Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.

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PurposeThe main purpose of this study was to investigate parasympathetic reactivation of the heart [evaluated through heart rate recovery (HRR) and HR variability (HRV)] after maximal cardiopulmonary exercise testing (CPET) using three different exercise modalities.MethodsTwenty healthy men, aged 17 to 28 yr, performed three maximal CPETs (cycling, walking, and running) separated by 72 h and in a randomized, counter-balanced order. HRR was determined from the absolute differences between HRpeak and HR at 1–3 min after exercise. The root mean square of successive R-R differences calculated for consecutive 30-s windows (rMSSD30s) was calculated to assess the parasympathetic reactivation after maximal CPET.ResultsLower HRpeak, VO2peak and energy expenditure were observed after the cycling CPET than the walking and running CPETs (P < 0.001). Both HRR and rMSSD30s were significantly greater during recovery from the cycling CPET compared to the walking and running CPETs (P < 0.001). Furthermore, Δ rMSSD (i.e. resting minus postexercise rMSSD every 30 s into the recovery period) was positively related to the resting high-frequency component (HF), rMSSD, and standard deviation of all normal R-R intervals (SDNN) (rs = 0.89 to 0.98; P < 0.001), and negatively related to the resting low-frequency component (LF) and sympathovagal balance (LF:HF ratio) after all exercise conditions (rs = −0.73 to −0.79 and −0.86 to −0.90, respectively; P < 0.001).ConclusionsThese findings support that parasympathetic reactivation after maximal CPET (as assessed by HRR and rMSSD30s) depends on exercise modality and cardiac autonomic control at rest.

Pyocyanin was the first natural phenazine described. The molecule is synthesized by about 95% of the strains of Pseudomonas aeruginosa. From discovery up to now, pyocyanin has been characterised by a very rich and avant-garde history, which includes its use in antimicrobial therapy, even before the discovery of penicillin opened the era of antibiotic therapy, as well as its use in electric current generation. Exhibiting an exuberant blue colour and being easy to obtain, this pigment is the subject of the present review, aiming to narrate its history as well as to unveil its mechanisms and suggest new horizons for applications in different areas of engineering, biology and biotechnology.

BackgroundGlycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion.ResultsGHs and accessory proteins were produced by representative brown- and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and β-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.ConclusionsThe extracellular proteins produced by wood decay fungi provide useful tools to improve commercial enzyme cocktails that are currently used for the saccharification of alkaline-pretreated lignocellulosic substrates. The relevant proteins encompass multiple glycoside hydrolase families, including the GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0525-y) contains supplementary material, which is available to authorized users.

IMT increases HRV, improves 6MWT distance and HRR.

BackgroundThe enzymatic degradation of lignocellulosic materials by fungal enzyme systems has been extensively studied due to its effectiveness in the liberation of fermentable sugars for bioethanol production. Recently, variants of the fungus Penicillium echinulatum have been described as a great producer of cellulases and considered a promising strain for the bioethanol industry.ResultsPenicillium echinulatum, wild-type 2HH and its mutant strain S1M29, were grown on four different carbon sources: cellulose, sugar cane bagasse pretreated by steam explosion (SCB), glucose, and glycerol for 120 h. Samples collected at 24, 96, and 120 h were used for enzymatic measurement, and the 96-h one was also used for secretome analysis by 1D-PAGE LC–MS/MS. A total of 165 proteins were identified, and more than one-third of these proteins belong to CAZy families. Glycosyl hydrolases (GH) are the most abundant group, being represented in larger quantities by GH3, 5, 17, 43, and 72. Cellobiohydrolases, endoglucanases, β-glycosidases, xylanases, β-xylosidases, and mannanases were found, and in minor quantities, pectinases, ligninases, and amylases were also found. Swollenin and esterases were also identified.ConclusionsOur study revealed differences in the two strains of P. echinulatum in several aspects in which the mutation improved the production of enzymes related to lignocellulosic biomass deconstruction. Considering the spectral counting analysis, the mutant strain S1M29 was more efficient in the production of enzymes involved in cellulose and hemicellulose degradation, despite having a nearly identical CAZy enzymatic repertoire. Moreover, S1M29 secretes more quantities of protein on SCB than on cellulose, relevant information when considering the production of cellulases using raw materials at low cost. Glucose, and especially glycerol, were used mainly for the production of amylases and ligninases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0476-3) contains supplementary material, which is available to authorized users.

An essential step in the conversion of lignocellulosic biomass to ethanol and other biorefinery products is conversion of cell wall polysaccharides into fermentable sugars by enzymatic hydrolysis. The objective of the present study was to understand the mode of action of hemicellulolytic enzyme mixtures for pretreated sugarcane bagasse (PSB) deconstruction and wheat arabinoxylan (WA) hydrolysis on target biotechnological applications. In this study, five hemicellulolytic enzymes-two endo-1,4-xylanases (GH10 and GH11), two α-L-arabinofuranosidases (GH51 and GH54), and one β-xylosidase (GH43)-were submitted to combinatorial assays using the experimental design strategy, in order to analyze synergistic and antagonistic effects of enzyme interactions on biomass degradation. The xylooligosaccharides (XOSs) released from hydrolysis were analyzed by capillary electrophoresis and quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Based on this analysis, it was possible to define which enzymatic combinations favor xylose (X1) or XOS production and thus enable the development of target biotechnological applications. Our results demonstrate that if the objective is X1 production from WA, the best enzymatic combination is GH11 + GH54 + GH43, and for xylobiose (X2) production from WA, it is best to combine GH11 + GH51. However, if the goal is to produce XOS, the five enzymes used in WA hydrolysis are important, but for PSB hydrolysis, only GH11 is sufficient. If the final objective is bioethanol production, GH11 is responsible for hydrolyzing 64.3 % of hemicellulose from PSB. This work provides a basis for further studies on enzymatic mechanisms for XOS production, and the development of more efficient and less expensive enzymatic mixtures, targeting commercially viable lignocellulosic ethanol production and other biorefinery products.