Martín Blasco

Molecular characterization of cyp11a1 and cyp11b1 and their gene expression profile in pejerrey (Odontesthes bonariensis) during early gonadal development

Sex steroids are known to be involved in gonadal differentiation in fish, but whether androgens are early mediators of testis differentiation remains unclear. We studied the sex-related developmental variations in the gene expression of two key enzymes involved in steroids and androgen synthesis (cyp11a1 and cyp11b1) in trunks and isolated gonads of pejerrey (Odontesthes bonariensis) larvae during and after the sex determination period. Also, and in order to have a better characterization of this process we studied the expression of Sertoli (dmrt1, amh, sox9) and Leydig (nr5a1 or sf-1) cell markers as well as a gene with higher expression in females (cyp19a1a). No clear differences were observed in the expression of cyp11a1 and cyp11b1 during the temperature-sensitive window in the trunk of pejerrey larvae. Nevertheless, a clear increase of cyp11b1 was observed in isolated gonads taken from fish reared at the male producing temperature. In these gonads we also confirmed the trends of genes with higher expression in males (dmrt1, amh) and females (cyp19a1a) as previously described in larval trunks of pejerrey. Our results showed that the expression of cyp11b1 was positively associated with the morphological differentiation of the testis. Nevertheless the involvement of 11-oxygenated androgens during the temperature-sensitive window could not be clearly established.

A quantitative HPLC-MS method for the simultaneous determination of testosterone, 11-ketotestosterone and 11-β hydroxyandrostenedione in fish serum.

A simple and novel HPLC-MS method for the simultaneous quantification of testosterone, 11-ketotestosterone, and 11beta-hydroxyandrostenedione in fish serum was developed and validated. Separation was achieved on a C-18 column using a water-acetonitrile mobile-phase with a cycle time of 12 min. Ion detection was performed using ESI positive SIM at [M+H] (m/z 303, 303, 289). The linear ranges (0.2-50 ng/ml), limits of detection (0.1-0.2 ng/ml) and quantification (0.2-0.5 ng/ml) were established. The method was validated by measuring the three androgens in goldfish sera, displaying comparable values to those reported by other analytical techniques (RIA, EIA).

Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter

The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.

Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method.

Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123-128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 μg of polygalacturonic acid, and 45 μg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.

Bioprocess and downstream optimization of recombinant bovine chymosin B in Pichia (Komagataella) pastoris under methanol-inducible AOXI promoter

A clone of the methylotrophic yeast Pichia pastoris strain GS115 transformed with the bovine prochymosin B gene was used to optimize the production and downstream of recombinant bovine chymosin expressed under the methanol-inducible AOXI promoter. Cell growth and recombinant chymosin production were analyzed in flask cultures containing basal salts medium with biodiesel-byproduct glycerol as the carbon source, obtaining values of biomass level and milk-clotting activity similar to those achieved with analytical glycerol. The effect of biomass level at the beginning of methanol-induction phase on cell growth and chymosin expression was evaluated, determining that a high concentration of cells at the start of such period generated an increase in the production of chymosin. The impact of the specific growth rate on chymosin expression was studied throughout the induction stage by methanol exponential feeding fermentations in a lab-scale stirred bioreactor, achieving the highest production of heterologous chymosin with a constant specific growth rate of 0.01h(-1). By gel filtration chromatography performed at a semi-preparative scale, recombinant chymosin was purified from exponential fed-batch fermentation cultures, obtaining a specific milk-clotting activity of 6400IMCU/mg of chymosin and a purity level of 95%. The effect of temperature and pH on milk-clotting activity was analyzed, establishing that the optimal temperature and pH values for the purified recombinant chymosin are 37°C and 5.5, respectively. This study reported the features of a sustainable bioprocess for the production of recombinant bovine chymosin in P. pastoris by fermentation in stirred-tank bioreactors using biodiesel-derived glycerol as a low-cost carbon source.

A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation

A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (E a), quotient energy (Q 10), K m, and V max were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.

Use of grape pomaces to produce biomass of aKomagataella pastoris strain expressing a bovine chymosin activity.

The use of agroindustrial wastes not only decreases bioprocesses and disposal costs but also contributes to the upgrading of the residues. An active recombinant methanol-inducible bovine chymosin has been expressed in our laboratory in the yeastKomagataella pastoris, and grape pomace extracts (GRE) were proposed as a convenient C-energy source for the biomass production of the genetically engineered strain. Carbon and nitrogen sources, growth factors, and initial pH conditions were selected by classical methodology; thereafter, growth conditions optimization was performed using statistical designed experiments (DoEs). In the presence of (in g·L−1) 67.0 monosaccharides (glucose and fructose) from GRE, 5.0 (NH4)2SO4, and 10.0 sugar cane molasses (CMz), a yield of 20.0 g·L−1 cell dry weight (CDW) was obtained aerobically after 60 h incubation at 28°C and pH 4.0. Applying a fed-batch strategy with methanol:sorbitol as the enzyme inducers, a chymosin production of 8.53 International Milk Clotting Units (IMCU) per mg protein was obtained in the supernatant. The results presented show that through a statistical design, a simple, cheap, and easy to prepare culture medium could be developed using two agroindustrial derivatives (GRE and CMz) to obtain a higher value added product.