-glutaminase enzyme from H. jecorina pure culture and composed PAA-Cu ²⁺– glutaminase enzyme triple complex for enhanced enzyme activity. After enzyme immobilization, the bioanalytical properties and characterizations of free and immobilized enzymes were compared. The free and immobilized L- glutaminase enzyme activities were determined by measuring ammonium ions released as a result of glutaminase enzyme reaction. The results of some quantitative parameters of free enzyme and PAA-Cu ²⁺–glutaminase triple complex are summarized in Table I.

Table I. Immobilization of glutaminase enzyme from Hypocrea Jecorina.

	NH4^+ amount liberated from L-Glutaminase reaction (mg/mL) ± s*	L-Glutaminase activity (U/mL) ± s*	Protein amount (mg/mL) ± s*	Specific activity (U/mg protein) ± s*
Free enzyme	4.42 ± 0.5	13.75 ± 0.9	0.34 ± 0.8	40.32 ± 0.5
PAA-Cu ^2+-glutaminase triple complex	3.86 ± 0.4	8.65 ± 0.6	0.24 ± 0.4	36.45 ± 0.5

*s: Standard deviation. The data were calculated from the results obtained from triplicated experiments as mentioned section.

It is seen in Table I, our produced glutaminase enzyme from H. Jecorina was immobilized to PAA-Cu ²⁺ complex at the ratio of 62.9%. Immobilized enzyme activity decreased 1.5-fold.

Effect of pH and temprature

The effect of pH on the activity of free and immobilized glutaminase enzyme from H. Jecorina was studied within the pH range of 5.0–9.0 at room temperature. The total activity of the enzyme obtained is presented in Figure 1.

Figure 1. Effect of pH on free and immobilized L-glutaminase enzyme produced from Hypocrea jecorina.

As seen in Figure 1, the activity of free and immobilized L-glutaminase enzyme increased progressively until the value of pH 8.0. After pH 8.0, the activity decreased very rapidly for both enzymes. Thus, optimum working pH of the free and immobilized enzymes obtained from H. jecorina was chosen as 8.0. It was reported that the pH optimum of L- glutaminase from Vibrio sp., Debaryomyces spp., Aspergillus sp. microorganisms were in the range of pH 7.0–8.5 (Parasanth Kumar et al. 2009, Jeya Prakash et al. 2010, Durá et al. 2004).

The temperature effect on the activities of free and immobilized L-glutaminase enzyme produced from H. Jecorina were tested at pH 8.0 and L-glutaminase activity as a function of temperature under our standard assay conditions were determined by using temperatures from 30 to 70°C for 30 min and the enzyme activities were determined. The results obtained is shown in Figure 2.

Figure 2. Effect of temperature on free and immobilized L-glutaminase enzyme produced from Hypocrea jecorina.

The maximum catalytic activity for free and immobilized enazymes was obtained at 50°C (Figure 2). L-glutaminase enzyme activity produced from Lactobacillus rhamnosus was also highest at 50°C (Weingand-Ziadé et al. 2003). That absence of any loss of enzyme activity at high temperatures is very important in terms of their analytical and industrial uses.

Thermal stability of L-glutaminase from H. jecorina

To determine thermal stability of free and immobilized L-glutaminase from H. Jecorina, the enzyme solution was incubated for various time intervals (15–45 min) at the specified temperature (25–70°C) and the activity was measured at room temperature. Temperature-stability profiles for free L-glutaminase are presented as relative activity, and shown in Figure 2.

Thermal stability experiments were carried out with L-glutaminase enzyme from H. Jecorina. The free enzyme and PAA-Cu ²⁺-enzyme triple complex were incubated separately in the absence of substrate at various temperatures. As it was shown in Figure 3 and 4, the both enzymes retained 85% of its activity during a 45 min of incubation period at 40°C and 50°C. Both L-glutaminase enzyme were lost about 5% of its original activity at 70°C for 45 min. It was shown any important activity lost at 60°C and 70°C after 45 min. It can be said that the enzyme was relatively high thermal stability. This is important feature for enzymes in respect to their bioanalytical and biosensor applications.

Figure 3. Thermal stability of free L-glutaminase from Hypocrea Jecorina.

Figure 4. Thermal stability of immobilized L-glutaminase from Hypocrea Jecorina.

It has been reported that the thermal stability of many enzyme was increased after its immobilization on a support because the support material is supposed to preserve the tertiary structure of the enzyme. In addition, it is pointed out that the thermal stability of an enzyme may indicate the efficiency of the immobilization method and reflect the delicate balance between the acquired conformational stability and the resulting microenvironment created around the enzyme (Arica 2000).

Enzyme kinetic studies

Km and Vmax values for the enzyme at optimal pH and temperature were determined by Lineweaver–Burk plot (Lineweaver and Burk 1934). The kinetic constants (Km and Vmax values) for free and immobilized glutaminase enzyme were determined by using glutamineas a substrate (Table II).

Table II. The kinetic properties of free and immobilized L-glutaminase from Hypocrea Jecorina.

Enzyme	Km (mM)	Vmax (U/L)
Free	0.49	13.86
İmmobilized	0.38	10.9

Km and Vmax values of both enzymes were calculated from the intercepts on x and y axes of the Lineweaver–Burk plots for the free and immobilized L-glutaminase enzyme, respectively. For the free enzyme Km was 0.49 mM and the apparent Km value of immobilized L-glutaminase enzyme was 0.38 mM. Both Km values almost equal. For the free enzyme, Vmax was 13.86 U/L, but Vmax little decreased to 10.9 U/L for PAA-Cu ²⁺–enzyme triple complex. The difference in kinetic parameters between free and immobilized enzymes were nearly same. This shows the enzyme do not lose its kinetic performance after immobilization procedure.

The Km value of L-glutaminase enzyme from Lactobacillus rhamnosus microorganism was 4.8 mM and Vmax value ot the same enzyme was found to be 13.86 U/L (Weingand-Ziadé et al. 2003). The Km value obtained in this study is lower than the other studies in the literature. Low Km value for L-glutaminase enzyme from H. jecorina can be an indicator that the enzyme's affinity against L-glutamine is very high.

Storage sability of L-glutaminase

In order to determine the storage stability of free and immobilized L-glutaminase, the enzyme extracts for free and immobilized enzyme produced from H. jecorina were kept in small erlenmeyer flasks at 4°C for 30 days. The total activity measurements were carried out taking samples in order to determine the stability during 30 days. Results are shown in Figure 5. After 30 days, free and immobilzed enzyme activities did not decrease. This is very important for bioanalytical enzyme studies.

Figure 5. Storage stability of free and immobilized L-glutaminase from Hypocrea Jecorina at + 4°C.

Conclusion

This study presents the immobilization of our produced glutaminase enzyme from H. jecorina pure culture. The characterization of free and immobilized enzymes was also studied. L-glutaminase enzyme produced from microbial sources is important in respect to their antileukaemic and as flavor-enhancing agents. Because of therapeutic and technological importance, L-glutaminase enzyme production and immobilization have biotechnological importance.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

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.