Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

Abstract

L-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). Km, Vmax, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg2+, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.

Expression, characterization, and activity optimization of a novel cellulase from the thermophilic bacteria Cohnella sp. A01

“…Parameters of the irreversible thermodynamic can be determined by the transition state (T) calculation of protein. It is assumed that irreversible denaturation of proteins is a two-step reaction: N ↔ U → I, that the transition state refers to a protein formed between N (native state) and U (reversible/partially unfolded state), or I (irreversible/inactivated state) 63 . A key factor in understanding an enzyme’s thermal capacity is calculating the activation energy needed for an enzyme’s irreversible thermal inactivation.…”

Enhanced salt-tolerance of Bacillus subtilis glutaminase by fusing self-assembling amphipathic peptides at its N-terminus

“…Glutaminase catalyzes the production of L-glutamic acid from L-glutamine, which could improve the umami taste of soy sauce. Due to the high salt content (15–18% NaCl) required by soy sauce fermentation, many studies focused on the screening of salt-tolerant glutaminases from bacteria ( Wakayama et al, 2005 ; Kumar et al, 2012 ; Mosallatpour et al, 2019 ; Ferreira et al, 2021 ) and fungi ( Sato et al, 1999 ; Masuo et al, 2005 ; Elshafei et al, 2014 ). However, there are very few studies on the sat-tolerance enhancement of glutaminase via molecular modification.…”

“…LKG-01 ( Kumar et al, 2012 ), Bacillus amyloliquefaciens ( Ye et al, 2013 ), Cohnella sp. A01 ( Mosallatpour et al, 2019 ), Bizionia argentinensis ( Ferreira et al, 2021 ). Among them, the Cohnella glutaminase retained 90% of the catalytic activity in a 25% NaCl solution ( Mosallatpour et al, 2019 ).…”

“…A01 ( Mosallatpour et al, 2019 ), Bizionia argentinensis ( Ferreira et al, 2021 ). Among them, the Cohnella glutaminase retained 90% of the catalytic activity in a 25% NaCl solution ( Mosallatpour et al, 2019 ). Moreover, there are many reports on the salt-resistant glutaminases from fungi, such as Cryptococcus nodaensis ( Sato et al, 1999 ), A. oryzae RIB40 ( Masuo et al, 2005 ), and Penicillium brevicompactum NRC 829 ( Elshafei et al, 2014 ).…”

Production of L-glutaminase From Agro-industrial Waste by Halomonas Elongata MM-5 Under Solid State Fermentation

“…L-glutaminase (EC. 3.5.1.2) is an important enzyme that catalyzes the hydrolysis of the amide bond in the side chain of L-glutamine and produces L-glutamic acid and ammonia and is present in nearly all organisms (Mosallatpour et al 2019). L-glutaminase is the key enzyme in the food industry which is responsible for producing umami taste in several oriental fermented foods like soy sauce, sufu, miso (Nandakumar et al 2003).…”