Biochemical characterization of extremozyme L-asparaginase from Pseudomonas sp. PCH199 for therapeutics

Abstract

L-asparaginase (L-ASNase) from microbial sources is a commercially vital enzyme to treat acute lymphoblastic leukemia. However, the side effects associated with the commercial formulations of L-ASNases intrigued to explore for efficient and desired pharmacological enzymatic features. Here, we report the biochemical and cytotoxic evaluation of periplasmic L-ASNase of Pseudomonas sp. PCH199 isolated from the soil of Betula utilis, the Himalayan birch. L-ASNase production from wild-type PCH199 was enhanced by 2.2-fold using the Response Surface Methodology (RSM). Increased production of periplasmic L-ASNase was obtained using an optimized osmotic shock method followed by its purification. The purified L-ASNase was a monomer of 37.0 kDa with optimum activity at pH 8.5 and 60 ℃. It also showed thermostability retaining 100.0% (200 min) and 90.0% (70 min) of the activity at 37 and 50 ℃, respectively. The K_m and V_max values of the purified enzyme were 0.164 ± 0.009 mM and 54.78 ± 0.4 U/mg, respectively. L-ASNase was cytotoxic to the K562 blood cancer cell line (IC₅₀ value 0.309 U/mL) within 24 h resulting in apoptotic nuclear morphological changes as examined by DAPI staining. Therefore, the dynamic functionality in a wide range of pH and temperature and stability of PCH199 L-ASNase at 37 ℃ with cytotoxic potential proves to be pharmaceutically important for therapeutic application.

Keywords: Cytotoxicity; Himalayan niche; Osmotic shock method; Periplasmic L-asparaginase; Protein purification.

Fig. 1

Graphical representation of periplasmic L-ASNase activity when PCH199 was cultured at different temperatures (4, 15, 20, and 28 ℃). PCH199 was grown at different temperatures and periplasmic L-ASNase activity was measured at different time intervals from the culture of PCH199 corresponding to each temperature

Fig. 2

Statistical optimization of production medium using RSM. Three-dimensional response surface plots showing the interactive effects of selective variable on L-ASNase activity, a L-asparagine (%, w/v) and buffer concentration (mM), b glucose (%, v/v) and buffer concentration (mM) and c L-asparagine (%, w/v), and glucose concentration (%, v/v)

Fig. 3

SDS-PAGE analysis of purified PCH199 L-ASNase. Proteins were separated on 10% SDS-PAGE and stained with silver stain. Lane 1 (C), cell-free periplasmic extract; Lane 2 (AS), ammonium sulfate precipitation extract; Lane 3 (FT), flow-through; Lane 4 and 5 (E1, E2), eluted fractions of anion-exchange chromatography; Lane 6 and 7 (E3, E4), eluted fractions of size-exclusion chromatography; Lane 8 (M), protein molecular weight marker (kDa). Arrow indicates homogenous purified protein

Fig. 4

Effect of different physicochemical parameters on PCH199 L-ASNase activity. Effect of a different buffer pH, b incubation temperature, c thermal stability at different temperatures (37, 50, 60, and 70 ℃) and d metal ions and protein modifying agents (1.0 mM) on purified PCH199 L-ASNase activity

Fig. 5

Graphical representation of kinetic study of PCH199 L-ASNase. Determination of K_m and V_max of purified L-ASNase for L-asparagine by non-linear regression analysis of experimental steady-state data. a Plot of the reaction velocities (V) versus substrate concentration (S: 0.1– 3.0 mM) fitted to the Michaelis–Menten equation. b The corresponding Lineweaver–Burk plot (K_m = 0.164 mM and V_max = 54.78 U/mg) of L-ASNase catalyzed reaction

Fig. 6

Cytotoxicity evaluation of purified L-ASNase against cancer cell lines. a Cytotoxic effect of purified L-ASNase from PCH199. b E. coli L-ASNase (Elspar) on K562 blood cancer cell line using MTT assay. c The cytotoxic effect of L-ASNase was also tested towards normal cancer cell line IEC-6. d DAPI staining. Treated cells stained with DAPI clearly showed DNA fragmentation. Control cells showed uniform, rounded nuclei. L-ASNase-C indicates E. coli L-ASNase