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Ficus carica produces, in addition to the cysteine protease ficin, a serine protease ( FSP ) . Here, we purified FSP to homogeneity from the fruit of F. carica cultivar Masui Dauphine. An 81-fold enrichment in specific activity of FSP with 2.1% recovery was attained. Three protein bands ( 70, 62, and 60 kDa ) were identified on SDS-PAGE. Each band was identified as a subtilisin-like protease ( 661 amino acids ) by trypsin digestion, LC-MS/MS analysis, and the partial N-terminal amino acid sequence analysis. Gelatin zymography revealed that the active FSP exists as a dimer. The optimum hydrolysis pH of FSP was 7.5, and the pHs at which the enzyme retained its initial activity by 70% in 24 h were 8.0–11.0. The optimum hydrolysis temperature of FSP was 50–60 ºC, and the temperature required to reduce the initial activity by 50% in 15 min was 70 ºC. These results will inform the industrial use of FSP. [ABSTRACT FROM AUTHOR]

Subtilisin-like proteases are widely distributed and reported to be required for virulence in pathogenic fungi. In chestnut blight fungus Cryphonectria parasitica, prb1, encoding a putative subtilisin-like protease, was expressed and recombinant Prb1 protein was shown to have a protease activity in vitro. prb1-deleted mutants exhibited reduced total protease activity by 60%. The Δ prb1 mutants showed a phenotype of reduced aerial hyphae, lower level of sporulation, and a significant reduction in virulence. Additionally, site-directed mutagenesis of Prb1 protein revealed that D195, H227, and S393 are critical for C. parasitica Prb1 function in vivo. Transcriptional analysis showed that deletion of prb1 also reduced the transcript accumulation levels for genes encoding key components of the heterotrimeric G-protein signaling pathway, including cpga1, cpgb1, cpgc1, and ste12. Furthermore, deletion of prb1 results in the accumulation of autophagic bodies in the fungus. Taken together, our results showed that prb1-encoded protease functions in the regulation of virulence, phenotypical traits, and autophagy in C. parasitica. [ABSTRACT FROM AUTHOR]

Aims Cloning, expression and characterization of a new cold-adapted protease with potential biotechnological application, isolated from Antarctic bacteria. Method and Results A subtilisin-like gene was isolated from several Antarctic bacterial genus using CODPEHOP-designed primers and a genome walking method. This gene encodes a precursor protein, which undergoes an autocatalytic cleavage resulting in a 34·6 kDa active cold-adapted protease with a maximum activity at 25-35°C and optimum p H of 8·0-9·0. It showed a higher catalytic efficiency at lower temperatures compared to its mesophilic counterpart. Heat-induced inactivation resulted in a very low melting point. Local packing analysis using the homology model indicated Ala284 as an important cold-adaptation determinant, which was corroborated by the site-directed mutagenesis. Conclusions A new thermolabile subtilisin-like protease has been successfully cloned and analysed, and an important hot spot in the evolution of the cold adaptation and substrate specificity of this enzyme was identified and tested. Significance and Impact of the Study This work reports a new cold-adapted protease with a vast representation amongst Antarctic genus, suggesting therefore its evolutionary success in this cold environment. Likewise, important sites for genetic potentiation have been identified, which are extrapolated to other enzymes of the same kind. [ABSTRACT FROM AUTHOR]

SPM1, encoding a putative subtilisin-like protease, is involved in pathogenicity of the rice blast fungus Magnaporthe oryzae, but its detailed function remains unknown. Here, we report that SPM1 encodes a vacuole-localized protease that is a critical component for autophagy during the infection process of M. oryzae. Detailed phenotypic analysis of targeted disruption mutants of SPM1 revealed that the mutants have pleiotropic defects in infection-related steps including germination, appressorium formation, host invasion and postinvasive growth, indicating the requirement of Spm1 function for the broad phase of infection. It has been shown that the Spm1 homolog of yeast functions in autophagy, the degradation machinery mediated by vacuoles, implying the involvement of Spm1 in autophagy in M. oryzae. In-gel protease activity assay of the recombinant Spm1 protein indicated that Spm1 had a protease activity. An Spm1–GFP fusion protein was detected inside vacuoles of fungal cells, indicating that Spm1 is a protease localized in vacuoles. Furthermore, degradation of putative autophagic bodies was retarded in vacuoles of the spm1 mutant. These data strongly suggest that SPM1-encoded protease functions in autophagy required for the pathogenicity of M. oryzae. [ABSTRACT FROM AUTHOR]

Trichoderma harzianum parasitizes a large variety of phytopathogenic fungi. Trichoderma harzianum mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. A gene ( SS10) encoding a subtilisin-like protease was cloned from T. harzianum T88, a biocontrol agent effective against soil-borne fungal pathogens. The full-length cDNA was isolated by 5′ and 3′ rapid amplification of the cDNA ends. The coding region of the gene is 1302 bp long, encoding 433 amino acids of a predicted protein with a molecular mass of 45 kDa and a pI of 6.1. Analysis of the deduced amino acid sequence revealed that this protein had homology to the serine proteases of the subtilisin-like superfamily (subtilases) (EC 3.4.21.) and had a predicted active site made up of the catalytic residues Asp 187, His 218 and Ser 376. Northern experiments demonstrated that SS10 was induced in response to different fungal cell walls. Subtilisin-like protease gene SS10 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (17.8 U mL−1) 60 h after induction with galactose. The optimal enzyme reaction temperature was 50 °C and the optimal pH was 8. The subtilisin-like protease exerted broad-spectrum antifungal activity against Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Cytospora chrysosperma. [ABSTRACT FROM AUTHOR]

We have isolated a bacterium (TP-6) from the Indonesian fermented soybean, Tempeh, which produces a strong fibrinolytic protease and was identified as Bacillus subtilis. The protease (TPase) was purified to homogeneity by ammonium sulfate fractionation and octyl sepharose and SP sepharose chromatography. The N-terminal amino acid sequence of the 27.5 kDa enzyme was determined, and the encoding gene was cloned and sequenced. The result demonstrates that TPase is a serine protease of the subtilisin family consisting of 275 amino acid residues in its mature form. Its apparent K m and V max for the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe- pNA were 259 μM and 145 μmol mg−1 min−1, respectively. The fibrinogen degradation pattern generated by TPase as a function of time was similar to that obtained with plasmin. In addition, N-terminal amino acid sequence analysis of the fibrinogen degradation products demonstrated that TPase cleaves Glu (or Asp) near hydrophobic acids as a P1 site in the α- and β-chains of fibrinogen to generate fragments D′, E′, and D′ similar to those generated by plasmin. On plasminogen-rich fibrin plates, TPase did not seem to activate fibrin clot lysis. Moreover, the enzyme converted the active plasminogen activator inhibitor-1 to the latent form. [ABSTRACT FROM AUTHOR]

A secreted 31.5-kDa keratinolytic subtilase (SUB3; AJ431180) is thought to be a Microsporum canis virulence factor and represents a candidate for vaccination trials. In this study, the recombinant keratinase (r-SUB3) was produced by the Pichia pastoris expression system and purified to homogeneity. Recombinant SUB3 displayed identical biochemical properties with the native protease. Experimentally cutaneously infected guinea pigs showed specific lymphoproliferative response towards r-SUB3, while no specific humoral immune response was induced except for one animal. The heterologous expression of SUB3 provides a valuable tool for addressing further investigations on the role of this keratinase in the specific cellular immune response and on its use in vaccination trials in the cat. [Copyright &y& Elsevier]

Differential screening of a cDNA library for mRNA species that specifically accumulate during auxin-induced lateral root formation in Arabidopsis thaliana led to the isolation of the AIR3 cDNA clone. The corresponding single copy gene consists of 10 exons which encode a protein that possesses all the characteristics of subtilisin-like proteases. The promoter of the AIR3 gene was fused to the gusA (β-glucuronidase) reporter gene and introduced into Arabidopsis. Expression was almost completely restricted to the outer layers of the parental root at sites of lateral root emergence and could be observed even before protrusion of the newly formed root tip. In the presence of external auxin, GUS activity was visible throughout the parts of the root that are competent for lateral root formation. By digesting structural proteins in the extracellular matrix of cells located above sites of lateral root formation, AIR3 might weaken cell-to-cell connections and thus facilitate lateral root emergence. [ABSTRACT FROM PUBLISHER]