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

Samaneh Mosallatpour,Reza Hajihosseini,Mehdi Shamsara,Saeed Aminzadeh

doi:10.1038/s41598-019-55587-9

Samaneh Mosallatpour, Reza Hajihosseini + Show 2 more

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https://doi.org/10.1038/s41598-019-55587-9

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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.

Highlights

L-glutaminase importance to use in the food industry and medicine has attracted much attention
The single band of rSAM after purification with molecular weight of 34 kDa was shown in Fig. 1B lane 8 and the total cellular expressed protein from E. coli BL21 (DE3) was shown in Fig. 1B lane 9
Enzyme qualitative assay. rSAM qualitative assay on Agar-phenol red plate which had L-glutamine as enzyme substrate showed changing in medium color surrounding the enzyme-containing well from yellow to red (Supplementary Fig S1A-well 3)

Summary

Introduction

L-glutaminase importance to use in the food industry and medicine has attracted much attention. Presence of high amounts of salt (14–20%) is a common condition in the soy sauce fermentation L-glutaminases which are capable to withstand low pH, high temperature and the presence of NaCl and ethanol are preferred to use for soy sauce production[12,13]. To tackle these problems and due to the importance of the enzymes with appreciable activity in harsh conditions which is the scientific world concerns, in the present research after heterologous expression of L-glutaminase from an indigenous thermophilic bacteria A01 PTCC No 1921) and full activity and structural characterization under different conditions, rSAM was found to have high resistance and rigid structure

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