Evidence of Antioxidant Activity of Novel L-Glutaminase Purified from L. Gasseri BRLHM

*Corresponding Author: Butheina A. Hasson 100235@uotechnology.edu.iq Abstract Probiotic strains have the potential to be used as bio-preservatives and functional radical scavenging treatments in the future. Antioxidant tests, including DPPH radical scavenging, were used to evaluate the antioxidant effects of extracellular LGlutaminase isolated from L. gasseri BRLHM. Parameters for the promoted production of the enzyme under minimal production media were optimized. The importance of this study lies in enhancing the production of the LGlutaminase isolated from L. gasseri BRLHM with a high activity using these L. gasseri bacterial as antioxidant activity. In ion-exchange chromatography, the specific activity was 14.7U/mg protein, with 58.8% yield and 4.6 purification folds. In the gel filtration, the specific activity was 23.4 U/mg protein, with a yield of 54 % and 4.6 purification folds. According to the findings, L-Glutaminase isolated from L .gasseri BRLHM exhibited good antioxidant properties. As the concentration rose, there was a remarkable proportionate increase in scavenging activity. The IC50 values for control and LGlutaminase were 36.09 1.12 and 619.8 gm/ml, respectively. The IC50 values were discovered to be 100 and 200 μg/ml, respectively. Conclusion: For the first time, the high of LGlutaminase isolated L. gasseri BRLHM was shown to exhibit antioxidant. This could be a promising discovery for future radical scavenging treatments and antioxidant prophylaxis with natural probiotics.

oxidative stress. Various malignancies have been found to have higher levels of oxidative DNA damage. Due to the increase of antimicrobial resistance, the inclusion of probiotics in food is a fascinating alternative to antibiotics that have piqued public interest [5]. Antioxidant activity of artificial antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and n-propyl gallate (PG) is powerful against a variety of oxidation processes. However, due to potential health hazards, the use of these antioxidants in food has been restricted in several countries. Because of the documented negative effects of some artificial antioxidants [6], natural antioxidants are likely to be more tolerable than those created chemically. the result, the hunt for natural, stable, safe antioxidant agents as alternatives to manufactured molecules have gained more attention. The aim of the study is to isolate L-glutaminases from Lactobacillus gasseri and their study antioxidant activity.

2.Experimental Procedure
Collection of samples78 vaginal swabs were isolated from healthy women. Lactobacillus spp. is diagnosed based on the findings of culture, microscopic, biochemical, and API 50 CHL testing [7]. Collected of Lactobacillus spp. from aged  years old from the Hospital AL-elweya of Obstetrics in Baghdad, Iraq, from December 2019 to March 2020. Detection, screening, and purification of L-Glutaminase purified from L. gasseri BRLHM Crude L. gasseri prepared by [8], and L-glutaminases was prepared according to [9] with a modification, local isolate L. gasseri purified by four steps: ammonium -sulfate precipitation at 80%, dialysis, ion-exchange using Sephadex-G100 and gel filtration chromatography [10], concentrated of protein by the Bradford method [11] at each step was measured. Study the effectiveness of L-Glutaminase purified from L. gasseri BRLHM as antioxidant.
 Determination of antioxidant activity of L-Glutaminase by DPPH: Using the free radical scavenging action of L-Glutaminase was investigated [13]. The L-Glutaminase solution (1.0 mg/ml) was diluted to five different concentrations: 12.5 , 25 , 50 , 100, and 200 mg/ml. Each concentration of L-Glutaminase (10 ) was mixed with ethanol (490) being and added with a (500) DPPH solution [12]. A residual amount of DPPH was calculated based on a reduction in absorbance at 517 nm after 30 minutes of incubation at room temperature. The % inhibition of DPPH was estimated using the equation formula [14], which is shown below.  L. gasseri isolate no.24 was more active in L-Glutaminase production according to the steps of the primary screening for L-Glutaminase production, thus was selected for the following steps of this current study. The selected isolates were characterized by their ability to produce heavy growth L-Glutamine-containing broth medium and had a red or pink color on L-Glutamine broth medium during semi-quantitative screening steps under optimum conditions for L-Glutaminase production, the initial pH (7), and incubation period (48) hr. at 37 o C. The L-Glutaminase activity indicated that the L. gasseri and specific activity reached 5 Unit/mg proteins.

Semi-Quantitative Screening
The L. gasseri BRLHM isolate no.24 isolate was cultured on an L-Glutamine broth medium containing L-Glutamine and monitored during 48 hr. of incubation, resulted from the reaction of ethanolic phenol red due to the variation in ammonia production between bacterial isolates since the active L-Glutaminase producing isolates increased NH3 and intensity of color production which indicated the activity of this L-Glutaminase screening method Figure 2. The 12 L. gasseri BRLHM isolates that grow on L-Glutamine broth during 24 hr. revealed pink color, while the other isolates formed pale yellowish color. The color intensity indicated bacterial activity to hydrolyze L-Glutamine and liberate glutamic acid and ammonia; this can be used as an indicator to produce Lglutamine. L .gasseri BRLHM isolate no.24 isolate was selected for further steps of this current study. This color change is due to changes in the pH of the medium, as L-Glutaminase causes the breakdown of the amide bond in L-Glutamine and liberates ammonia the L-Glutaminase activity present in Lactobacillus. Lactobacillus screening is based on the qualitative approach that Gulati et al., [20] identified. The change of color of the medium from yellow to pink is an indicator of the extracellular production of L-Glutaminase. This color shift is attributable to a change in the pH of the medium. Phenol red is yellow at acidic pH and turns pink at alkaline pH, thereby forming a pink zone around the bacterial colonies. The findings were those in agreement with (Savitha et al., [21].

L-glutaminases Purification
Dialysis, Ion exchange technique using Sephadex-G100 column, with 80% ammonium sulfate precipitation saturation have been used to L-glutaminases purification was eluted using a linear gradient of 0.05-0.1 M NaCl.
The first peak was found between 0.05 and 0.1 M, while the second peak was L-glutaminases, which eluted in 0.4-0.5 M NaCl. L-glutaminases that eluted in 5-10% of 1 M NaCl, and L-glutaminases that eluted in 40-50% of 1 M NaCl, are the first and second peaks, respectively. In ion-exchange chromatography, the specific activity of L-glutaminases was 14.7U/mg protein, with 58.8% yield and 4.6 purification folds. figure. 3. In the gel filtration the specific activity of L-glutaminase was 23.4 U/mg protein, with a yield of 54 percent and 4.6 purification folds. figure 4. This study introduced a new L-glutaminase which belonged to the l-glutaminases group and was termed L-glutaminase BRLHM because it differed from other L-glutaminase identified by lactobacillus. Purification of L-Glutaminase was critical for gaining a better knowledge of the enzyme's function [15].

Antioxidant activity of L-Glutaminase by using DPPH
The results of my study of L-Glutaminase purified from L. gasseri BRLHM isolate showed the DPPH root was displaced immediately proportional to the increase in concentration. The scavenged effect is assigned a concentration of (12.5,25,50,100, and 200) g/ml, as shown in figure 5. P-value test ascorbic acid was < 0.0012 at concentration 200 μg/ml. while the P-value test for purified L-Glutaminase was < 0.0001 at a concentration of 200 μg/ml. The IC50 value of control and L-Glutaminase purified from L. gasseri BRLHM isolate are (36.09± 1.12 and 619.8 µg/ml respectively. For DPPH radicals, the IC50 values were determined to be 100 and 200 g/ml, respectively. as indicated in table (1). The intact cells of L-Glutaminase purified L. gasseri isolate displayed powerful antioxidant activity in vitro, which was like the antioxidant activity of L .plantarum by [16]. These findings backed with confirmed of [17], who found that proteins isolated from Bifidobacterium animalis cells have antioxidant activity in vitro. One of the most often utilized tests is the scavenged effect test. Pure L-Glutaminases and a standardized antioxidant molecule are used to demonstrate the DPPH radical scavenging capability of purified L-Glutaminases. When antioxidant compounds returned an electron and a proton, the hue shifted to yellow [14]. Table 1. IC50 value for antioxidant activity of L-Glutaminase Glutamine can influence redox homeostasis, bioenergetics, nitrogen balance, and other processes, being a precursor to glutathione, the most important non-enzymatic cellular antioxidant, and it is convert glutamine to glutamate, which is then converted to alpha-ketoglutarate for further metabolism [18]. L-Glutaminase utilized scavenge free radicals generated in vitro by donating their H, which is the most widely used technique for evaluating the antioxidant capacity of pure substances. As a result, discovering antioxidants from natural sources is becoming increasingly popular [19].

Conclusion
My study demonstrated for the first time that the high-level of L-Glutaminase was purified L. gasseri BRLHM to have antioxidant effects. This could be a promising discovery for future radical scavenging therapies and antioxidant prophylaxis with natural probiotics.