Recombinant<scp>l</scp>-glutaminase obtained from<i>Geobacillus thermodenitrificans</i>DSM-465: characterization and<i>in silico</i>elucidation of conserved structural domains

Shah, Luqman Ali; Nadeem, Muhammad Shahid; Khan, Jalaluddin A.; Zeyadi, Mustafa; Zamzami, Mazin A.; Mohammed, Kaleemuddin

doi:10.1039/c8ra04740e

Cited by 3 publications

(3 citation statements)

References 40 publications

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“…The kinetics parameters of BA glutaminase were compared with available data of characterized L-glutaminases calculated upon similar conditions and using information deposited in BRENDA database [40] (Supplementary Table S1). The K m of BA glutaminase for the substrate L-glutamine (1.1 mM) is located in the lower range of K m reported for bacterial L-glutaminases up to date, whose values range between 0.104 and 109.5 mM for the enzymes from G. thermodenitrificans [42] and Anabaena sp. [43], respectively.…”

Section: Purification and Characterizationmentioning

confidence: 62%

“…PCC 7120 and Bacillus sp./G. thermodenitrificans, respectively [15,42,43] (Supplementary Table S1). Compared to mesophilic counterparts, the L-glutamine glutaminase from Aspergillus oryzae, whose optimum temperature is 30 °C, retained only 40% of maximum activity at 25 °C [47], whereas the L-glutamine glutaminase from E. coli showed low level of activity at 4 °C [48].…”

Section: Effect Of Temperature On Enzyme Activity and Stabilitymentioning

confidence: 99%

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Characteristics of a Cold-Adapted L-glutaminase with Potential Applications in the Food Industry

Ferreira

Herrmann-Andrade

Binolfi

et al. 2021

Appl Biochem Biotechnol

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L-glutaminases are enzymes that catalyze the hydrolysis of L-glutamine, producing L-glutamate and ammonium, and they have promising applications in pharmaceutical and food industries. Several investigations have focused on thermotolerant L-glutaminases; however, studies on cold-adapted L-glutaminases have not been reported. These enzymes could be useful in the food industry because they display high catalytic activity at low and room temperatures, a valuable feature in processes aimed to save energy. Besides, they can be easily inactivated by warming and are suitable to prevent decomposition of thermo-labile compounds. The objectives of this work were to characterize the L-glutaminase from the Antarctic bacterium Bizionia argentinensis and analyze its capability as flavor enhancer of protein hydrolysates. The enzyme was heterologously expressed and purified from Escherichia coli, obtaining optimum and homogeneous yields. Kinetic parameters K m and V max were located at the lower and upper range of values reported for L-glutaminases, suggesting high catalytic efficiency. Optimum temperature was 25 °C, and the enzyme conserved around 90% of maximum activity at 0 °C and in presence of 15% (v/v) ethanol and methanol. In saline conditions, the enzyme conserved around 80% of maximum activity in 3 M NaCl. Analysis of structural model suggested cold-adaptation features such as low Arg/ (Arg+Lys) ratio and fewer intramolecular interactions than mesophilic and thermo-tolerant L-glutaminases. This work provides a novel cold-adapted L-glutaminase with promising features in the food industry.

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Section: Purification and Characterizationmentioning

confidence: 62%

Section: Effect Of Temperature On Enzyme Activity and Stabilitymentioning

confidence: 99%

Characteristics of a Cold-Adapted L-glutaminase with Potential Applications in the Food Industry

Ferreira

Herrmann-Andrade

Binolfi

et al. 2021

Appl Biochem Biotechnol

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“…One unit was defined as the amount of enzyme that can release 1 µmole of ammonia per minute under above assay conditions. Glutaminase activity of enzyme was determined by the procedure adopted from the recent literature (Shah et al, 2019).…”

Section: Measurement Of Enzyme Activity and Protein Contentmentioning

confidence: 99%

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

et al. 2022

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L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G – 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

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Molecular characterization and computational analysis of a highly specific L-glutaminase from a marine bacterium Bacillus australimaris NIOT30

Thenmozhi Kulasekaran,

Vanlalrovi,

Subramanian

et al. 2024

Sci Rep

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An alkaline active L-glutaminase (BALG) producing bacterium was screened and identified from seamount sediment samples of the Arabian Sea. The isolate was confirmed to be Bacillus australimaris NIOT30 based on morphological characteristics and 16 S rRNA gene sequencing. The glutaminase gene, balg was PCR amplified, cloned and expressed in E. coli BL21 (DE3) host. The molecular weight of purified BALG was estimated to be 36 kDa and the enzyme showed a specific activity of 507 ± 27 Umg −1 against L-glutamine under optimal assay conditions of pH 7.0 and temperature at 37 °C for 15 min. The enzyme showed maximum activity at pH 7 and retained 95% activity at pH 10. BALG retained a relative activity of about 82% and 45% at 45 °C and 60 °C respectively. The kinetic parameters of BALG, Km and Kcat/Km were determined to be of 210 ± 11 mM and 4.4 × 10 2 M s −1 respectively. Homology modeling and substrate ligand interaction studies revealed the stability of the enzyme-substrate complex. The present study highlights the characterization of a highly active L-glutaminase from B. australimaris NIOT30. Further, mutational analyses of ligand binding residues would show insights into the affinity of L-Glutaminase.

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

Abstract: Glutaminase (GLS) is an enzyme essential for amino acid metabolism; in particular, it acts as a catalyst in glutaminolysis, a reaction exploited by the malignant cells to meet the nutrient requirements for their accelerated growth and proliferation.

Cited by 3 publications

References 40 publications

Characteristics of a Cold-Adapted L-glutaminase with Potential Applications in the Food Industry

Characteristics of a Cold-Adapted L-glutaminase with Potential Applications in the Food Industry

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

Molecular characterization and computational analysis of a highly specific L-glutaminase from a marine bacterium Bacillus australimaris NIOT30

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