Dastmalchi Mahrokh, Hamzeh-Mivehroud Maryam, Rezazadeh Hassan, Farajollahi Mohammad M, Dastmalchi Siavoush
Department of Medical Biotechnology, Faculty of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran.
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Adv Pharm Bull. 2024 Oct;14(3):675-685. doi: 10.34172/apb.2024.048. Epub 2024 Jun 22.
L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from bacteria with Y176F/S241C mutations.
The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in , and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler's reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme.
The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218 (mM/min), 273.9 (min), 237.8 (IU/mg) and 19.62 (mM min), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively.
The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications.
自20世纪60年代引入以来，L-天冬酰胺酶已被广泛认为是治疗各种类型淋巴增生性疾病的关键成分。然而，在某些情况下使用它会导致过敏反应，使治疗无法继续进行。因此，人们一直考虑从其他来源开发L-天冬酰胺酶或生产工程酶。本研究旨在生产和评估一种基于细菌L-天冬酰胺酶序列设计的新型酶，该酶具有Y176F/S241C突变。
Y176F/S241C突变型L-天冬酰胺酶在大肠杆菌中成功表达为GST融合蛋白，然后进行亲和色谱和尺寸排阻色谱。使用奈斯勒试剂，根据底物水解产生的氨释放量来测定纯化酶的活性。在存在浓度不断增加的氯化胍的情况下进行化学变性实验，以确定纯化酶的折叠稳定性。
突变酶的纯化效率为77倍，但回收率较低，仅为0.7%。测定的动力学参数Km、Vmax、kcat、比活性和催化效率分别为13.96（mM）、2.218（mM/min）、273.9（min）、237.8（IU/mg）和19.62（mM·min）。此外，通过氯化胍诱导变性测定的突变型和市售L-天冬酰胺酶的解折叠自由能分别为8421 J/mol和5274 J/mol。
突变酶显示出比野生型更好的稳定性。尽管表达水平和回收率较低，但突变型L-天冬酰胺酶表现出有前景的活性和稳定性，具有潜在的临床和工业应用价值。
