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Thermostability enhancement and insight of L-asparaginase from Mycobacterium sp. via consensus-guided engineering.

Huibing ChiXiaoyu ZhuJuan ShenZhaoxin LuFengxia LuYunbin LyuPing Zhu
Published in: Applied microbiology and biotechnology (2023)
Acrylamide alleviation in food has represented as a critical issue due to its neurotoxic effect on human health. L-Asparaginase (ASNase, EC 3.5.1.1) is considered a potential additive for acrylamide alleviation in food. However, low thermal stability hinders the application of ASNase in thermal food processing. To obtain highly thermal stable ASNase for its industrial application, a consensus-guided approach combined with site-directed saturation mutation (SSM) was firstly reported to engineer the thermostability of Mycobacterium gordonae L-asparaginase (GmASNase). The key residues Gly97, Asn159, and Glu249 were identified for improving thermostability. The combinatorial triple mutant G97T/N159Y/E249Q (TYQ) displayed significantly superior thermostability with half-life values of 61.65 ± 8.69 min at 50 °C and 5.12 ± 1.66 min at 55 °C, whereas the wild-type was completely inactive at these conditions. Moreover, its T m value increased by 8.59 °C from parent wild-type. Interestingly, TYQ still maintained excellent catalytic efficiency and specific activity. Further molecular dynamics and structure analysis revealed that the additional hydrogen bonds, increased hydrophobic interactions, and favorable electrostatic potential were essential for TYQ being in a more rigid state for thermostability enhancement. These results suggested that our strategy was an efficient engineering approach for improving fundamental properties of GmASNase and offering GmASNase as a potential agent for efficient acrylamide mitigation in food industry. KEY POINTS: • The thermostability of GmASNase was firstly improved by consensus-guided engineering. • The half-life and T m value of triple mutant TYQ were significantly increased. • Insight on improved thermostability of TYQ was revealed by MD and structure analysis.
Keyphrases
  • human health
  • wild type
  • risk assessment
  • molecular dynamics
  • climate change
  • mycobacterium tuberculosis
  • heavy metals
  • clinical practice
  • density functional theory
  • molecular dynamics simulations