Purification and kinetic characterisation of extracellular glutaminase from Priestia aryabhattai B8W22 with deamidation activity on wheat gluten

Abstract

Importance of the work: Glutaminase, an enzyme catalyzing the conversion of glutamine to
glutamate and ammonia, has garnered considerable interest due to its role in enhancing the physical
and functional properties of proteins in food processing industries.
Objectives: To isolate and characterize an extracellular glutaminase from strain B1, evaluate its
enzymatic properties and assess its effect on improving the functionality of gluten proteins.
Materials and Methods: An extracellular glutaminase was isolated from a bacterial strain
(designated B1), based on 16S rRNA gene sequencing and showed 97% homology to the strain
Priestia aryabhattai B8W22. Screening was carried out for influential factors for glutaminase
production (incubation time, temperature, pH, carbon and nitrogen sources, concentration of
glutamine). Partial glutaminase purification using ammonium sulfate was performed based on 10 kDa
molecular weight cut-off (MWCO) dialysis followed by 3 kDa MWCO concentration. The molecular
weight was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Glutaminase activity and kinetic parameters (Km and Vmax) were measured under various
temperature and pH conditions. The glutaminase’s deamidation ability was evaluated by treating
wheat gluten and using Fourier-transform infrared spectroscopy (FTIR) analysis, followed by analysis
of the functional properties (solubility, foaming capacity, water-holding and oil-holding capacities).
Results: Glutaminase displayed maximal activity ± SE of 161.571 ± 0.704 U/mL. Optimal catalysis
occurred at 40°C and pH 7, with a Km value of 0.9611 mM and a Vmax value of 193.3 U/mL.
The molecular weight of the partially purified glutaminase was approximately 60 kDa, based on
SDS-PAGE. Based on the FTIR results, there was a substantial influence of glutaminase in all the
gluten samples. Compared to the untreated controls, the wheat gluten treated with partially purified
glutaminase had significantly enhanced solubility (33.4-fold), foaming capacity (163.2-fold),
water-holding capacity (3.1-fold) and oil-holding capacity (28.0-fold), outperforming conventional
chemical treatments.
Main finding: Partial purified glutaminase was an effective biocatalyst for wheat gluten modification,
offering promising applications to improve protein functionality in food processing industries.