A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

Bacillus thuringiensis-Based Bioproduct: Characterization and Performance Against Spodoptera frugiperda Strains in Maize Under Different Environmental Temperatures.

Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is an important pest in several regions being the use of Bacillus thuringiensis-based bioproducts an alternative for its control. Firstly, 3 L of an aqueous bioproduct suspension was produced and characterized. Its 50% lethal concentration against molecularly identified corn and rice S. frugiperda strains using an artificial diet were 77.01% (95% CL, 68.16-90.47) and 2.22% (95% CL, 0.01-6.68), respectively. The next objective of this work was to evaluate the performance of this bioproduct in maize against S. frugiperda strains under different simulated agrological regions mimicking their corresponding periodic day/night temperatures. Thus, the impact of environmental temperature on the bioproduct efficacy (E) was studied. It was observed that a warmer scenario (35 °C day/30 °C night) could favor the tolerance of corn S. frugiperda strain to the bioproduct (E = 56.36 ± 0.61%) maintaining a high efficacy (92.44 ± 6.55%) when it was tested against rice S. frugiperda strain. Conversely, under temperate conditions, efficacy values ranged from 84 to 95% for both S. frugiperda strains. On the other hand, based on a foliar feeding damage analysis, our bioproduct displayed a significant foliar protection in maize plants infested with either corn or rice S. frugiperda strains.

Industrial biotransformations catalyzed by microbial lipases: screening platform and commercial aspects.

The successfulness of a lipase-catalyzed industrial process depends on a proper lipase selection. In this work, an alternative screening platform for industrially important biotransformations catalyzed by microbial lipases was proposed. Thus, the reactivity of sixty lipase activities from spore-forming microorganisms towards hydrolytic and transesterification reactions by using p-nitrophenyl palmitate as a chromogenic acyl donor substrate was explored. Only three biocatalysts were capable of catalyzing all reactions tested. Fourteen biocatalysts did not show hydrolytic activity at all; however, they displayed transesterification activities using ethanol, starch, low-methoxyl (LM) pectin, high-methoxyl (HM) pectin, or vitamin C as acyl acceptors. Using heat-treated biocatalysts, hydrolytic activities were not highly correlated with the corresponding transesterification activities using ethanol (r = -0.058, p = 0.660), starch (r = 0.431, p = 0.001), LM pectin (r = -0.010, p = 0.938), HM pectin (r = 0.167, p = 0.202), and vitamin C (r = -0.048, p = 0.716) as acyl acceptor. In addition, to the best of our knowledge, several transesterification activities produced from microorganisms of the genus Bacillus, Brevibacillus, Lysinibacillus, Geobacillus, or Sporosarcina were reported for first time. Finally, the global lipase market was presented and segmented by date, application, geography and player highlighting the commercial contribution of microbial lipases.

Orchestration an extracellular lipase production from Aspergillus niger MYA 135: biomass morphology and fungal physiology.

The impact of biomass morphology and culture conditions on fungal fermentation was widely reviewed in the literature. In this work, we presented three independent experiments in order to evaluate the influence of some of those input factors on a lipase production separately by using the Aspergillus niger MYA 135 and the two-stage fermentation technique. Regarding the culture modality, the biomass was pre-grown in a first reactor. Then, the washed mycelium was transferred to a second reactor to continue the study. Firstly, linear effects of fungal morphology and several physiological parameters on a lipase production were explored using the Plackett-Burman design. The dispersed fungal morphology was confirmed as a proper quality characteristic for producing an extracellular lipase activity. Concerning the impact of the carbon source on the biomass pre-growth, the sucrose (E = 9.923, p < 0.001) and the L-arabinose (E = 4.198, p = 0.009) presented positive and significant effects on the enzyme production. On the contrary, the supplementation of 0.05 g/L CaCl2 displayed a highly negative and significant effect on this process (E = - 7.390, p < 0.001). Secondly, the relationship between the enzyme production and the input variables N:C ratio, FeCl3 and olive oil was explored applying the central composite design. Among the model terms, the N:C ratio of the production medium had the most negative and significant influence on the enzyme synthesis. Thus, it was concluded that a low N:C ratio was preferable to increase its production. In addition, the bifunctional role of FeCl3 on this fungus was presented. Thirdly, a prove of concept assay was also discussed.

Highly reusable invertase biocatalyst: Biological fibrils functionalized by photocrosslinking.

Here we report a novel strategy for the immobilization of invertase using amyloid-like fibrils as a support. Optimal conditions to get Tyr-Tyr covalent binding between invertase and the support were determined using a photocrosslinking approach. The biological fibrils with invertase activity turn into microstructured catalysts according to electron microscopy outcomes. Thermal and storage stability as well as optimal pH and temperature of the enzyme were conserved. Moreover, the immobilized enzyme recovered by low g-force centrifugation retained 83% of its initial enzymatic activity after 15 reuse cycles. Considering that enzyme cost is the most significant part of the overall fee of enzymatic biomass conversion, the highly efficient recovery/reuse strategy described herein becomes relevant. Besides, it can also be applied to the immobilization of other enzymes for industrial biocatalysis.

Tailoring fungal morphology of Aspergillus niger MYA 135 by altering the hyphal morphology and the conidia adhesion capacity: biotechnological applications.

Current problems of filamentous fungi fermentations and their further successful developments as microbial cell factories are dependent on control fungal morphology. In this connection, this work explored new experimental procedures in order to quantitatively check the potential of some culture conditions to induce a determined fungal morphology by altering both hyphal morphology and conidia adhesion capacity. The capacity of environmental conditions to modify hyphal morphology was evaluated by examining the influence of some culture conditions on the cell wall lytic potential of Aspergillus niger MYA 135. The relative value of the cell wall lytic potential was determined by measuring a cell wall lytic enzyme activity such as the mycelium-bound ß-N-acetyl-D-glucosaminidase (Mb-NAGase). On the other hand, the quantitative value of conidia adhesion was considered as an index of its aggregation capacity. Concerning microscopic morphology, a highly negative correlation between the hyphal growth unit length (lHGU) and the specific Mb-NAGase activity was found (r = -0.915, P < 0.001). In fact, the environment was able to induce highly branched mycelia only under those culture conditions compatible with specific Mb-NAGase values equal to or higher than 190 U gdry.wt-1. Concerning macroscopic morphology, a low conidia adhesion capacity was followed by a dispersed mycelial growth. In fact, this study showed that conidia adhesion units per ml equal to or higher than 0.50 were necessary to afford pellets formation. In addition, it was also observed that once the pellet was formed the lHGU had an important influence on its final diameter. Finally, the biotechnological significance of such results was discussed as well.

Mycelium-bound lipase production from Aspergillus niger MYA 135, and its potential applications for the transesterification of ethanol.

The potential biotechnological applications of both constitutive and inducible lipase sources from Aspergillus niger MYA 135 were evaluated. To this end, the effect of environmental conditions on mycelium-bound lipase production from this strain was studied, when cultured either in the absence or presence of 2% olive oil. It was previously reported that mycelium-bound lipase from Aspergillus niger MYA 135 possess high stability in reaction mixtures containing ethanol; which could be especially important for their use in biodiesel synthesis. In this connection, the performance of the lipase sources produced in the transesterification of ethanol using p-nitrophenyl palmitate as acyl donor was also explored. Under our assay conditions, hydrolytic and synthetic activity of the mycelia produced in the absence or presence of olive oil were not highly correlated. While the hydrolytic activity was strongly increased by the addition of lipid to the culture medium, the best performance in the transesterification reactions of ethanol were associated with mycelia produced in absence of olive oil. Interestingly, the supplementation of the culture medium with Fe(+3) increased the transesterification activity by 71%, as compared to the activity previously reported for this strain. Therefore, the constitutive lipase sources from Aspergillus niger MYA 135 are considered to be promising for industrial biodiesel-fuel production.

Effect of environmental conditions on extracellular lipases production and fungal morphology from Aspergillus niger MYA 135.

Under the current assay conditions, lipase production in mineral medium was only detected in the presence of vegetable oils, reaching the highest specific activity with olive oil. In this way, effect of different environmental conditions on fungal morphology and olive oil-induced extracellular lipases production from Aspergillus niger MYA 135 was studied. It was observed that addition of 1.0 g l(-1) FeCl(3)to the medium encouraged filamentous growth and increased the specific activity 6.6 fold after 4 days of incubation compared to the control. However, major novelty of this study was the satisfactory production of an acidic lipase at initial pH 3 of the culture medium (1.74 +/- 0.06 mU microg(-1)), since its potencial applications in food and pharmaceutical industry are highly promising.