Raunel Tinoco-Valencia

Toward an understanding of the effects of agitation and aeration on growth and laccases production by Pleurotus ostreatus

Mycelial growth and laccase production by Pleurotus ostreatus CP50 cultured in a 10-L mechanically agitated bioreactor were assessed through a 2(3) factorial experimental design. The main effects and interactions of three factors (agitation, aeration and copper induction) over five responses (μ, αLacc, βLacc, maximal volumetric laccase activity and maximal biomass concentration) were analyzed. P. ostreatus growth was significantly improved when culturing was conducted with high agitation (5.9kW/m(3)s) and aeration flow (0.5vvm) rates. Under the experimental conditions evaluated, no evidence of hydrodynamic stress affecting fungal growth was observed. However, the high agitation and aeration conditions were detrimental for the growth-associated laccase production constant (αLacc), leading to a very complex optimization of the process. The maximal laccase volumetric activity (1.2 and 3.8U/ml for non-induced and copper-induced cultures, respectively) was observed when the culturing was performed at a low agitation rate (0.9kW/m(3)s) and a high aeration flow rate (0.5vvm). Laccase proteolysis may explain the complex interactions observed between agitation and aeration and the effects of these factors on the laccase volumetric activity observed in the cultures.

Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP)

With the goal of understanding the chitinolytic mechanism of the potential biological control strain Serratia marcescens CFFSUR-B2, genes encoding chitinases ChiA, ChiB and ChiC, chitobiase (Chb) and chitin binding protein (CBP) were cloned, the protein products overexpressed in Escherichia coli as 6His-Sumo fusion proteins and purified by affinity chromatography. Following affinity tag removal, the chitinolytic activity of the recombinant proteins was evaluated individually and in combination using colloidal chitin as substrate. ChiB and ChiC were highly active while ChiA was inactive. Reactions containing both ChiB and ChiC showed significantly increased N-acetylglucosamine trimer and dimer formation, but decreased monomer formation, compared to reactions with either enzyme alone. This suggests that while both ChiB and ChiC have a general affinity for the same substrate, they attack different sites and together degrade chitin more efficiently than either enzyme separately. Chb and CBP in combination with ChiB and ChiC (individually or together) increased their chitinase activity. We report for the first time the potentiating effect of Chb on the activity of the chitinases and the synergistic activity of a mixture of all five proteins (the three chitinases, Chb and CBP). These results contribute to our understanding of the mechanism of action of the chitinases produced by strain CFFSUR-B2 and provide a molecular basis for its high potential as a biocontrol agent against fungal pathogens.

Enhancing methyl parathion degradation by the immobilization of Burkholderia sp. isolated from agricultural soils

Organophosphate pesticides are of great interest for research because they are currently the most commonly used pesticides. In this study, a bacterial strain capable of completely degrading methyl parathion (MP) was isolated from agricultural soils in central Mexico. This strain was designated strain S5‐2 and was identified as Burkholderia cenocepacia. To increase degradation yields, cells were immobilized on three different supports: powdered zeolite and Opuntia sp. and Agave sp. fibers. The results indicated a significant increase in MP hydrolysis and p‐nitrophenol (PNP) degradation with immobilized cells compared to free cell cultures. Furthermore, immobilized cells were capable of withstanding and degrading higher concentrations of PNP compared to cell suspension cultures. The cell viability in the free cell cultures, as well as PNP degradation, was affected at concentrations greater than 25 mg/L. In contrast, cells immobilized on Opuntia sp. and Agave sp. fibers completely degraded PNP at concentrations of 100 mg/L. To verify that MP solution toxicity was decreased by B. cenocepacia strain S5‐2 via pesticide degradation, we measured the acetylcholinesterase activity, both before and after treatment with bacteria. The results demonstrate that the activity of acetylcholinesterase was unaffected after MP degradation by bacteria.

Diffusional and transcriptional mechanisms involved in laccases production by Pleurotus ostreatus CP50

The independent effects of hydrodynamic stress (assessed as the Energy Dissipation/Circulation Function, EDCF) and dissolved oxygen tension (DOT) on the growth, morphology and laccase production by Pleurotus ostreatus CP50 were studied using a 3(2) factorial design in a 10L reactor. A bell-shape function for fungus growth between 8 and 22% DOT was observed, as well as a significant negative effect on laccase production and the expression of poxc, the gene encoding for the most abundant laccase produced by P. ostreatus CP50. Increasing EDCF from 1 to 21 kW/m(3)s, had a positive effect on fungus growth, whereas no effect on poxc gene expression was observed. However, the increase in EDCF favored the specific laccase production due to the generation of smaller pellets with less diffusional limitations and increased metabolically active biomass. The results show, for the first time, that hydrodynamic effects on growth and laccase production are mainly physical and diffusional, while the influence of the dissolved oxygen is at transcriptional level.

Improved production, purification and characterization of biosurfactants produced by Serratia marcescens SM3 and its isogenic SMRG-5 strain

In this study, the biosurfactants (Bs) production of two Serratia marcescens strains (SM3 and its isogenic SMRG‐5 strain) was improved and the tenso‐active agents were purified and characterized. A 23 factorial design was used to evaluate the effect of nitrogen and carbon sources on the surface tension (ST) reduction and emulsion index (EI24) of the produced Bs. Optimum Bs production by SM3 was achieved at high concentrations of carbon and nitrogen, reducing ST to 26.5 ± 0.28 dynes/cm, with an EI24 of 79.9 ± 0.2%. Meanwhile, the best results for SMRG‐5 were obtained at low concentrations, reducing the ST to 25.2 ± 0.2 dynes/cm, with an EI24 of 89.7 ± 0.28%. The optimal conditions for Bs production were scaled up in a 2‐L reactor, yielding 4.8 and 5.2 g/L for SM3 and SMRG‐5, respectively. Gas Chromatography–Mass Spectrometry (GC–MS) analysis revealed the presence of two different lipopeptides (hidrofobic fractions: octadecanoic and hexadecanoic acid for SM3 and SMRG5, respectively). Both strains were capable of benzo [a] pyrene removal (59% after 72 H of culture).

Validation of a general method for activity estimation of cyanide evolving oxidoreductases.

Ethylene is a key molecule in organic synthesis currently produced by steam cracking of fossil hydrocarbons. In nature, ethylene is produced in higher plants by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). Biocatalytic alternatives for ethylene production are still far from being competitive with traditional production plants. Furthermore, data dispersion shown in the literature adds uncertainty to the introduction of ACCO as a biocatalyst, especially when larger numbers of isoforms or mutants are to be compared. Here we propose a new method for measuring ACCO activity based on cyanide detection. Data provided here indicate that cyanide detection is more precise, more responsive, and much more stable than any other method tested for ACCO activity estimation so far. Briefly, enzymatically produced cyanide can be detected by its derivatization with naphthalene-2,3-dicarboxyaldehide (NDA) to generate 1-cyanobenz[f]isoindole (CBI), which is further detected by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector. Cyanide can be detected in the range between 0.99 and 60.17pmol, which is three orders of magnitude more sensitive than the currently used ethylene estimation method.