PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF MICROBIAL L-GLUTAMINASE

Abstract

A significant protein L-glutaminases discovers potential applications in various divisions running from the nourishment industry to remedy and cure. L-glutaminase is the principal enzyme that changes L-glutamine to L-glutamate and is generally distributed in bacteria, actinomycetes, fungi, and higher organisms. The present study was focused on the isolation and characterization of glutaminase from microbial sources. Different bacterial and fungal strains were isolated. One bacterial strain RSHG1 was isolated from an expired glutamine sample, while 4bacterial strains were isolated from different soil samples. One endophytic fungal strain Epicocum sp. NFW1 previously isolated was also included in the study. Selected bacterial strains were identified as Achromobacter xylosoxidans RSHG1, Bacillus subtilis RSHU1, Bacillus halotolerance RSHQ1, Stenotrophomonas maltophilia RSHU3, and Alcaligenes sp. RSHS3 by 16S rDNA sequence analyses. All the bacterial strains were processed for the production of glutaminase, and different parameters were optimized. Results indicated that A. xylosoxidans RSHG1 showed maximum production of L-glutaminase at pH 9, 30˚C, sorbitol, and glutamine as carbon source and nitrogen source, respectively. The optimum pH for glutaminase production from other bacterial strains was between 6-8. Alcaligenes sp. RSHS3 achieved the highest L-glutaminase production at 25˚C, A xylosoxidans RSHG1, and S. maltophilia RSHU3, at 30˚C while for B. subtilis RSHU1 and B. halotolerans RSHQ1 optimum temperature was 37˚C. Sorbitol was the best carbon source for L-glutaminase production for S. maltophilia RSHU3 while B. subtilis RSHU1, and Alcaligenes sp. RSHS3 and B. halotolerans RSHQ1 showed maximum production with glucose sucrose as a carbon source, respectively. L-Glutamine was the best nitrogen source for Lglutaminase production by A. xylosoxidans RSHG1 and B. subtilis RSHU1, and 1.5 % glutamine was inducer for glutaminase production by all the strains. Optimization of enzyme production for fungal strain Epicoccum sp. NFW1 showed the best production at pH 7, 30˚C, sucrose as carbon source and glutamine as nitrogen source and inducer. Enzymes from all the bacterial strains and fungal isolate were purified using ammonium sulfate precipitation and size exclusion column chromatography. Further purified enzyme (B. subtilis RSHU1, A xylosoxidans RSHG1, and Epicoccum sp. NFW1) and partially purified enzyme (B. halotolerans RSHQ1, S. maltophilia RSHU3, and Alcaligenes sp. RSHS3) were characterized for their kinetic behavior. L-Glutaminase xv from B. subtilis RSHU1 showed the highest Vmax 522U/mg among all the selected strains and best affinity for its substrate with Km of 0.0591mM. While Vmax of other enzymes from A. xylosoxidans RSHG1, B. halotolerans RSHQ1, S. maltophilia RSHU3, Alcaligenes sp. RSHS3, and Epicoccum sp. NFW1 was 443U/mg, 343.6 U/mg, 351 U/mg, 232 U/mg, and 450 U/mg, respectively. The L-glutaminases from A xylosoxidans RSHG1, B. halotolerans RSHQ1, S. maltophilia RSHU3, Alcaligenes sp. RSHS3, and Epicoccum sp. NFW1 showed Km values of 0.235mM, 0.37mM, 0.166mM, 0.186mM, and 0.424mM, respectively. Optimum pH L-glutaminase produced by A. xylosoxidans RSHG1, Alcaligenes sp. RSHS3, and Epicoccum sp. NFW1 was 7 while the enzyme from B. subtilis RSHU1 and B. halotolerans RSHQ1 were active at pH 6-6.5, and optimum pH8 was observed for the enzyme from S. maltophilia. At temperature 40˚C, the best activities were recorded for L-glutaminase produced by all the strains except for B. halotolerans RSHQ1, which showed the best activity at 50˚C. All the L-glutaminases were inhibited by EDTA, MnSO4, FeSO4 while A. xylosoxidans RSHG1, B. subtilis RSHU1, and Epicoccum sp. NFW1 were also inhibited by HgCl and activated by 20 mM CoCl2, CaCl2, BaCl2, ZnSO4, KCl, MgSO4, and NaCl. B. halotolerans RSHQ1 L-glutaminase was also inhibited by CaCl2, and increased activities were observed in the presence of CoCl2, BaCl2, ZnSO4, KCl, MgSO4, and NaCl. S. maltophilia RSHU3 L-glutaminase was additionally inhibited by MgSO4, and raised activities were observed by CoCl2, BaCl2, KCl, ZnSO4, and NaCl. Alcaligenes sp. RSHS3 L-glutaminase activity was additionally decreased by ZnSO4 and improved by CoCl2, BaCl2, KCl, MgSO4, and NaCl is shown in Figures 4.54, 4.69, 5.8. A. xylosoxidans RSHG1 L-glutaminase activity was enhanced 64.4% by 8% NaCl, S. maltophilia RSHU3 and Alcaligenes sp. RSHS3 using 12% NaCl with a rise of 32 % and 44 %, respectively, and B. subtilis RSHU1, B. halotolerans RSHQ1, and Epicoccum sp. NFW1 L-glutaminase activity enhanced by 16 % NaCl 65%, 53%, and 64.9% (Figure 5.9). The molecular weight of A. xylosoxidans RSHG1, B. subtilis RSHU1, and Epicoccum sp. NFW1 L-glutaminases were 40, 60, and 38KDa approximately (Figure 4.71 & 4.72). Partially purified L-glutaminase produced by A. xylosoxidans RSHG1, B. subtilis RSHU1, and Epicoccum sp. NFW1 L-glutaminases were immobilized on 3.6% agar. The immobilized L-glutaminase showed stability up to 3 weeks. L-Glutaminase, produced by local indigenous bacterial strain A. xylosoxidans RSHG1, was stable at xvi wide pH and temperature conditions, with a high substrate affinity. Salt-tolerant bacteria may produce halophilic L-glutaminases, which can be used in the food industry and tolerate high salt concentrations. The bacterial and fungal strains produced relatively stable L-glutaminase, which have great potential to be used in the food and pharmaceutical industry, particularly as an antileukemic agent and as biosensors. Keywords: Achromobacter xylosoxidans RSHG1; B. subtilis RSHU1; Characterization; Epicoccum sp NFW1; L Glutaminase production; Molecular identification; Optimization; Purification