Enzymatic production process of capric acid-rich structured lipids: Development of formulation as a new therapeutic approach.

The present work reports an optimization of the synthesis of MLM-type (medium, long, medium) structured lipids (SL) through an acidolysis reaction of grape seed oil with capric acid catalyzed by Rhizopus oryzae lipase immobilized. At first, tests were carried out by preparing the biocatalysts using enzyme loadings (0.15 to 1 g of enzymatic powder) for each gram of support. Enzyme loading was used 0.3 g of enzymatic powder, and hydrolytic activity of 1860 ± 23.4 IU/g was reached. Optimized conditions determined by the Central Composite Rotatable Design (CCRD) revealed that the acidolysis reaction reached approximately 59 % incorporation degree (%ID) after 24 h, in addition to the fact that the biocatalyst could maintain the incorporation degree in five consecutive cycles. From this high incorporation degree, cell viability assays were performed with murine fibroblast cell lines and human cervical adenocarcinoma cell lines. Concerning the cytotoxicity assays, the concentration of MLM-SL to 1.75 and 2 % v/v were able to induce cell death in 56 % and 64 % of adenocarcinoma cells, respectively. Human cervical adenocarcinoma cells showed greater sensitivity to the induction of cell death when using emulsions with MLM-SL > 1.75 % v/v compared to emulsions with lower content indicating a potential for combating carcinogenic cells.

Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.

Xylanase Production by Talaromyces amestolkiae Valuing Agroindustrial Byproducts.

In general, agroindustrial byproducts can be easily assimilated by several microorganisms due to their composition, which is rich in carbohydrates. Therefore, they could be appropriate for use as raw materials in a sustainable refinery concept, including the production of hydrolytic enzymes with industrial applicability. In this work, xylanase production by the filamentous fungi Talaromyces amestolkiae in submerged culture was evaluated using five agroindustrial byproducts, namely, wheat bran, citrus pulp, rice bran, peanut skin, and peanut shell. Firstly, the aforementioned byproducts were characterized in terms of cellulose, xylan, lignin, and extractives. Next, production studies were performed, and wheat bran generated the highest enzymatic activity (5.4 U·mL-1), probably because of its large amount of xylan. Subsequently, a factorial design was performed to evaluate the independent variables yeast extract, wheat bran, K2HPO4, and pH, aiming to improve the variable response, xylanase activity. The condition that promoted the highest production, 13.02 U·mL-1 (141% higher than the initial condition), was 20 g·L-1 wheat bran, 2.5 g·L-1 yeast extract, 3 g·L-1 K2HPO4, and pH 7. Thus, industrial byproducts with a high content of xylan can be used as a culture medium to produce xylanase enzymes with a Talaromyces strain through an economical and sustainable approach.

L-asparaginase production review: bioprocess design and biochemical characteristics.

In the past decades, the production of biopharmaceuticals has gained high interest due to its great sensitivity, specificity, and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-asparaginase (L-asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production. KEY POINTS: • Microbial enzyme applications as biopharmaceutical and in food industry • Biosynthesis process: from the microorganism to bioreactor technology • Enzyme activity and kinetic properties: crucial for the final application.

Critical applications of Mucor circinelloides within a biorefinery context.

The establishment of an efficient and feasible biorefinery model depends on, among other factors, particularly the selection of the most appropriate microorganism. Mucor circinelloides is a dimorphic fungus species able to produce a wide variety of hydrolytic enzymes, lipids prone to biodiesel production, carotenoids, ethanol, and biomass with significant nutritional value. M. circinelloides also has been selected as a model species for genetic modification by being the first filamentous oleaginous species to have its genome fully characterized, as well as being a species characterized as a potential bioremediation agent. Considering the potential of replacing several nonrenewable feedstocks is widely dependent on fossil fuels, the exploitation of microbial processes and products is a desirable solution for promoting a green and sustainable future. Here, we introduce and thoroughly describe the recent and critical applications of this remarkable fungus within the context of developing a fungal-based biorefinery.

Sustainable enzymatic approaches in a fungal lipid biorefinery based in sugarcane bagasse hydrolysate as carbon source.

Single cell oil (SCO) was produced from enzymatically hydrolysed sugarcane bagasse by Mucor circinelloides. The fungus was cultured in the hydrolysate medium rich in glucose and xylose being able to assimilate both sugars simultaneously, attaining satisfactory values of lipid accumulation (25 wt%). The main concepts addressed herein were the utilization of these lipids for the production of (i) ethyl esters of fuel grade, and (ii) concentrate of polyunsaturated fatty acids for nutraceutical applications. It was noticed that the fungal lipids also contained carotenoids and that the fungal biomass presented lipolytic activity. The concept of integrating an M. circinelloides-based biorefinery into the sugarcane energy matrix may, thus, present a relevant alternative for the production of high value-added products.