Rational Design of a Thermostable 2′-Deoxyribosyltransferase for Nelarabine Production by Prediction of Disulfide Bond Engineering Sites

Abstract

One of the major drawbacks of the industrial implementation of enzymatic processes is the low operational stability of the enzymes under tough industrial conditions. In this respect, the use of thermostable enzymes in the industry is gaining ground during the last decades. Herein, we report a structure-guided approach for the development of novel and thermostable 2′-deoxyribosyltransferases (NDTs) based on the computational design of disulfide bonds on hot spot positions. To this end, a small library of NDT variants from Lactobacillus delbrueckii (LdNDT) with introduced cysteine pairs was created. Among them, LdNDTS104C (100% retained activity) was chosen as the most thermostable variant, displaying a six- and two-fold enhanced long-term stability when stored at 55 °C (t1/255 °C ≈ 24 h) and 60 °C (t1/260 °C ≈ 4 h), respectively. Moreover, the biochemical characterization revealed that LdNDTS104C showed >60% relative activity across a broad range of temperature (30–90 °C) and pH (5–7). Finally, to study the potential application of LdNDTS104C as an industrial catalyst, the enzymatic synthesis of nelarabine was successfully carried out under different substrate conditions (1:1 and 3:1) at different reaction times. Under these experimental conditions, the production of nelarabine was increased up to 2.8-fold (72% conversion) compared with wild-type LdNDT.

Thermostable enzyme research advances: a bibliometric analysis

“…Biochemistry, genetics and molecular biology Study on the isolation and characterization [42][43][44][45], expression and purification [46][47][48][49][50], gene cloning, structural function analysis, improving catalytic efficiency, site-directed mutagenesis, protein crystallization, computational simulations, rational engineering to improve enzymes activity [51][52][53][54][55][56] preservation of enzymes, indigenous thermophilic exploration, cell-free enzymatic, polymerase synthesis, marine thermophiles, enzymatic purification and biodegradation [57][58][59]. The most important enzymes: Cellulose, xylanase, lipase and amylase.…”

Enhanced Activity and Stability of an Acetyl Xylan Esterase in Hydrophilic Alcohols through Site-Directed Mutagenesis

“…Directed evolution and rational design are the widely applied protein engineering strategies generating enzyme variants with modified catalytic or physiological properties [9][10][11]. In a rational design approach, the structure-function link of the target enzyme is exploited in formulating hypotheses, identifying hotspots for mutagenesis such as flexible regions, specific modification of the enzyme surface, or the modification of the catalytic site, thereby generating the desired genetic diversity [12][13][14]. For example, improved stability of a lipase from Geobacillus stearothermophilus in methanol was achieved by a rational design incorporating bulky aromatic residues to occupy solvent channels and induce aromatic interaction.…”