Use In Biotechnological Processes Research Articles

Antimicrobial activity and chemical constituents of essential oils and oleoresins extracted from eight pepper species

ABSTRACT: Essential oils are the most important compounds produced during secondary metabolism in aromatic plants. Essential oils are volatile, have characteristic odor and are used as defensive agents by plants. In pepper, it is possible to say that essential oils are the “flavor fingerprint” of each species. In the present article, eight species of pepper were studied in order to extract their essential oils and oleoresins, test their antibacterial and antifungal activities and also to identify the compounds present in the most bioactive samples. Results demonstrated that two essential oils [Pimenta dioica (L.) Merr. and Schinus terebinthifolius] and three oleoresins (Schinus terebinthifolius and Piper nigrum white and black) recorded significant antimicrobial activity. These active essential oils and oleoresins are interesting for use in biotechnological processes employed in food, pharmaceutical and other industries.

Exogenous enzymes in dairy technology: acidic proteases from processing discards of shrimp Pleoticus muelleri and their use as milk‐clotting enzymes for cheese manufacture

SummaryArgentina is the seventh largest world producer of cheese. Owing to the profitability of this industry, more than a thousand of dairy companies and farms exclusively produce cheeses, and, thus, this activity utilises 42% of total raw milk production of the country. Due to the increase in cheese demand, and because calf rennet is relatively expensive, this has motivated the study of new sources of proteases due to the potential use in biotechnological processes. Previously, crustacean proteases have been considered as good rennet substitutes for milk coagulation during the cheese making process. The aim of this study was to evaluate and characterise acidic proteases obtained from Pleoticus muelleri discards and their proteolytic capability as well as their potential use as a milk‐clotting agent. The activity of aspartic proteases and the satisfactory acceptation by the taste panel for cheese made with shrimp's enzymes suggest that P. muelleri proteins offer a high potential for use in dairy biotechnological processes with potential use as milk‐clotting agents for cheese making.

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

The application of enzymes for the removal of dyes from textile effluents

Dwindling water resources is a global problem. The effective effluent treatment is an important step towards conserving our water resources. Some pollutants such as dyestuffs resist degradation by conventional treatment methods and persist in the environment. Dye wastes represent one of the most problematic groups of pollutants because they can be easily identified by the human eye and are not easily biodegradable. The use of biotechnological processes in textile dye effluents treatment has attracted considerable attention and achieved interesting results in recent years. This work describes the use of enzymes as an alternative method for the treatment of such pollutants. Noteworthy processes for the treatment of dye waste include a biological treatment, filtration, catalytic oxidation, a sorption process and the combination of treatments. This emphasizes the need for current and future research with the focus on developing economically feasible and environmentally sustainable wastewater treatment practices.

Produção e caracterização de biomassa extracelular obtida por fermentação submersa usando Lasidioplodia theobromae isolado do cacau

Gums produced by microorganisms are used as food improvement agents and the residual biomass is used for the isolation of their constituents. The objective of this work was to produce and characterize fractions of extracellular biomass in submerged fermentation with Lasidioplodia theobromae isolated from cocoa of Bahia with descending death. We tested four carbon sources fermented at 28 oC, 180 rpm for 72 h. The one resulting in greater production of biomass was evaluated with regard to changes in concentration and pH. The fractions obtained were characterized by infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA), percentage composition and monosaccharide. The submerged fermentation by L. theobromae with commercial sucrose at 40 g.L-1 and pH 4.0 led to a maximum production of fraction I (8.64 g.L-1), while the fermentation at 50 g.L-1 and pH 5.0 yielded maximum production of fraction II (23.69 g.L-1). The FTIR spectra indicated the presence of amino groups, polyols and esters. Three weight loss events with different intensities were observed in the thermogravimetric analysis. The fractions have the same composition, but with different proportions, being sources of protein (19.88-29.45%), lipids (11.07-28.79%), ash (3.55-3.88%), and carbohydrates (30.16-37.96%) made only of glucose and mannose, i.e. they are both glucomannans. The fractions have desirable properties for wide use in biotechnological processes of high scientific relevance.

Canola Cake as a Potential Substrate for Proteolytic Enzymes Production by a Selected Strain of Aspergillus oryzae: Selection of Process Conditions and Product Characterization.

Oil cakes have excellent nutritional value and offer considerable potential for use in biotechnological processes that employ solid-state fermentation (SSF) for the production of high value products. This work evaluates the feasibility of using canola cake as a substrate for protease production by a selected strain of Aspergillus oryzae cultivated under SSF. The influences of the following process parameters were considered: initial substrate moisture content, incubation temperature, inoculum size, and pH of the buffer used for protease extraction and activity analysis. Maximum protease activity was obtained after cultivating Aspergillus oryzae CCBP 001 at 20°C, using an inoculum size of 107 spores/g in canola cake medium moistened with 40 mL of water to 100 g of cake. Cultivation and extraction under selected conditions increased protease activity 5.8-fold, compared to the initial conditions. Zymogram analysis of the enzymatic extract showed that the protease molecular weights varied between 31 and 200 kDa. The concentrated protease extract induced clotting of casein in 5 min. The results demonstrate the potential application of canola cake for protease production under SSF and contribute to the technological advances needed to increase the efficiency of processes designed to add value to agroindustrial wastes.

English

A total of 842 spore-forming strains were isolated from 221 animal feces samples, in which a new feather-degrading bacterium identified as Bacillus sp. 50-3 based on morphological, biochemical and 16S rDNA tests was isolated from Calotes versicolor (an agamid lizard) feces. The bacterium can degrade native feather completely at mild conditions and in much shorter time (36 h) when using chicken feather as the sole carbon and nitrogen source. It presented optimum growth and maximum keratinase activity (680±25 U/ml, 36 h) at 37°C and pH 7.0 in feather meal medium. The keratinase of Bacillus sp. 50-3 was active on feather keratin as substrate and presented optimum additive quantity of 1.0% (w/v). So the high efficiency of Bacillus sp. 50-3 on feather-degradation suggested its potential use in biotechnological processes, especially in decreasing the environment pollution. Key words: Animal feces, Bacillus, chicken feathers, keratinase, optimum conditions.

Isolation and Characterization of Amylase Producing Yeasts and Improvement of Amylase Production

Objectives: Because most of the yeasts from nature are not harmful as compared to bacteria, interest in yeasts with potential use in biotechnological processes has increased in recent years. Therefore, in this study, we aimed to isolate and characterize amylase producing yeasts and investigate the effects of different parameters on the production of amylase. Methods: Five different sources were used for the isolation of the yeasts. For screening of the amylase production, the isolates were incubated in Amylase Activity Medium at 30°C for 3 days. The amylase activity was determined by dinitrosalicylic acid method. Molecular char- acterization of the amylase producing yeasts was performed with restriction fragment length polymorphism (RFLP) of the 5.8S and 18S rRNA regions, and sequence analysis of D1/D2 domain of the 26S rRNA region. Results: In total, twenty five yeast isolates were obtained from five different sources. Fol- lowing the incubation in medium containing starch for the screening of amylase production, it was found that 12 yeast isolates produce amylase, and among the isolates, three of them showed the highest amylase activity. The isolates (19-3, 19-6 and 19-7) having the highest activity were identified as S. fibuligera. The strains showed the highest amylase production at 30°C and pH 5.5. Among additives, sodium chloride, polyethylene glycol, sodium dodecyl sulfate, Triton X-100 and Tween 80 stimulated the production of amylase. Other additives showed negative influence on the production of enzyme. Conclusion: The study revealed that favorable fermentation conditions and the selection of suitable growth parameters played key roles in the production of amylase by S. fibuligera.

New Trends in Biotechnological Processes to Increase the Environmental Protection

A sustainable development can be achieved by deepening into more effective and eco-friendly products and technologies. From this point of view, the development of biotechnological processes to increase the environmental protection could be included in the best available techniques reference documents, the so-called BREFs, that cover, as far as practicable, the industrial activities to achieve an integrated pollution prevention and control. Members of the European Union are required to take these documents into account when determining the best available techniques, generally or in specific cases under the European Commission Directives. In order to include biotechnological processes into the BREFs, for example, for obtaining food and pharmaceutical additives, these products have to be cost competitive with those synthesized by chemical ways. Biotechnological processes are advantageous compared to the chemical ones since various metabolites can be obtained simultaneously in the same process, and these metabolites are more eco-friendly than their chemical counterparts. Additionally, this feature also matches the increasing demand of consumers for natural products, which has intensified the biotechnological production of natural additives. This special issue reports advances in the use of biotechnological processes for the treatment of contaminated soil or water as well as the revalorization of agroindustrial residues through the production of valuable metabolites such as biosurfactants or antioxidants, with applications in biomedicine, food industry, pharmaceutical industry, or environmental bioremediation. Therefore, to avoid or at least to reduce the use of synthetic additives, it is necessary to identify natural alternative sources of food and pharmaceutical additives. For instance, in the recent years, there has been a growing interest in the use of natural antioxidants in the food industry not only for application as preservatives but also due to their benefits to human health. Moreover, the brewery industry generates waste that could be used to produce natural extracts containing bioactive phenolic compounds, which can be further used as natural antioxidants with potential applications in the food, cosmetic, and pharmaceutical industries. On the other hand, water and soil pollution has become a significant environmental issue around the world. Toxic substances, such as endocrine disruptors, heavy metals, and excessive inflows of phosphorus, nitrogen, and other elements, all contribute to the water and soil pollution. Biotechnological processes, as well as metabolites extracted from plants, can be used to treat those contaminations enabling the use of mild operating conditions, as compared with other physical and chemical treatments, thus permitting a sustainable development. Accordingly, anammox bacteria can be used for the elimination of ammonium in wastewater, whereas phosphorus can be removed from water by using transgenic plants, which hyperaccumulate inorganic phosphate. In addition, in this special issue it has been showed that epipsammic biofilms can play an important role in the environmental quality of river systems, increasing arsenic and phosphorous retention by the system, especially in environments where both arsenic and phosphorus occur simultaneously, whereas vegetables and vegetable residues can be used as sources of enzymes capable of removing colored compounds from water. Regarding soil bioremediation, biosurfactants produced by Lactobacillus pentosus were found to improve the solubilization of hydrocarbons in the water phase of soil, leading to even better results than those reached with common chemical surfactants like sodium dodecyl sulfate.

Bioprocessing of "Hair Waste" by Paecilomyces lilacinus as a Source of a Bleach-Stable, Alkaline, and Thermostable Keratinase with Potential Application as a Laundry Detergent Additive: Characterization and Wash Performance Analysis.

Paecilomyces lilacinus (Thom) Samson LPS 876, a locally isolated fungal strain, was grown on minimal mineral medium containing “hair waste,” a residue from the hair-saving unhairing process, and produced a protease with keratinolytic activity. This enzyme was biochemically characterized. The optimum reaction conditions, determined with a response surface methodology, were 60°C and pH 6.0. It was remarkably stable in a wide range of pHs and temperatures. Addition of Ca2+, Mg2+, or sorbitol was found to be effective in increasing thermal stability of the protease. PMSF and Hg2+ inhibited the proteolytic activity indicating the presence of a thiol-dependent serine protease. It showed high stability toward surfactants, bleaching agents, and solvents. It was also compatible with commercial detergents (7 mg/mL) such as Ariel, Skip, Drive, and Ace, retaining more than 70% of its proteolytic activity in all detergents after 1 h of incubation at 40°C. Wash performance analysis revealed that this protease could effectively remove blood stains. From these properties, this enzyme may be considered as a potential candidate for future use in biotechnological processes, as well as in the formulation of laundry detergents.

Biodegradation of a Keratin Waste and the Concomitant Production of Detergent Stable Serine Proteases from Paecilomyces lilacinus

Paecilomyces lilacinus (LPS 876) efficiently degraded keratin in chicken feather during submerged cultivation producing extracellular proteases. Characterization of crude protease activity was done including its compatibility in commercial detergents. Optimum pH and temperature were 10.0 and 60 °C, respectively. Protease activity was enhanced by Ca²⁺ but was strongly inhibited by PMSF and by Hg²⁺ suggesting the presence of thiol-dependent serine proteases. The crude protease showed extreme stability toward non-ionic (Tween 20, Tween 85, and Triton X-100) and anionic (SDS) surfactants, and relative stability toward oxidizing agent (H₂O₂ and sodium perborate). In addition, it showed excellent stability and compatibility with various solid and liquid commercial detergents from 30 to 50 °C. The enzyme preparation retained more than 95% of its initial activity with solid detergents (Ariel™ and Drive™) and 97% of its original activity with a liquid detergent (Ace™) after pre-incubation at 40 °C. The protective effect of polyols (propylene glycol, PEG 4000, and glycerol) on the heat inactivation was also examined and the best results were obtained with glycerol from 50 to 60 °C. Considering its promising properties, P. lilacinus enzymatic preparation may be considered as a candidate for use in biotechnological processes (i.e., as detergent additive) and in the processing of keratinous wastes.

Вплив іонів важких металів і цинкового комплексу на динаміку росту бактерій роду Pseudomonas

Проблема очищення стічних вод – одне з найактуальніших питань із початку століття. Особливе місце важких металів серед забруднювачів довкілля пов’язане з їх накопиченням в організмі та передачею трофічними ланцюгами, високою їх токсичністю. Розроблено сучасні технології очищення стічних вод: перспективи мають природні та найдешевші біологічні методи очищення, що являють собою інтенсифікацію природних процесів розкладання органічних сполук мікроорганізмами в аеробних або анаеробних умовах. Одні з основних – процеси нітрифікації та денітрифікації. Велика увага приділяється мікроорганізмам, здатним виконувати ці процеси. Процеси взаємодії між іонами важких металів і мікроорганізмами мають велике значення не тільки для фундаментальної науки, а і як можливе застосування у біотехнологічних процесах.

Production and properties of keratinolytic proteases from three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils

The keratinolytic potential and protease properties of three novel Gram-negative feather-degrading bacteria isolated from Brazilian soils was described. Aeromonas hydrophila K12, Chryseobacterium indologenes A22 and Serratia marcescens P3 were able to degrade feather meal, producing high amounts of soluble proteins and forming thiol groups. The proteases of strains K12, A22 and P3 had optimal pH of 8.0, 7.5 and 6.0, respectively; this last is an uncommon feature for bacterial keratinases. The optimal temperature was in the range 45-55°C. All three proteases were active towards azokeratin and were inhibited by EDTA, suggesting that they are keratinolytic metalloproteases. The proteolytic activity of K12 was stimulated by organic solvents and the detergent SDS, suggesting its potential application for detergent formulations and peptide synthesis. Strains A22, K12 and P3 have great potential for use in biotechnological processes involving hydrolysis of keratinous byproducts.

In-depth analysis of the chicken egg white proteome using an LTQ Orbitrap Velos

BackgroundHen's egg white has been the subject of intensive chemical, biochemical and food technological research for many decades, because of its importance in human nutrition, its importance as a source of easily accessible model proteins, and its potential use in biotechnological processes. Recently the arsenal of tools used to study the protein components of egg white has been complemented by mass spectrometry-based proteomic technologies. Application of these fast and sensitive methods has already enabled the identification of a large number of new egg white proteins. Recent technological advances may be expected to further expand the egg white protein inventory.ResultsUsing a dual pressure linear ion trap Orbitrap instrument, the LTQ Orbitrap Velos, in conjunction with data analysis in the MaxQuant software package, we identified 158 proteins in chicken egg white with two or more sequence unique peptides. This group of proteins identified with very high confidence included 79 proteins identified in egg white for the first time. In addition, 44 proteins were identified tentatively.ConclusionsOur results, apart from identifying many new egg white components, indicate that current mass spectrometry technology is sufficiently advanced to permit direct identification of minor components of proteomes dominated by a few major proteins without resorting to indirect techniques, such as chromatographic depletion or peptide library binding, which change the composition of the proteome.

Characterization of detergent stable and feather degrading serine proteases from Bacillus mojavensis A21

The present study describes the characterization of crude protease from Bacillus mojavensis A21 and its evaluation in detergent and chicken feathers hydrolysis. The strain was found to produce at least six major extracellular proteases as shown by casein-zymography. The optimum pH and temperature for proteolytic activity were 8.0–11.0 and 60 °C, respectively. The crude protease showed extreme stability towards non-ionic (5% Tween 80 and 5% Triton X-100) and anionic (1% SDS) surfactants, and relative stability towards oxidizing agents. Additionally, it showed excellent stability and compatibility with various solid (7 mg/ml) and liquid (1%; v/v) detergents at temperatures from 30 to 50 °C. In the presence of solid detergents, the enzyme preparation retained 100% of its initial activity after pre-incubation for 1 h at 40 °C with Axion and Ariel, followed by Nadhif (87%), Dixan (85%) and New Det (82%). With liquid detergents, the enzyme preparation retained 100% of its original activity after pre-incubation for 1 h at 40 °C with Dixan and Nadhif. Wash performance analysis revealed that A21 crude protease could effectively remove blood-stains. In addition, B. mojavensis A21 proteolytic preparation showed important feather degrading activity. Considering its promising properties, B. mojavensis A21 enzymatic preparation may be considered a potential candidate for future use in biotechnological processes, particularly in detergent and in the processing of poultry waste.

Estudios de Caracterización Cinética y Fisicoquímica de una Proteinasa Aspártica Aislada de Frutos Maduros de Salpichroa origanifolia

A new proteolytic enzyme with milk-clotting activity was isolated from ripe fruits of Salpichroa origanifolia. Crude extract was obtained by dissolving the ethanolic powder of the fruits in 50 mM phosphate buffer pH 7.0. The enzyme presented proteolytic activity toward denatured protein substrates. Hemoglobin and casein hydrolysis were optimal in the pH range 3.5 to 6.5 at temperatures between 40 and 45°C. In addition, the crude extract showed high stability at moderate temperatures (40°C) and the proteinase activity was activated by cations such as calcium and magnesium. Degradation pattern of the bovine casein brought about by the crude extract of Salpichroa origanifolia produced higher hydrolysis degree than the commercial rennet. According to these results, it is concluded that the aspartic proteinase isolated from ripe fruits of Salpichroa origanifolia presents appropriate milk-clotting activity and thermostability properties for its use in biotechnological processes.

Isolation and characterization of a new Bacillus sp. 50-3 with highly alkaline keratinase activity from Calotes versicolor faeces

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkali-stable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.

Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening

Molecular evolution is a powerful tool for improving or changing activities of enzymes for their use in biotechnological processes. Cytochromes P450 are highly interesting enzymes for biotechnological purposes because they are able to hydroxylate a broad variety of substrates with high regio- and stereoselectivity. One promising steroid hydroxylating cytochrome P450 for biotechnological applications is CYP106A2 from Bacillus megaterium ATCC 13368. It is one of a few known bacterial cytochromes P450 able to transform steroids such as progesterone and 11-deoxycortisol. CYP106A2 can be easily expressed in Escherichia coli with a high yield and can be reconstituted using the adrenal redox proteins, adrenodoxin and adrenodoxin reductase. We developed a simple screening assay for this system and performed random mutagenesis of CYP106A2, yielding variants with improved 11-deoxycortisol and progesterone hydroxylation activity. After two generations of directed evolution, we were able to improve the k (cat)/K (m) of the 11-deoxycortisol hydroxylation by a factor of more than four. At the same time progesterone conversion was improved about 1.4-fold. Mapping the mutations identified in catalytically improved CYP106A2 variants into the structure of a CYP106A2 model suggests that these mutations influence the mobility of the F/G loop, and the interaction with the redox partner adrenodoxin. The results show the evolution of a soluble steroid hydroxylase as a potential new catalyst for the production of steroidogenic compounds.

Bacterial Keratinases: Useful Enzymes for Bioprocessing Agroindustrial Wastes and Beyond

Keratin-rich wastes in the form of feathers, hair, nails, and horn are highly available as byproducts of agroindustrial processing. The increased needs for energy conserving and recycling, summed with the huge increase in poultry industry, have strongly stimulated the search for alternatives for the management of recalcitrant keratinous wastes. Keratinases, which are produced by several bacteria that have been often isolated from soils and poultry wastes, show potential use in biotechnological processes involving keratin hydrolysis. Although these isolates are mostly restricted to the genera Streptomyces and Bacillus, the diversity of keratinolytic bacteria is significantly greater. Bacterial keratinases are mostly serine proteases, although increased information about keratinolytic metalloproteases, particularly from Gram-negative bacteria, became available. These enzymes are useful in processes related with the bioconversion of keratin waste into feed and fertilizers. Other promising applications have been associated with keratinolytic enzymes, including enzymatic dehairing for leather and cosmetic industry, detergent uses, and development of biopolymers from keratin fibers. The use of keratinases to enhance drug delivery in some tissues and hydrolysis of prion proteins arise as novel outstanding applications for these enzymes.

New Feather-Degrading Filamentous Fungi

Among 106 filamentous fungi isolated from poultry farm waste, 13 species belonging to seven genera (Aspergillus, Acremonium, Alternaria, Beauvaria, Curvularia, Paecilomyces, and Penicillium) were able to grow and produce keratinase in stationary cultures using poultry feather powder as the only substrate. The four most efficient keratinase producers were selected for a comparative study of keratinase production in submerged and stationary conditions. The highest keratinolytic activities were produced after 4-6 days of cultivation in submerged conditions: 53.8 +/- 6.1 U/mL (Alternaria tenuissima), 51.2 +/- 5.4 U/mL (Acremonium hyalinulum), 55.4 +/- 5.2 U/mL (Curvularia brachyspora), and 62.8 +/- 4.8 U/mL (Beauveria bassiana). These novel nondermatophytic keratinolytic fungi have potential use in biotechnological processes involving keratin hydrolysis. The results of this work contribute to show that keratinolytic activity is relatively widespread among common filamentous fungi and may have an important rule in feather decomposition in natural settings.