A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

Abstract

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0–6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

“…1,2 This enzyme displays great potential for a wide range of applications in the food and pharmaceutical industries, such as reducing the formation of carcinogenic acrylamide in fried foods and treating acute lymphoblastic leukemia (ALL). 3 Due to the short half-life, commercial L-ASNase coming from Escherichia coli (EcA) and Erwinia chrysanthemi (ErA) have a high-dose-dependent disadvantage for ALL treatment, 4 causing severe toxic effects on patients. 5 In addition, the low thermal stability of L-ASNase hinders acrylamide mitigation efficiency in thermally processed food, 6,7 which also increases the risk of microbial contamination and overall production costs.…”

“…l -Asparaginase ( l -ASNase, amidohydrolase enzyme, EC3.5.1.1) can catalyze the production of non-essential amino acid l -asparagine (L-ASN) to l -aspartic acid (L-ASP) and ammonia. , This enzyme displays great potential for a wide range of applications in the food and pharmaceutical industries, such as reducing the formation of carcinogenic acrylamide in fried foods and treating acute lymphoblastic leukemia (ALL) . Due to the short half-life, commercial l -ASNase coming from Escherichia coli (EcA) and Erwinia chrysanthemi (ErA) have a high-dose-dependent disadvantage for ALL treatment, causing severe toxic effects on patients .…”

Highly Active Thermophilic L-Asparaginase from Melioribacter roseus Represents a Novel Large Group of Type II Bacterial L-Asparaginases from Chlorobi-Ignavibacteriae-Bacteroidetes Clade

“…It appears that adaptation at high environmental temperatures involves an increase in K M and k kat for thermophilic L-ASNases [53]. This characteristic feature allows optimiz-ing catalytic efficiency by reaching a balance between substrate binding and the rate of product release.…”

Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions

“…The same doubt was expressed about the type I enzyme of archaea from Pyrococcus horikoshi , which acts as a dimer rather than a tetramer [ 33 ]. L-ASNases of some extremophile bacterial organisms [ 61 ], for example, Thermus thermophilus [ 62 ], Thermococus sibiricus [ 63 ] or Melioribacter roseus [ 64 ], can act as hexamers (trimers of dimers), but there is no structural evidence to support this hypothesis. It is believed that the dimer of L-ASNase II, which has two active centers, is not able to cleave L-asparagine.…”

“…Zuo et al [ 152 ] reported the cloning and expression of a new thermostable L-ASNase from Thermococcus zilligii that showed maximum activity at pH 8.5 and a temperature of 90 °C, the highest ever observed. A later L-ASNase from Thermococus sibiricus demonstrated similar thermal stability and was 86% active after 20 min of incubation at 90 °C [ 63 ].…”