RESUMEN
A novel extracellular protease called MPDZ was purified and characterized from Pseudomonas fluorescens strain TBS09. The enzymatic properties of MPDZ were investigated using biochemical and biophysical methods. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that it was a monomer with a molecular mass of 50013.17Da. The NH2-terminal 27 amino acid sequence of MPDZ showed high homology with those of Pseudomonas-proteases of the serralysin family. MPDZ showed optimal activity at pH 7 and 60°C. It was totally inhibited by EGTA, EDTA, and 1,10-phenanthroline, suggesting its belonging to the metalloprotease family. Because of the interesting properties, the mpDZ gene encoding MPDZ was cloned, sequenced, and expressed in E. coli. The deduced amino acid sequence showed a strong homology with other Pseudomonas-metalloproteases. The highest sequence identity value (97%) was obtained with AprX from P. fluorescens strain CY091, with only 12 different amino acid residues. The physico-chemical properties of the extracellular purified recombinant enzyme (rMPDZ) were similar to those of MPDZ. Overall, MPDZ is bestowed with a number of promising biochemical properties that might give new opportunities for its biocatalytic applications. These data constitute an essential first step towards an understanding of the properties of MPDZ enzyme.
Asunto(s)
Metaloproteasas/química , Metaloproteasas/genética , Pseudomonas fluorescens/enzimología , Secuencia de Aminoácidos/genética , Clonación Molecular , Escherichia coli/genética , Metaloproteasas/aislamiento & purificación , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Pseudomonas fluorescens/genética , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
An alkaline proteinase (STAP) was produced from strain TN650 isolated from a Tunisian off-shore oil field and assigned as Streptomyces koyangensis strain TN650 based on physiological and biochemical properties and 16S rRNA gene sequencing. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 45125.17-Da. The enzyme had an NH2-terminal sequence of TQSNPPSWGLDRIDQTTAFTKACSIKY, thus sharing high homology with those of Streptomyces proteases. The results showed that this protease was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), diiodopropyl fluorophosphates (DFP), and partially inhibited by 5,5-dithio-bis-(2-nitro benzoic acid) (DTNB), which strongly suggested its belonging to the serine thiol protease family. Using casein as a substrate, the optimum pH and temperature values for protease activity were pH 10 and 70 °C, respectively. The protease was stable at pH 7-10 and 30-60 °C for 24 h. STAP exhibited high catalytic efficiency, significant detergent stability, and elevated organic solvent resistance compared to the SG-XIV proteases from S. griseus and KERAB from Streptomyces sp. AB1. The stap gene encoding STAP was isolated, and its DNA sequence was determined. These properties make STAP a potential candidate for future application in detergent formulations and non-aqueous peptide biocatalysis.
