f Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a "last-resort" antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 g/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120¡Ala and Trp73¡Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus ␤-lactam combinations and continue to question the DAP breakpoint of 4 g/ml.T he surge of Enterococcus faecium as an important nosocomial pathogen has been associated with an expanding pandemic caused by a hospital-associated (HA) genetic clade (1, 2). Indeed, isolates belonging to this genetic lineage are frequently multidrug resistant (MDR) with high MICs of ampicillin and vancomycin (3). Daptomycin (DAP) is a cell membrane (CM)-targeting lipopeptide that has in vitro bactericidal activity against MDR E. faecium and, due to the paucity of other bactericidal options, is often used as first-line therapy despite lacking U.S. Food and Drug Administration approval for these organisms. However, one of the major problems when using DAP against enterococci is the emergence of resistance during therapy (4-6).The mechanisms of DAP resistance in enterococci are not fully understood, but recent evidence suggests that there are several genetic pathways involved and that resistance results from a sequential and ordered mutational pathway (7-9). In Enterococcus faecalis, we have previously shown that emergence of resistance during therapy involves substitutions in three proteins: (i) LiaF, a member of the three-component regulatory system LiaFSR which, in Bacillus subtilis and other Gram-positive bacteria (10), has been shown to orchestrate the cell envelope response to stress; (ii) GdpD, a glycerophosphory...

Please cite this article as: Cavello IA, Hours RA, Rojas NL, Cavalitto SF, Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876, Process Biochemistry (2013), http://dx.doi.org/10. 1016/j.procbio.2013.03.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 of 32 A c c e p t e d M a n u s c r i p t Highlights ►This is the first report of a serine protease with keratinolytic activity from P.lilacinus LPS #876. ► Enzyme stability in broad pH range, and up to 65 °C, suggests its suitability as a detergent additive.► Oxidant/detergent stability strengthens the enzyme's potential application as laundry additive.►The production of this enzyme could be an alternative for solid waste management processes, an added valued product for tanneries

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The pg1 gene from the filamentous fungus Aspergillus kawachii, which codifies for an acid polygalacturonase, was cloned into the pYES2 expression vector, giving rise to the pYES2:pg1ΔI construct. Engineered Saccharomyces cerevisiae, transformed with pYES2:pg1ΔI construct, both expressed and exported an active polygalacturonase with a MW of ~60 kDa and an isoelectric point of 3.7, similar to those reported for the wild-type enzyme. The recombinant enzyme has the ability to hydrolyze polygalacturonic acid at pH 2.5. Heterologous PG1 production was studied under controlled conditions in batch and fed-batch systems. A simultaneous addition of glucose and galactose was found to be the most suitable feeding strategy assayed, resulting in a final PG1 production of 50 U/ml. The production process proposed in this study could be applied for the industrial production of a novel and useful polygalacturonase.