RRC ID 60352
Author Xie G, Shao Z, Zong L, Li X, Cong D, Huo R.
Title Heterologous expression and characterization of a novel subtilisin-like protease from a thermophilic Thermus thermophilus HB8.
Journal Int. J. Biol. Macromol.
Abstract Subtilisins are a family of serine proteases used widely throughout the detergent, leather and food industries, with the identification and development of new enzymes holding much potential value. Thermus thermophilus HB8 was examined for serine proteases and found TTHA0724 gene. Sequence analysis of this putative serine protease placed it within the subtilisin family. To obtain active T. thermophilus HB8 subtilisins, three genes encoding prepro-subtilisin, pro-subtilisin and mature-subtilisin were cloned and expressed in Escherichia coli Transetta (DE3). Although direct expression of the mature-subtilisin gene was found to produce inactive inclusion bodies, expression of the pro-subtilisin gene resulted in active mature-subtilisin, indicating that the pro-sequence of translated pro-subtilisin underwent autoproteolysis. The resulting mature-subtilisin exhibited maximal activity between 65 and 85 °C at pH 7.5. The mature-subtilisin showed good stability, maintaining 50% activity after 48 h at 75 °C and >78% activity across the pH range 5.0-9.5. Furthermore, the mature-subtilisin demonstrated broad substrate specificity, with no requirement for the presence of metal ions which are essential for other subtilisin enzymes. Despite this Cu2+ was able to increase enzyme activity, while Ca2+ partially inhibited the activity. These properties suggest that T. thermophilus HB8 mature-subtilisin has potential value in its application in many industries.
Volume 138
Pages 528-535
Published 2019-10-1
DOI 10.1016/j.ijbiomac.2019.07.101
PII S0141-8130(19)31167-5
PMID 31323269
MeSH Amino Acid Sequence Computational Biology / methods Enzyme Stability Gene Expression* Hydrogen-Ion Concentration Hydrolysis Ions / chemistry Metals / chemistry Models, Molecular Mutation Phylogeny Protein Conformation Recombinant Proteins* Serine Endopeptidases / chemistry Serine Endopeptidases / genetics* Serine Endopeptidases / isolation & purification Substrate Specificity Subtilisin / genetics Thermus thermophilus / classification Thermus thermophilus / genetics*
IF 4.784
Resource
General Microbes JCM10941