Biochemical characterization of glutaminase-free L-asparaginases from Himalayan Pseudomonas and Rahnella spp. for acrylamide mitigation.

L-asparaginase having low glutaminase activity is important in clinical and food applications. Herein, glutaminase-free L-asparaginase (type I) coding genes from Pseudomonas sp. PCH182 (Ps-ASNase I) and Rahnella sp. PCH162 (Rs-ASNase I) was amplified using gene-specific primers, cloned into a pET-47b(+) vector, and plasmids were transformed into Escherichia coli (E. coli). Further, affinity chromatography purified recombinant proteins to homogeneity with monomer sizes of ~37.0 kDa. Purified Ps-ASNase I and Rs-ASNase I were active at wide pHs and temperatures with optimum activity at 50 °C (492 ± 5 U/mg) and 37 °C (308 ± 4 U/mg), respectively. Kinetic constant K and V for L-asparagine (Asn) were 2.7 ± 0.06 mM and 526.31 ± 4.0 U/mg for Ps-ASNase I, and 4.43 ± 1.06 mM and 434.78 ± 4.0 U/mg for Rs-ASNase I. Circular dichroism study revealed 29.3 % and 24.12 % α-helix structures in Ps-ASNase I and Rs-ASNase I, respectively. Upon their evaluation to mitigate acrylamide formation, 43 % and 34 % acrylamide (AA) reduction were achieved after pre-treatment of raw potato slices, consistent with 65 % and 59 % Asn reduction for Ps-ASNase I and Rs-ASNase I, respectively. Current findings suggested the potential of less explored intracellular L-asparaginase in AA mitigation for food safety.