Glycosylation and charge distribution orchestrates the conformational ensembles of a biotechnologically promissory phytase in different pHs - a computational study.

Phytases [myo-inositol(1,2,3,4,5,6) hexakisphosphate phosphohydrolases] are phytate-specific phosphatases not present in monogastric animals. Nevertheless, they are an essential supplement to feeding such animals and for human special diets. It is crucial, hence, the biotechnological use of phytases with intrinsic stability and activity at the acid pHs from gastric environments. Here we use Metadynamics (METADY) simulations to probe the conformational space of the Aspergillus nidulans phytase and the differential effects of pH and glycosylation in this same space. The results suggest that strategic combinations of pH and glycosylation affect the stability of native-like conformations and alternate these structures from a metastable to a stable profile. Furthermore, the protein segments previously reported as more thermosensitive in phytases from this family present a pivotal role in the conformational changes at different conditions, especially H2, H5-7, L8, L10, L12, and L17. Also, the glycosylations and the pH-dependent charge balance modulate the mobility and interactions at these same regions, with consequences for the surface solvation and active site exposition. Finally, although the glycosylations have stabilized the native structure and improved the substrate docking at all the studied pHs, the data suggest a higher phytate receptivity at catalytic poses for the unglycosylated structure at pH 6.5 and the glycosylated one at pH 4.5. This behavior agrees with the exact change in optimum pH reported for this enzyme, expressed on low or high glycosylating systems. We hope the results and insights presented here will be helpful in future approaches for rational engineering of technologically promising phytases and intelligent planning of their heterologous expression systems and conditions for use.Communicated by Ramaswamy H. Sarma.

Recent Patents on the Industrial Application of Biosurfactants.

BACKGROUND: Biosurfactants are natural bioactive compounds produced from fungi, bacteria and plants. These molecules have several properties that enable them to be involved in various industrial applications. The surface-active properties of biosurfactants allow their use in various sectors, such as agriculture, health, cosmetics, bioremediation/ petroleum, mining, and bioprocess. OBJECTIVE: The objective of this study was to analyze the patents deposited in intellectual property databases for applications of biosurfactants in the areas of agriculture, health, cosmetics, bioremediation/petroleum, mining, and bioprocesses, with the research period consisting of the last six years. METHODS: This study targeted the use of biosurfactants in various industrial sectors. The patent search was carried out using the Google Patents platform, a platform that is commonly used for this purpose and freely accessible. RESULTS: During the search for patents related to applications of biosurfactants in industry filed in the last 6 years, results were found to be distributed in the following areas: agriculture (70), cosmetics (2311), bioremediation/petroleum (179), health (1794), mining (5), and bioprocess (133). Six patents were selected from each area, except for health and mining, for which the search resulted in only 5 and 4 patents, respectively, to be discussed and provide information on the biotechnological applications of biosurfactants in the industry. CONCLUSION: The present study showed a wide area of application of biosurfactants in industry. The interest in the inclusion of biosurfactants in the industry is directly related to the need for more sustainable solutions to solve real market problems. The cosmetics sector presented the most patents that employ the use of biosurfactants.

Development and validation of an analytical method by HPLC-DAD for determination of caffeine in products based on guarana extracts (Paullinia cupana)

Abstract Guarana (Paullinia cupana) is a native plant from the Amazon whose seeds contain a high concentration of caffeine. Aqueous extract of guarana is widely used in the world. In this study, the objective was to develop and validate a High-Performance Liquid Chromatography method for the determination of caffeine in extracts and commercial beverages based on guarana. A sensitive, simple, and viable high performance liquid chromatographic method without the need of an analyte extraction procedure was developed and validated according to Brazilian and international requirements. The method presented high performance, fulfilling Brazilian and international requirements, in addition to allowing product compliance tests. Results confirmed high selectivity and linearity (>0.999) between 5 to 135 ug/mL, with no significant matrix effect. Detection and quantification limits were 0.02 µg/mL and 2 µg/mL, respectively. Precision was less than 4 %, and accuracy varied from 99.9-120 %. Applicability of the method was demonstrated by conducting a limited evaluation in products containing caffeine. Commercial extracts showed quite different caffeine levels, while carbonated drinks follow Brazilian and American recommendations. Our results indicate that the developed method can be used to evaluate the quality of the guarana extract and of products containing caffeine

Optimization of bioprocess of Schleiferilactobacillus harbinensis Ca12 and its viability in frozen Brazilian berries (Açai, Euterpe oleracea Mart.).

Amazonian palm berries (açaí, Euterpe oleracea Mart.) are fruits with high nutritional value and antioxidant activity and have aroused the interest of consumers, popularizing fruit pulps enriched with probiotics. Amazonian palm berries (açaí, Euterpe oleracea Mart.) are fruits with high nutritional potential, providing a source of carbohydrates, fibers, proteins, lipids, vitamins, and minerals. Furthermore, açai provides several health benefits, including antioxidant activity. Nutritionally enhanced foods have aroused the interest of consumers, popularizing fruit pulps enriched with probiotics. Probiotics are dietary supplements consisting of live, beneficial microorganisms in the host which improve the intestinal microbiota. The objective of this study was to isolate, identify, and characterize the probiotic potential of an isolated Schleiferilactobacillus harbinensis strain (dubbed Ca12) and provide an optimized bioprocess for its production, using the complete factorial and central rotational compound design to supplement the frozen açai pulp. The isolated strain S. harbinensis Ca12 presented adequate resistance to gastric juice and bile salts, microbial activity against different Candida strains, self-aggregation and coaggregation properties, high adhesion in HT-29 cells, and 35% inhibition of Salmonella in HT-29 cells. When optimized, the cellular biomass production of the S. harbinensis Ca12 strain was approximately 600% higher than the unsupplemented whey, with a production of 3.6 × 1010 CFU mL-1. The S. harbinensis Ca12 strain's viability in the creamy and traditional frozen açai pulp was shown to be stable for up to 6 months at 20 °C. The impact of this study involved for the first time the S. harbinensis Ca12 described in the Brazilian cocoa pulp with activity against Candida albicans of clinical importance, creating the potential of a new functional food with important benefits to human health as prevention for candidiasis.

Recent Patents on Impact of Lipopeptide on the Biofilm Formation onto Titanium and Stainless Steel Surfaces.

BACKGROUND: Numerous causes of infection in arthroplasties are related to biofilm formation on implant surfaces. In order to circumvent this problem, new alternatives to prevent bacterial adhesion biosurfactants-based are emerging due to low toxicity, biodegradability and antimicrobial activity of several biosurfactants. We revised all patents relating to biosurfactants of applicability in orthopedic implants. METHODS: This work aims to evaluate the capability of a lipopeptide produced by Bacillus subtilis ATCC 19659 isolates acting as inhibitors of the adhesion of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 onto titanium and stainless steel surfaces and its antimicrobial activity. RESULTS: The adhesion of the strains to the stainless-steel surface was higher than that of titanium. Preconditioning of titanium and stainless-steel surfaces with 10 mg mL-1 lipopeptide reduced the adhesion of E. coli by up to 93% and the adhesion of S. aureus by up to 99.9%, suggesting the strong potential of lipopeptides in the control of orthopedic infections. The minimal inhibitory concentration and minimum bactericidal concentration were 10 and 240 µg mL-1 for E. coli and S. aureus, respectively. CONCLUSION: The lipopeptide produced by Bacillus subtilis ATCC 19659 presented high biotechnological application in human health against orthopedic implants infections.

Anti-nutritional factors and digestibility of protein in Cayocar brasiliense seeds

Abstract The Brazilian Cerrado presents a wide variety of natural products, including the Pequi (Caryocar brasiliense). The important factor that determines the dietary and nutritional ideality of protein is degestitibility. This work aims to evaluate the protein digestibility of Pequi seeds and the presence of anti-nutritional factors. The protein Pequi almonds were extracted, toasted and untoasted. Evaluation digestibility was structurally characterized by SDS-PAGE 15%, which can be used to analyze simulated gastric fluid (SFG) and digestion in intestinal fluid (SFI) in the presence of pepsin and trypsin/chymotrypsin. The extract from Pequi almonds showed inhibitory activity and was not detected hemagglutination. The intensity of the bands according to 2S albumins, after heat treatment, did not decrease in comparison to the condition native, significantly. In the crisp crude extract, which was incubated under SGF and SIF, the intensity of the corresponding bands at 8 and 3 kDa was resistant to SGF and indigestible after 4 hours under SIF. The characterization of Pequi almonds demonstrated that the product of the crude extract has anti-nutritional factors, which were confirmed by evaluating digestibility.

Overproduction of polygalacturonase by Penicillium griseoroseum recombinant strains and functional analysis by targeted disruption of the pgg2 gene.

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production.

Bioconversion of biodiesel refinery waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2.

BACKGROUND: The microbial bioemulsifiers was surface active compounds, are more effective in stabilizing oil-in-water emulsions. The yeasts have been isolated to produce bioemulsifiers from vegetable oils and industrial wastes. RESULTS: Trichosporon mycotoxinivorans CLA2 is bioemulsifier-producing yeast strain isolated from effluents of the dairy industry, with ability to emulsify different hydrophobic substrates. Bioemulsifier production (mg/L) and the emulsifying activity (E24) of this strain were optimized by response surface methodology using mineral minimal medium containing refinery waste as the carbon source, which consisted of diatomaceous earth impregnated with esters from filters used in biodiesel purification. The highest bioemulsifier production occurred in mineral minimal medium containing 75 g/L biodiesel residue and 5 g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58 g/L biodiesel refinery residue and 4.6 g/L ammonium sulfate, and under these conditions, the model estimated an emulsifying activity of 85%. Gas chromatography and mass spectrometry analysis suggested a bioemulsifier molecule consisting of monosaccharides, predominantly xylose and mannose, and a long chain aliphatic groups composed of octadecanoic acid and hexadecanoic acid at concentrations of 48.01% and 43.16%, respectively. The carbohydrate composition as determined by GC-MS of their alditol acetate derivatives showed a larger ratio of xylose (49.27%), mannose (39.91%), and glucose (10.81%). 1 H NMR spectra confirmed by COSY suggested high molecular weight, polymeric pattern, presence of monosaccharide's and long chain aliphatic groups in the bioemulsifier molecule. CONCLUSIONS: The biodiesel residue is an economical substrate, therefore seems to be very promising for the low-cost production of active emulsifiers in the emulsification of aromatics, aliphatic hydrocarbons, and kerosene.

Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum

Protoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and pectin lyase production (1.2-fold).

Fusões de protoplastos entre linhagens mutantes auxotróficas e morfológicas complementares de Penicillium griseoroseum e P. expansum foram induzidas por polietilenoglicol e íons cálcio (Ca2+). Fusionantes foram obtidos em meio mínimo e uma linhagem prototrófica, possivelmente diplóide, foi selecionada para a haploidização com o fungicida benomil. Diferentes linhagens recombinantes foram isoladas e caracterizadas quanto à presença de mutações auxotróficas e a produção de enzimas pectinolíticas. O fusionante prototrófico não apresentou maior atividade de pectinases em relação às linhagens parentais, entretanto, entre 29 recombinantes analisados, quatro apresentaram maiores atividades enzimáticas. O recombinante RGE27, o qual possui as mesmas mutações auxotróficas e morfológicas que a linhagem parental de P. griseoroseum, apresentou um aumento considerável na produção de poligalacturonase (3 vezes) e de pectina liase (1,2 vezes).