Methods for Cultivation of Bacteroides thetaiotaomicron and Analysis of Heme Metabolism by Mass Spectrometry and Spectroscopic Approaches.

Traditional studies of cellular metabolism have relied on the use of radioisotopes. These have clear disadvantages associated with safety and waste generation. Furthermore, detection of the labeled species by scintillation counting provides only a quantification of its presence or absence. The use of stable isotopes, by contrast, allows the application of powerful, orthogonal spectroscopic approaches such as nuclear magnetic resonance spectroscopy (NMR) and various mass spectrometric methods. Using stable isotope labeling to study heme metabolism requires integrating methods for (a) generating the heme in labeled forms, (b) cultivating and quantifying the organism of choice in chemically defined media, to which labeled compounds can be added, (c) recovering cellular components and/or spent growth media, and (d) analyzing these materials for the labeled species using spectroscopic and mass spectrometric methods. These methods are summarized here in the context of Bacteroides thetaiotaomicron, a generally nonpathogenic anaerobe and heme auxotroph.

Fungal endo and exochitinase production, characterization, and application for Candida biofilm removal.

Chitinases are promising enzymes for a multitude of applications, including chitooligosaccharide (COS) synthesis for food and pharmaceutical uses and marine waste management. Owing to fungal diversity, fungal chitinases may offer alternatives for chitin degradation and industrial applications. The rapid reproduction cycle, inexpensive growth media, and ease of handling of fungi may also contribute to reducing enzyme production costs. Thus, this study aimed to identify fungal species with chitinolytic potential and optimize chitinase production by submerged culture and enzyme characterization using shrimp chitin. Three fungal species, Coriolopsis byrsina, Trichoderma reesei, and Trichoderma harzianum, were selected for chitinase production. The highest endochitinase production was achieved in C. byrsina after 168 h cultivation (0.3 U mL- 1). The optimal temperature for enzyme activity was similar for the three fungal species (up to 45 and 55 ºC for endochitinases and exochitinases, respectively). The effect of pH on activity indicated maximum hydrolysis in acidic pH (4-7). In addition, the crude T. reesei extract showed promising properties for removing Candida albicans biofilms. This study showed the possibility of using shrimp chitin to induce chitinase production and enzymes that can be applied in different industrial sectors.

The degradation of chicken feathers by Ochrobactrum intermedium results in antioxidant and metal chelating hydrolysates and proteolytic enzymes for staphylococcal biofilm dispersion.

The increase in the generation of chicken feathers, due to the large production of the poultry industry, has created the need to search for ecologically safer ways to manage these residues. As a sustainable alternative for recycling keratin waste, we investigated the ability of the bacterium Ochrobactrum intermedium to hydrolyze chicken feathers and the valorization of the resulting enzymes and protein hydrolysate. In submerged fermentation with three different inoculum sizes (2.5, 5.0, and 10.0 mg of bacterial cells per 50 mL of medium), the fastest degradation of feathers was achieved with 5.0 mg cells, in which a complete decomposition of the substrate (96 h) and earlier peaks of keratinolytic and caseinolytic activities were detected. In the resulting protein hydrolysate, we noticed antioxidant and Fe2+ and Cu2+ chelating activities. ABTS scavenging, Fe3+-reducing ability and metal chelating activities of the fermentative samples followed the same trend of feather degradation; as feather mass decreased in the media, these activities increased. Furthermore, we noticed about 47% and 60% dispersion of established 7-day biofilms formed by S. aureus after enzymatic treatment for 5 h and 24 h, respectively. These findings highlight the potential use of this bacterium as an environmentally friendly alternative to treat this poultry waste and offer valuable products.

Xylose consumption and ethanol production by Pichia guilliermondii and Candida oleophila in the presence of furans, phenolic compounds, and organic acids commonly produced during the pre-treatment of plant biomass.

For 2G ethanol production, pentose fermentation and yeast tolerance to lignocellulosic hydrolyzate components are essential to improve biorefinery yields. Generally, physicochemical pre-treatment methodologies are used to facilitate access to cellulose and hemicellulose in plant material, which consequently can generate microbial growth inhibitory compounds, such as furans, weak acids, and phenolic compounds. Because of the unsatisfactory yield of wild-type Saccharomyces cerevisiae during pentose fermentation, the search for xylose-fermenting yeasts tolerant to microbial growth inhibitors has gained attention. In this study, we investigated the ability of the yeasts Pichia guilliermondii G1.2 and Candida oleophila G10.1 to produce ethanol from xylose and tolerate the inhibitors furfural, 5-hydroxymethylfurfural (HMF), acetic acid, formic acid, ferulic acid, and vanillin. We demonstrated that both yeasts were able to grow and consume xylose in the presence of all single inhibitors, with greater growth limitation in media containing furfural, acetic acid, and vanillin. In saline medium containing a mixture of these inhibitors (2.5-3.5 mM furfural and HMF, 1 mM ferulic acid, 1-1.5 mM vanillin, 10-13 mM acetic acid, and 5-7 mM formic acid), both yeasts were able to produce ethanol from xylose, similar to that detected in the control medium (without inhibitors). In future studies, the proteins involved in the transport of pentose and tolerance to these inhibitors need to be investigated.

Application of a recombinant GH10 endoxylanase from Thermoascus aurantiacus for xylooligosaccharide production from sugarcane bagasse and probiotic bacterial growth.

Xylooligosaccharides (XOs) are a promising class of prebiotics capable of selectively stimulating the growth of the beneficial intestinal microbiota against intestinal pathogens. They can be obtained from xylan present in residual lignocellulosic material from agriculture. Thus, in this study we produced XOs by extracting xylan from sugarcane bagasse and hydrolyzing it using the GH10 xylanase from Thermoascus aurantiacus expressed by Pichia pastoris. An alkaline method to extract xylan is described, which resulted in 83.40% of xylan recovery and low amounts of cellulose and lignin. The enzymatic hydrolysate exhibited a mixture of XOs containing mainly xylobiose, xylotriose and xylotetraose. These oligosaccharides stimulated the growth of Lactobacillus casei, L. rhamnosus, L. fermentum and L. bulgaricus strains, which were able to produce organic acids, especially acetic acid. These findings demonstrate the possibility to redirect crop by-products to produce XOs and their use as a supplement to stimulate the growth of probiotic strains.

Functional properties and potential application of ethanol tolerant ß-glucosidases from Pichia ofunaensis and Trichosporon multisporum yeasts.

ß-Glucosidases have been extensively investigated to integrate the enzyme complex for cellulose fiber saccharification and for improving the aroma of wine. To produce these enzymes, greater attention has been given to filamentous fungi and bacteria, and few investigations have targeted the potential applications of enzymes secreted by yeasts. Addressing this issue, in this study, ß-glucosidases were produced by the Pichia ofunaensis and Trichosporon multisporum yeasts, via solid state fermentation with wheat bran as a substrate. When using p-Nitrophenyl ß-d-glucopyranoside (pNPG) as an enzyme substrate, maximum ß-glucosidase activities were detected at pH 5.5-6.0 and 50-60 °C for P. ofunaensis, and pH 5-6 and 55 °C for T. multisporum. Both enzymes were able to hydrolyze cellobiose and exhibited stability over a wide range of pH (3.5-9.0) for 24 h at 4 °C, thermostability up to 50 °C for 1 h and tolerance to 10 mM phenolic compounds. Negative modulation on enzyme activity was observed in the presence of Cu2+, Fe3+, Zn2+, Al3+ and Hg2+, while both ß-glucosidases were tolerant to 30% methanol, isopropanol and acetone. In the presence of ethanol and glucose, enzymes from P. ofunaensis were the more active and stable of the two. These enzymes, especially the P. ofunaensis ß-glucosidases, could be tested in enology for improving the aroma of wine and for integrating a cellulolytic complex to produce 2G ethanol.

Enzymatic hydrolysis of non-animal proteins for improving nutritional and sensory properties of foods.

Taste and aroma of foods are recognized properties that contribute to increasing food acceptance and consumption, and methods for improving these attributes are highly sought after. For this, natural compounds are widely investigated and include peptides from fungi and plant proteins, which are alternative sources of protein in vegan and vegetarian diets. According to the specificity of peptidases and the protein substrates, a variety of peptides can be produced from enzymatic hydrolysis, which is an important advantage compared with chemical hydrolysis. Therefore, the following article discusses the use of proteolytic enzymes to produce protein hydrolysate from nonanimal sources for improving the sensory properties and nutritional value of foods. PRACTICAL APPLICATIONS: The purpose of this letter is to discuss the use of proteolytic enzymes as an eco-friendly alternative to produce protein hydrolysate from nonanimal sources for improving the sensory properties and nutritional value of foods.

Industrial sustainability of microbial keratinases: production and potential applications.

Keratinases are proteolytic enzymes with a particular ability to cleave peptide bonds in keratin, and in other proteins. Due to their broad-spectrum of activity, keratinases are considered viable substitutes for chemical and thermal treatments of protein-rich industrial by-products. Among these protein residues, special attention has been given to keratinous materials (feathers, hair, horns, etc.), which disposal through harsh conditions methods, such as acid/alkaline hydrolysis or incineration, is not considered ecologically safe. Microbial keratinolytic enzymes allow for keratin degradation under mild conditions, resulting in keratin hydrolysates containing undamaged amino acids and peptides. In this review article, we offer perspectives on the relevance of these unique biocatalysts and their revolutionary ascent in industries that generate keratin-rich wastes. Additionally, we share insights for applications of keratinases and protein hydrolysates in agriculture, animal feed, cosmetics, phamaceuticals, detergent additives, leather processing, and others. Due to the scientific importance of keratinases and their potential use in green technologies, searching for bacterial and fungal species that efficiently produce these enzymes may contribute to the sustainability of industries.

Keratinases from Coriolopsis byrsina as an alternative for feather degradation: applications for cloth cleaning based on commercial detergent compatibility and for the production of collagen hydrolysate.

OBJECTIVES: Keratinases are proteolytic enzymes that emerge as an alternative for dealing with the disposal of chicken feathers. In this study, we aimed to investigate the keratin-degrading enzymes secreted by the fungus Coriolopsis byrsina and their partial biochemical characterization to adapt their use for keratin decomposition, detergent additive applications, and collagen degradation. RESULTS: We observed the secretion of different proteolytic enzymes that possessed caseinolytic activity that peaked at pH 7.0-9.0 and 60-70 °C and at pH 10.5 and 55-60 °C, and keratinolytic activity that reached a maximum at pH 7.0-7.5 and 40-55 ºC and at pH 9.0 and 55 °C. Keratinolytic activity was maintained at approximately 63% of residual activity for 1 h at 50 °C. The caseinolytic activity at pH 10.5 remains stable until 1 h at 50 °C, and this is in contrast to the activity at pH 8.5, where the residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was inhibited by PMSF and EDTA. When investigating the application of C. byrsina peptidases as an additive to commercial detergent, we observed an egg stain removal performance that was similar to that demonstrated by the commercial detergent. CONCLUSIONS: Based on their activity and stability at alkaline pH, these enzymes appear to be attractive candidates for use in the detergent industry. Additionally, the collagenolytic activity of these enzymes potentially allows for their use in a wide array of industrial sectors that require collagenolytic enzymes, such as for the production of collagen hydrolysates from residues derived from the meat industry.

Citrobacter diversus-derived keratinases and their potential application as detergent-compatible cloth-cleaning agents.

Currently, poultry farming is one of the sectors that have a significant impact on the global economy. In recent years, there has been an increase in the production of broilers, inflicting this segment of the industry to generate tons of keratin due to huge disposal of chicken feathers. This points to the need to degrade these chicken feathers, as they have emerged as a major threat to the environment. Thus, in this study we aimed to identify keratinases that are produced by the bacterium Citrobacter diversus and further investigate the biochemical characteristics of these keratin-degrading enzymes. In a submerged medium, the bacterium was capable of degrading chicken feathers almost completely after 36 h of fermentation. We found a maximum caseinolytic activity at pH 9-10.5 and 50-55 °C, and keratinolytic activity at pH 8.5-9.5 and 50 °C. Thus, given its stability at higher temperatures, upon incubation of this enzyme extract for 1 h at 50 °C, it showed approximately 50% of the keratinolytic and 100% of the caseinolytic activity. Further, under pH stability for 48 h at 4 °C, the enzyme extract maintained greater residual activity in the pH range 6-8. Caseinolytic activity was inhibited by EDTA and PMSF, whereas the keratinolytic activity was inhibited only by EDTA. Additionally, due to its alkaline activity and detergent compatibility, this enzyme extract could improve washing performance when added to a commercial detergent formulation. Using application tests, we could demonstrate a potential use of this bacterial enzyme extract as an additive in detergents to remove egg stains from cloth.

A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris.

Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH 5.0 and 50 °C, and pH 5.0 and 60 °C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH 6.0 and 6.5 for 24 h at 4 °C, and sensitive to pepstatin A, ß-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.

Prospecting for L-arabinose/D-xylose symporters from Pichia guilliermondii and Aureobasidium leucospermi.

With the strong trend toward sustainable technologies, such as the gradual substitution of fossil fuel consumption, improvement in the utilization of sugars from lignocellulosic biomass appears to be an alternative for bioenergy. However, from a number of C5 sugars, few are used in fermentative processes for ethanol production. One of the reasons is because wild-type Saccharomyces cerevisiae is unable to efficiently co-utilize hexoses and pentoses via specific transporters for each type of sugar. Thus, a system of pentose uptake that is not modulated by D-glucose is required. Here, we were able to identify the presence of sugar/H+ symporters for D-xylose and L-arabinose, especially for Pichia guilliermondii, where an uptake of D-glucose via symporter was not detected. The best D-xylose uptake route in P. guilliermondii exhibited a KM of 48 mM and VMAX of 0.48 mmol h-1 g-1 at the early stationary phase (24 h). For L-arabinose, the best route of uptake exhibited a KM of 109 mM and VMAX of 0.8 mmol h-1 g-1 on log phase (12 h). The highest kinetic uptake was observed when the final pH of the medium was below 7. In general, an alkaline medium limited the expression of symporters. The results obtained in this study will help in the further investigation of these symporters through their overexpression in engineered S. cerevisiae.

Evaluation of the milk clotting properties of an aspartic peptidase secreted by Rhizopus microsporus.

Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by Rhizopus microsporus. The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 °C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 °C. Proteolytic activity was significantly reduced in the presence of Cu2+ and Al3+. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 ± 155 mM-1.s-1), Abz-KLRSSNQ-EDDnp (3,514 ± 130 mM-1.s-1), and Abz-KLRQSKQ-EDDnp (3,068 ± 386 mM-1.s-1). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca2+ and broad pH range of 4-7, in addition to its ability to efficiently clot milk.

Controlling proteolysis of Clp peptidase: a possible target for combating mitochondrial diseases.

Some mechanisms of cellular stress, aging, and apoptosis are related to proteolysis. With respect to ClpP, little is known about the mechanical manner in which the substrate is hydrolyzed in and released from the degradation chamber. Furthermore, what would be the real influence of ClpP in mammalian UPRmt?

Heterologous expression, purification and biochemical characterization of a new xylanase from Myceliophthora heterothallica F.2.1.4.

Myceliophthora heterothallica is a thermophilic fungus potentially relevant for the production of enzymes involved in the degradation of plant biomass. A xylanase encoding gene of this species was identified by means of RT-PCR using primers designed based on a xylanase coding sequence (GH11) of the fungus M. thermophila. The obtained gene was ligated to the vector pET28a(+) and the construct was transformed into Escherichia coli cells. The recombinant xylanase (r-ec-XylMh) was heterologously expressed, and the highest activity was observed at 55 °C and pH 6. The enzyme stability was greater than 70% between pH 4.5 and 9.5 and the inclusion of glycerol (50%) resulted in a significant increase in thermostability. Under these conditions, the enzyme retained more than 50% residual activity when incubated at 65 °C for 1 h, and approximately 30% activity when incubated at 70 °C for the same period. The tested cations did not increase xylanolytic activity, and the enzyme indicated significant tolerance to several phenolic compounds after 24 h, as well as high specificity for xylan, with no activity for other substrates such as CMC (carboxymethylcellulose), Avicel, pNPX (p-nitrophenyl-ß-D-xylopyranoside) and pNPA (p-nitrophenyl-α-L-arabinofuranoside), and is thus, of potential relevance in pulp bleaching.