Biotransformation of Phytosterols into Androstenedione-A Technological Prospecting Study.

Androstenedione (AD) is a key intermediate in the body's steroid metabolism, used as a precursor for several steroid substances, such as testosterone, estradiol, ethinyl estradiol, testolactone, progesterone, cortisone, cortisol, prednisone, and prednisolone. The world market for AD and ADD (androstadienedione) exceeds 1000 tons per year, which stimulates the pharmaceutical industry's search for newer and cheaper raw materials to produce steroidal compounds. In light of this interest, we aimed to investigate the progress of AD biosynthesis from phytosterols by prospecting scientific articles (Scopus, Web of Science, and Google Scholar databases) and patents (USPTO database). A wide variety of articles and patents involving AD and phytosterol were found in the last few decades, resulting in 108 relevant articles (from January 2000 to December 2021) and 23 patents of interest (from January 1976 to December 2021). The separation of these documents into macro, meso, and micro categories revealed that most studies (articles) are performed in China (54.8%) and in universities (76%), while patents are mostly granted to United States companies. It also highlights the fact that AD production studies are focused on "process improvement" techniques and on possible modifications of the "microorganism" involved in biosynthesis (64 and 62 documents, respectively). The most-reported "process improvement" technique is "chemical addition" (40%), which means that the addition of solvents, surfactants, cofactors, inducers, ionic liquids, etc., can significantly increase AD production. Microbial genetic modifications stand out in the "microorganism" category because this strategy improves AD yield considerably. These documents also revealed the main aspects of AD and ADD biosynthesis: Mycolicibacterium sp. (basonym: Mycobacterium sp.) (40%) and Mycolicibacterium neoaurum (known previously as Mycobacterium neoaurum) (32%) are the most recurrent species studied. Microbial incubation temperatures can vary from 29 °C to 37 °C; incubation can last from 72 h to 14 days; the mixture is agitated at 140 to 220 rpm; vegetable oils, mainly soybean, can be used as the source of a mixture of phytosterols. In general, the results obtained in the present technological prospecting study are fundamental to mapping the possibilities of AD biosynthesis process optimization, as well as to identifying emerging technologies and methodologies in this scenario.

Palm oil wastes as feedstock for lipase production by Yarrowia lipolytica and biocatalyst application/reuse.

Palm oil production chain generates a greasy residue in the refining stage, the Palm Oil Deodorizer Distillate (PODD), mainly composed of free fatty acids. Palm oil is also used industrially to fry foods, generating a residual frying oil (RFO). In this paper, we aimed to produce lipase from palm agro-industrial wastes using an unconventional yeast. RFO_palm, from a known source, consisted of 0.11% MAG + FFA, 1.5% DAG, and 97.5 TAG, while RFO_commercial, from a commercial restaurant, contained 6.7% of DAG and 93.3% of TAG. All palm oil wastes were useful for extracellular lipase production, especially RFO_commercial that provided the highest activity (4.9 U/mL) and productivity (465 U/L.h) in 75 h of processing time. In 48 h of process, PODD presented 2.3 U/mL of lipase activity and 48.5 U/L.h of productivity. RFO_commercial also showed the highest values for lipase associated to cell debris (843 U/g). This naturally immobilized biocatalyst was tested on hydrolysis reactions to produce Lipolyzed Milk Fat and was quite efficient, with a hydrolysis yield of 13.1% and 3-cycle reuse. Therefore, oily palm residues seem a promising alternative to produce lipases by the non-pathogenic yeast Y. lipolytica and show great potential for industrial applications.

Waste soybean frying oil for the production, extraction, and characterization of cell-wall-associated lipases from Yarrowia lipolytica.

The lipolytic yeast Yarrowia lipolytica produces cell-wall-associated lipases, namely Lip7p and Lip8p, that could have interesting properties as catalyst either in free (released lipase fraction-RLF) or cell-associated (cell-bound lipase fraction-CBLF) forms. Herein, a mixture of waste soybean frying oil, yeast extract and bactopeptone was found to favor the enzyme production. Best parameters for lipase activation and release from the cell wall by means of acoustic wave treatment were defined as: 26 W/cm2 for 1 min for CBLF and 52 W/cm2 for 2 min for RLF. Optimal pH and temperature values for lipase activity together with storage conditions were similar for both the free enzyme and cell-associated one: pH 7.0; T = 37 °C; and > 70% residual activity for 60 days at 4, - 4 °C and for 15 days at 30 °C.

Catalytic and physical features of a naturally immobilized Yarrowia lipolytica lipase in cell debris (LipImDebri) displaying high thermostability.

Lipase activity (337 U/g dry weight of cell debris) was detected in cell debris after ultrasound treatment of Yarrowia lipolytica cells cultivated in residual frying palm oil. It is a naturally immobilized lipase with protein content of 47%, herein called LipImDebri. This immobilized biocatalyst presents low hydrophobicity (8%), that can be increased adjusting pH and buffer type. Despite apparent intact cells, electron microscopy showed a shapeless and flat surface for LipImDebri and optical microscopy revealed no cell viability. Besides, an inferior mean diameter (3.4 mm) in relation to whole cells reveals structure modification. A high negative zeta potential value (- 33.86 mV) for pH 6 and 25 °C suggests that LipImDebri is a stable suspension in aqueous solution. Fourier Transform Infrared Spectra (FTIR) expose differences between LipImDebri and extracellular lipase extract signaling a physical interaction between enzyme and cell debris, which is possibly the reason for the high thermostability (k d = 0.246 h-1; t 1/2 = 2.82 h at 50 °C, pH 7.0). A good adjustment of LipImDebri kinetic data with Hill equation (R 2 = 0.95) exposes an allosteric behavior related to the presence of more than one lipase isoform. These features reveal that LipImDebri can be a good catalyst for industrial applications.

Yarrowia lipolytica Adhesion and Immobilization onto Residual Plastics.

Research in cell adhesion has important implications in various areas, such as food processing, medicine, environmental engineering, biotechnological processes. Cell surface characterization and immobilization of microorganisms on solid surfaces can be performed by promoting cell adhesion, in a relatively simple, inexpensive, and quick manner. The adhesion of Yarrowia lipolytica IMUFRJ 50682 to different surfaces, especially potential residual plastics (polystyrene, poly(ethylene terephthalate), and poly(tetrafluoroethylene)), and its use as an immobilized biocatalyst were tested. Y. lipolytica IMUFRJ 50682 presented high adhesion to different surfaces such as poly(tetrafluoroethylene) (Teflon), polystyrene, and glass, independent of pH, and low adhesion to poly(ethylene terephthalate) (PET). The adhesion of the cells to polystyrene was probably due to hydrophobic interactions involving proteins or protein complexes. The adhesion of the cells to Teflon might be the result not only of hydrophobic interactions but also of acid-basic forces. Additionally, the present work shows that Y. lipolytica cell extracts previously treated by ultrasound waves (cell debris) maintained their enzymatic activity (lipase) and could be attached to polystyrene and PET and used successfully as immobilized biocatalysts in hydrolysis reactions.

Green (Detox) juice physicochemical properties and stabilization effect of naturals emulsifiers / Propriedades físico-químicas do suco verde (detox) e efeito de estabilização de emulsificantes naturais

ABSTRACT: Green or "detox" juice is a mixture of fruit juice with vegetables, which has been used intensively by consumers seeking for healthy food. Physicochemical properties of Green juice were accessed in the present research, which brings new insights for the use of this beverage in human diet. A total phenolic content of 2833.60 mg GAE (Gallic acid equivalent)/ g of juice and a Total Antioxidant Capacity by FRAP of 323.62 µM Fe2SO4 / g of juice and by ABTS•+ of 333.11 µM Trolox/ g of juice, indicated good antioxidant properties. Low energy and reducing sugar content indicate its use for low calorie diet, but low carbohydrate and protein content prove that Green juice cannot be used as meal replacement. The addition of a microbial biosurfactant (YlBio) and chia gel as bioemulsifiers was tested in the Green juice formulation to reduce solid decantation and increase consistency. YlBio and chia gel were able to change the Newtonian behavior of the Green juice to a Pseudoplastic behavior due to stabilization properties and also increase consistency, without the need to add synthetic stabilizers.

RESUMO: O suco verde ou "detox" é uma mistura de suco de frutas com vegetais que tem sido intensamente utilizado por consumidores que buscam alimentos saudáveis. As propriedades físico-químicas do suco verde foram avaliadas no presente trabalho, o que traz novas perspectivas para o uso dessa bebida na dieta humana. Um conteúdo fenólico total de 2833,60 mg de EAG (equivalente em ácido gálico) / g de suco) e uma capacidade antioxidante total por FRAP de 323,62 µM de Fe2SO4 / g de suco e por ABTS•+ de 333,11 µM de Trolox / g de suco, indicam boas propriedades antioxidantes. Um baixo teor de energia e açúcar redutor indica seu uso em dietas de baixa caloria, mas o baixo teor de carboidratos e proteínas prova que o suco verde não pode ser usado como substituto de refeição. A adição de um biossurfactante microbiano (YlBio) e do gel de chia no suco foi testada na formulação do suco verde, para reduzir a decantação de sólidos e aumentar a consistência. YlBio e o gel de chia foram capazes de mudar o comportamento do suco de fluido Newtoniano para um fluido pseudoplástico devido às propriedades estabilizantes, e também aumentaram a consistência do suco, sem a necessidade de adição de estabilizantes sintéticos.

High Catalytic Activity of Lipase from Yarrowia lipolytica Immobilized by Microencapsulation.

Microencapsulation of lipase from Yarrowia lipolytica IMUFRJ 50682 was performed by ionotropic gelation with sodium alginate. Sodium alginate, calcium chloride and chitosan concentrations as well as complexation time were evaluated through experimental designs to increase immobilization yield (IY) and immobilized lipase activity (ImLipA) using p-nitrophenyl laurate as substrate. To adjust both parameters (IY and ImLipA), the desirability function showed that microcapsule formation with 3.1%(w/v) sodium alginate, 0.19%(w/v) chitosan, 0.14 M calcium chloride, and 1 min complexation time are ideal for maximal immobilization yield and immobilized lipase activity. A nearly twofold enhancement in Immobilization yield and an increase up to 280 U/g of the lipase activity of the microcapsules were achieved using the experimental design optimization tool. Chitosan was vital for the high activity of this new biocatalyst, which could be reused a second time with about 50% of initial activity and for four more times with about 20% of activity.

Use of Yarrowia lipolytica Lipase Immobilized in Cell Debris for the Production of Lipolyzed Milk Fat (LMF).

Lipase immobilized on Yarrowia lipolytica cell debris after sonication of yeast cells (LipImDebri) was used in hydrolysis reaction as a novel strategy to produce lipolyzed milk fat (LMF). Extracellular (4732.1 U/L), intracellular (130.0 U/g), and cell debris (181.0 U/g) lipases were obtained in a 4 L bioreactor using residual frying oil as inducer in 24 h fermentation process. LipImDebri showed a good operational stability retaining 70% of lipolytic activity after the second cycle and 40% after the fourth. The highest degree of hydrolysis (28%) was obtained with 500 mg LipImDebri for 6 h of lipolysis of anhydrous milk fat. LMF produced with LipImDebri presented high contents of oleic (35.2%), palmitic (25.0%), and stearic (15.4%) acids and considerable amounts of odor-active short and medium chain fatty acids (C:4⁻C:10) (8.13%).

Structural characterization of a neuroblast-specific phosphorylated region of MARCKS.

MARCKS (Myristoylated Alanine-Rich C Kinase substrate) is a natively unfolded protein that interacts with actin, Ca(2+)-Calmodulin, and some plasma membrane lipids. Such interactions occur at a highly conserved region that is specifically phosphorylated by PKC: the Effector Domain. There are two other conserved domains, MH1 (including a myristoylation site) and MH2, also located in the amino terminal region and whose structure and putative protein binding capabilities are currently unknown. MH2 sequence contains a serine that we described as being phosphorylated only in differentiating neurons (S25 in chick). Here, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy were used to characterize the phosphorylated and unphosphorylated forms of a peptide with the MARCKS sequence surrounding S25. The peptide phosphorylated at this residue is recognized by monoclonal antibody 3C3 (mAb 3C3). CD and NMR data indicated that S25 phosphorylation does not cause extensive modifications in the peptide structure. However, the sharper lines, the absence of multiple spin systems and relaxation dispersion data observed for the phosphorylated peptide suggested a more ordered structure. Surface Plasmon Resonance was employed to compare the binding properties of mAb 3C3 to MARCKS protein and peptide. SPR showed that mAb 3C3 binds to the whole protein and the peptide with a similar affinity, albeit different kinetics. The slightly ordered structure of the phosphorylated peptide might be at the origin of its ability to interact with mAb 3C3 antibody, but this binding did not noticeably modify the peptide structure.