ABSTRACT
A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.
Subject(s)
Fusarium/enzymology , Industrial Microbiology , Subtilisins/isolation & purification , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Coloring Agents , Detergents/pharmacology , Electrophoresis, Polyacrylamide Gel , Fusarium/drug effects , Hydrogen Peroxide/pharmacology , Hydrogen-Ion Concentration , Kinetics , Mass Spectrometry , Molecular Sequence Data , Oxidants/pharmacology , Proteolysis/drug effects , Substrate Specificity/drug effects , Subtilisins/chemistry , Subtilisins/genetics , Temperature , Trichoderma/enzymologyABSTRACT
The Pseudomonas aeruginosa genome encodes a variety of different proteolytic enzymes several of which play an important role as virulence factors. Interestingly, only two of these proteases are predicted to belong to the subtilase family and we have recently studied the physiological role of the subtilase SprP. Here, we describe the functional overexpression of SprP in Escherichia coli using a novel expression and secretion system. We show that SprP is autocatalytically activated by proteolysis and exhibits optimal activity at 50°C in a pH range of 7-8. We also demonstrate a significant increase in sprP promoter activity upon growth of P. aeruginosa at 43°C indicating a role for SprP in heat shock response.
Subject(s)
Pseudomonas aeruginosa/enzymology , Subtilisins/isolation & purification , Subtilisins/metabolism , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Expression Profiling , Hydrogen-Ion Concentration , Proteolysis , Pseudomonas aeruginosa/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Subtilisins/chemistry , Subtilisins/genetics , TemperatureABSTRACT
The open reading frame PA1242 in the genome of Pseudomonas aeruginosa PAO1 encodes a putative protease belonging to the peptidase S8 family of subtilases. The respective enzyme termed SprP consists of an N-terminal signal peptide and a so-called S8 domain linked by a domain of unknown function (DUF). Presumably, this DUF domain defines a discrete class of Pseudomonas proteins as homologous domains can be identified almost exclusively in proteins of the genus Pseudomonas. The sprP gene was expressed in Escherichia coli and proteolytic activity was demonstrated. A P. aeruginosa ∆sprP mutant was constructed and its gene expression pattern compared to the wild-type strain by genome microarray analysis revealing altered expression levels of 218 genes. Apparently, SprP is involved in regulation of a variety of different cellular processes in P. aeruginosa including pyoverdine synthesis, denitrification, the formation of cell aggregates, and of biofilms.
