Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.
Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of β-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16 fused NHase in the N-terminus of β-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of β-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50°C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Yi
Y
Liu
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Wenjing
W
Cui
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Zhongmei
Z
Liu
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Youtian
Y
Cui
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Yuanyuan
Y
Xia
Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Michihiko
M
Kobayashi
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China. Electronic address: zhmzhou@jiangnan.edu.cn.
Zhemin
Z
Zhou
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D001426:Bacterial Proteins; D010455:Peptides; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006836:Hydro-Lyases; C048831:nitrile hydratase
Active inclusion body; Nitrile hydratase; Product tolerance; Self-assembling peptide; Thermo-stability