Darnal Sanyukta, Patial Vijeta, Kumar Virender, Kumar Subhash, Kumar Vijay, Padwad Yogendra S, Singh Dharam
Molecular and Microbial Genetics Lab, Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, 176 061, India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201 002, India.
AMB Express. 2023 Feb 24;13(1):22. doi: 10.1186/s13568-023-01521-2.
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 and V 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 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.
微生物来源的L-天冬酰胺酶（L-ASNase）是治疗急性淋巴细胞白血病的一种具有重要商业价值的酶。然而，L-ASNase商业制剂所伴随的副作用促使人们去探索具有高效且理想药理酶学特性的产品。在此，我们报告了从喜马拉雅桦木（Betula utilis）土壤中分离出的假单胞菌属（Pseudomonas sp.）PCH199周质L-ASNase的生化及细胞毒性评估。使用响应面法（RSM）使野生型PCH199的L-ASNase产量提高了2.2倍。采用优化的渗透休克法提高了周质L-ASNase的产量，随后对其进行纯化。纯化后的L-ASNase是一种37.0 kDa的单体，在pH 8.5和60℃时具有最佳活性。它还表现出热稳定性，在37℃和50℃时分别保留100.0%（200分钟）和90.0%（70分钟）的活性。纯化酶的K和V值分别为0.164±0.009 mM和54.78±0.4 U/mg。L-ASNase对K562血癌细胞系具有细胞毒性（IC值为0.309 U/mL），在24小时内导致凋亡核形态变化，这通过DAPI染色检测得到。因此，PCH199 L-ASNase在广泛的pH和温度范围内具有动态功能，且在37℃具有稳定性和细胞毒性潜力，这在治疗应用中被证明具有重要的药学意义。
