Applied Microbiology and Biotechnology

, Volume 102, Issue 18, pp 7891–7900 | Cite as

Evidence for the participation of an extra α-helix at β-subunit surface in the thermal stability of Co-type nitrile hydratase

  • Xiaolin Pei
  • Jiapao Wang
  • Yifeng Wu
  • Xiaoting Zhen
  • Manman Tang
  • Qiuyan Wang
  • Anming Wang
Biotechnologically relevant enzymes and proteins


Nitrile hydratase (NHase) has attracted considerable attention since it can efficiently catalyze the hydration of nitriles to valuable amides. However, the poor stability of NHase is one of the main drawbacks in the industrial application. In this study, we compared the structural difference between Fe-type and Co-type NHase and found that an extra α helix existed at the β-subunit surface of Co-type NHase (defined as the β-6th helix). Then, the effects of the β-6th helix were investigated on the thermal stability and the catalytic kinetics of a Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). When the β-6th helix was deleted or disrupted, the thermal stability of NHase1229 was reduced to 17.6 and 12.9% of that of wild NHase1229, respectively. Thus, the β-6th helix is important for the thermal stability of Co-type NHase. Based on the structural characteristics of Co-type NHase, the β-6th helix may be interacted with another helix at the α-subunit (defined as the α-2nd helix) by hydrophobic network just as a “magnetic suction buckle” on the enzyme surface to stabilize the binding of α- and β-subunits. The β-6th helix is located at the mouth of the substrate and product tunnel, so it plays crucial roles in catalytic process. Furthermore, the β-6th helix in NHase1229 was swapped with a thermophilic NHase fragment from Pseudonocardia thermophila JCM3095 (NHase1229-Swap). The thermal stability of NHase1229-Swap was significantly improved, and the half-life was approximately 2.4-fold at 40 °C than that of the wild NHase1229. The knowledge is useful for improving the stability of NHases by restriction fragment swapping.


Nitrile hydratase Thermal stability Thermal-sensitive region Fragment swapping Chimeric enzyme 


Funding information

This study was funded by the Natural Science Foundation of Zhejiang Province (No. LY15B060011, LY17C050001, LY18B060009, Q12B06007), the China Postdoctoral Science Foundation Funded Project (No. 2016M601966), the National Natural Science Foundation of China (Nos. 21576062, 21206024), and the Hangzhou Science and Technology Project (Nos. 20130533B17, 20151232I34).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9191_MOESM1_ESM.pdf (989 kb)
ESM 1 (PDF 989 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Material, Chemistry and Chemical EngineeringHangzhou Normal UniversityHangzhouPeople’s Republic of China
  2. 2.Collaborative Innovation Center of Yangtze River Delta Region Green PharmaceuticalsZhejiang University of TechnologyHangzhouPeople’s Republic of China
  3. 3.College of MedicineHangzhou Normal UniversityHangzhouPeople’s Republic of China

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