Optimization and purification of anticancer enzyme L-glutaminase from Alcaligenes faecalis KLU102

Abstract

L-Glutaminase, an amidohydrolase, is gaining importance on account of its potential anticancer activity. L-Glutaminase produced by Alcaligenes faecalis KLU102, isolated from the marine realm (Bay of Bengal), exhibits potential anticancer activity. Response surface methodology was employed for optimizing the medium composition. The concentrations of the various constituents were as follows: arabinose (2%), skim milk (4%), and salts viz. K2HPO4, KH2PO4, MgSO4, and NaCl (2%). The bacterium grown in the optimized medium yielded an enzyme activity of 1.34 ± 0.07 IU/mg in shake-flask cultures and this doubled (2.77 ± 0.35 IU/mg) when scale-up studies were conducted using a 3-L fermenter. The enzyme was purified to homogeneity using ion-exchange chromatography, and the purified enzyme was found to have a specific activity of 54.72 IU/mg, with a molecular weight of 37 kDa. Immobilization of the enzyme on PEG-PHB nanoparticles improved the stability of the enzyme significantly. Purified L-glutaminase exhibited cytotoxic activity against HeLa cells, as assayed by the MTT assay, with an IC50 value of 12.5 µg/mL.

Studies on optimization of L-glutaminase production under submerged fermentation from marine Bacillus subtilis JK-79

“…Suresh et al (2013) reported optimization of medium components through OFAT approach and FCCCD for the submerged production of L-glutaminase by Serratia marcescens. Pandian et al (2014) reported the statistical optimization of medium composition for the production of glutaminase from Alcaligenes faecalis KLU102. RSM was used for optimization and the bacterium grown in the optimized medium [arabinose (2%), skim milk (4%) and sodium chloride (2%)] yielded L-glutaminase activity of 1.34 IU/mg.…”

“…Supplementation of sucrose and yeast extract as carbon and nitrogen source improved Lglutaminase production by Zygosaccharomyces rouxii while higher L-glutaminase production was noticed in Providencia species with glucose and urea as carbon and nitrogen source, respectively. Recently, there are several reports which confirm the fact that the carbon and nitrogen sources significantly affect the production of Lglutaminase by microorganisms (Sathish and Prakasham, 2013;Krishnakumar et al, 2011;Pandian et al, 2014;Jesuraj et al, 2017).…”

“…In recent years, cancer therapy is highly relied on enzymes as they are low molecular weight protein molecules specific in their action and have less or no toxic effects. The enzymatic approach was reported to be more promising cancer therapy, due to the discovery of several enzymes with effective anti-cancer activity on various types of cancer (Vellard, 2003;Pandian et al, 2014).…”

Recombinantl-glutaminase obtained fromGeobacillus thermodenitrificansDSM-465: characterization andin silicoelucidation of conserved structural domains

“…The activity of glutaminase was determined by the modied Nesslerization method adopted from the literature. 7 Briey, 2800 mL reaction mixture contained 3.5 mM of glutamine solution prepared in 50 mM Tris-HCl buffer, pH 9.0. To this mixture, 100 mL (100 mg) of the enzyme was added, and the mixture was incubated for 10 min at 70 C. The reaction was terminated by the addition of 100 mL of 500 mM trichloroacetic acid aer the incubation period.…”

“…5,6 Thus, Lglutaminase is an attractive target to block the energy route for the proliferation of glutamine-dependent tumour cells and responsible for their selective death. 7,8 Several other applications of the enzyme have been reported due to its effective antiretroviral activity; 9 moreover, the enzyme is used to improve the taste and aroma of the food products. 10 L-Glutaminase also has a signicant application in biosensors where it is used to monitor the level of glutamine in mammalian and hybridoma cells.…”