title: Enhanced thermal stability and hydrolytic ability of Bacillus subtilis aminopeptidase by removing the thermal sensitive domain in the non-catalytic region.
abstract: Besides the catalytic ability, many enzymes contain conserved domains to perform some other physiological functions. However, sometimes these conserved domains were unnecessary or even detrimental to the catalytic process for industrial application of the enzymes. In this study, based on homology modeling and molecular dynamics simulations, we found that Bacillus subtilis aminopeptidase contained a thermal sensitive domain (protease-associated domain) in the non-catalytic region, and predicted that deletion of this flexible domain can enhance the structure stability. This prediction was then verified by the deletion of protease-associated domain from the wild-type enzyme. The thermal stability analysis showed that deletion of this domain improved the T50 (the temperature required to reduce initial activity by 50% in 30 min) of the enzyme from 71 °C to 77 °C. The melting temperature (Tm) of the enzyme also increased, which was measured by thermal denaturation experiments using circular dichroism spectroscopy. Further studies indicated that this deletion did not affect the activity and specificity of the enzyme toward aminoacyl-p-nitroanilines, but improved its hydrolytic ability toward a 12-aa-long peptide (LKRLKRFLKRLK) and soybean protein. These findings suggested the possibility of a simple technique for enzyme modification and the artificial enzyme obtained here was more suitable for the protein hydrolysis in food industry than the wild-type enzyme.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Xinxing
authors.initials: X
authors.lastname: Gao
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Zhongmei
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D055162:Biocatalysis; D004795:Enzyme Stability; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013025:Glycine max / Q000378:metabolism; D013696:Temperature
chemical_list: D010940:Plant Proteins; D000626:Aminopeptidases