TY - JOUR
T1 - Biochemical characterization of extremozyme L-asparaginase from Pseudomonas sp. PCH199 for therapeutics
AU - Darnal, Sanyukta
AU - Patial, Vijeta
AU - Kumar, Virender
AU - Kumar, Subhash
AU - Kumar, Vijay
AU - Padwad, Yogendra S
AU - Singh, Dharam
N1 - © 2023. The Author(s).
PY - 2023/2/24
Y1 - 2023/2/24
N2 - 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 (IC
50 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.
AB - 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 (IC
50 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.
KW - Cytotoxicity
KW - Himalayan niche
KW - Osmotic shock method
KW - Periplasmic L-asparaginase
KW - Protein purification
UR - http://www.scopus.com/inward/record.url?scp=85149108868&partnerID=8YFLogxK
U2 - 10.1186/s13568-023-01521-2
DO - 10.1186/s13568-023-01521-2
M3 - Journal article
C2 - 36828987
SN - 2191-0855
VL - 13
SP - 22
JO - AMB Express
JF - AMB Express
IS - 1
M1 - 22
ER -