A sequential design strategy was applied to optimize the secretion of pectinases by a Saccharomyces cerevisiae strain, from Brazilian sugarcane liquor vat, on passion fruit residue flour (PFRF), through solid-state fermentation (SSF). A factorial design was performed to determine the influence variables and two rotational central composite designs were executed. The validated experimental result was of 7.1 U mL−1 using 50% PFRF (w/w), pH 5, 30 °C for 24 h, under static SSF. Polygalacturonase, pectin methyl esterase, pectin–lyase and pectate–lyase activities were 3.5; 0.08; 3.1 and 0.8 U mL−1, respectively. Shotgun proteomics analysis of the crude extract enabled the identification of two pectin–lyases, one pectate–lyase and a glucosidase. The crude enzymatic extract maintained at least 80% of its original activity at pH values and temperatures ranging from 2 to 8 and 30 to 80 °C, respectively, over 60 min incubation. Results revealed that PFRF might be a cost-effective and eco-friendly substrate to produce pectinases. Statistical optimization led to fermentation conditions wherein pectin active proteins predominated. To the extent of our knowledge, this is the first study reporting the synthesis of pectate lyase by S. cerevisiae.
Yeasts play a fundamental role in nature, and due to their biochemical and physiological versatility, many of them can have their metabolic routes regulated and optimized for the biosynthesis of specific products. In view of this potentiality, new contributions that dedicate to the study of fermentative processes involving yeasts as protagonists, especially for the production of value-added products from lost vegetables and their residues, show importance. Among these products, particularly in this brief review, the production of pectinase and yeast probiotic potential in alternative culture media is mentioned as a way to make these processes more economically and environmentally viable.
Effects of heat pretreatment on the yield and bioactivity of powdered egg whites hydrolysates by three different proteases. Efeito do pré-tratamento térmico sobre o rendimento e a bioatividade de hidrolisados de clara de ovo em pó por três diferentes proteases.
Staphylococcus lugdunensis produces lugdulysin, a metalloprotease that may contribute to its virulence. This study aimed to evaluate the biochemical aspects of lugdulysin and investigate its effect on Staphylococcus aureus biofilms. The protease was isolated and characterized for its optimal pH and temperature, hydrolysis kinetics, and influence of metal cofactor supplementation. The protein structure was determined via homology modelling. The effect on S. aureus biofilms was assessed by the micromethod technique. The protease optimal pH and temperature were 7.0 and 37°C, respectively. EDTA inhibited protease activity, confirming it as a metalloprotease. Lugdulysin activity was not recovered by divalent ion supplementation post-inhibition and supplementation with divalent ions did not change enzymatic activity. The isolated enzyme was stable for up to 3 hours. Lugdulysin significantly inhibited the formation and disrupted pre-established protein-matrix MRSA biofilm. This preliminary study indicates that lugdulysin has a potential role as a competition mechanism and/or modulation of staphylococcal biofilm.
Background
Staphylococcus lugdunensis is a commensal skin microorganism that, unlike other coagulase-negative staphylococci, presents increasing clinical importance. This species yields a metalloprotease called lugdulysin that may contribute to its higher degree of virulence. This study aimed to determine the biochemical characterization of the lugdulysin produced by S. lugdunensis clinical isolates and investigate its effect on the formation and disruption of biofilm of Staphylococcus aureus isolates. The protease was isolated and characterized for its optimal pH and temperature, activity in the presence of inhibitors and enzymatic kinetics. The influence of metal cofactor supplementation on proteolysis was also evaluated, with and without inhibitors. Finally, the protease capacity to inhibit and disrupt biofilms of different S. aureus lineages and biofilm matrix was analyzed.
Results
The protease optimal pH and temperature were 7.0 and 37° C, respectively. EDTA inhibited the protease, and the activity was not recovered by divalent ion supplementation. In addition, divalent ions did not change enzymatic activity without inhibitors, which was stable for up to 3 hours. Its structure was determined via homology modelling. The protease significantly inhibited the formation and disrupted established biofilms of S. aureus isolates with protein biofilm.
Conclusions
This study confirmed features of the lugdulysin metalloprotease and showed that this S. lugdunensis virulence factor may be a new competition mechanism and/or modulation of the staphylococcal biofilm.
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