Improvement of catalytic efficiency and thermostability of recombinant Streptomyces griseus trypsin by introducing artificial peptide.
Streptomyces trypsin is one of the serine proteinases in Streptomyces griseus and acts as a key mediator during cell growth and differentiation. S. griseus trypsin (SGT) could be successfully expressed in Pichia pastoris by engineering the natural propeptide APNP. In this study, the recombinant Exmt with peptide YVEF and the wild-type SGT were comparatively investigated in detail. The recombinant Exmt showed significantly increased thermostability which t(½) value was 3.89-fold of that of the SGT at 40 °C. Moreover, the catalytic efficiency (referring to the specificity constant, k cat/K m) and pH tolerance of Exmt were also improved. In silico modeling analysis uncovered that introduction of the peptide YVEF resulted in a broadened substrate binding pocket and closer catalytic triad (His⁵⁷, Asp¹⁰² and Ser¹⁹⁵). The intramolecular Hydrogen bonds and the cation π-interactions were also dramatically increased. The results indicated that engineering of the N-terminus with artificial peptides might be an effective approach for optimizing the properties of the target enzymes.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Zhenmin
Z
Ling
Zhen
Z
Kang
Yi
Y
Liu
Song
S
Liu
Jian
J
Chen
Guocheng
G
Du
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D004795:Enzyme Stability; D007700:Kinetics; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013305:Streptomyces griseus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014357:Trypsin