Comparative oil extraction from mutt (Myliobatis goodei) liver by enzymatic hydrolysis: free versus immobilized biocatalyst.

BACKGROUND: The development and fine-tuning of biotechnological processes for fish oil extraction constitute a very important focus to contribute to the development of a food industry based on fish consumption. This work lies in a comparative analysis of the oil extraction yield of Myliobatis goodei livers using free and immobilized enzymes. RESULTS: An immobilized biocatalyst was designed from the cell-free extract of a Bacillus sp. Mcn4. A complete factorial design was used to study the components of the bacterial culture medium and select the condition with the highest titers of extracellular enzymatic activities. Wheat bran had a significant effect on the culture medium composition for enzymatic production. The immobilized biocatalyst was designed by covalent binding of the proteins present in the cocktail retaining a percentage of different types of enzymatic activities (Mult.Enz@MgFe2 O4 ). Among the biocatalyst used, Alcalase® 2.4 L and Purazyme® AS 60 L (free commercial proteases) showed extraction yields of 87.39% and 84.25%, respectively, while Mult.Enz@MgFe2 O4 achieved a better one of 89.97%. The oils obtained did not show significant differences in their physical-chemical properties while regarding the fatty acid content, the oil extracted with Purazyme® AS 60 L showed a comparatively lower proportion of polyunsaturated fatty acids. CONCLUSIONS: Our results suggest that the use of by-products of M. goodei is a valid alternative and encourages the use of immobilized multienzyme biocatalysts for the treatment of complex substrates in the fishing industry. © 2023 Society of Chemical Industry.

Interfacial Hyperactivation of Candida rugosa Lipase onto Ca2Fe2O5 Nanoparticles: pH and Ionic Strength Fine-Tuning to Modulate Protein-Support Interactions.

The current study provides a comprehensive look of the adsorption process of Candida rugosa lipase (CRL) on Ca2Fe2O5 iron oxide nanoparticles (NPs). Protein-support interactions were identified across a broad range of pH and ionic strengths (mM) through a response surface methodology, surface charge determination, and spectroscopic and in silico analyses. The maximum quantity of immobilized protein was achieved at an ionic strength of 50 mM and pH 4. However, this condition did not allow for the greatest hydrolytic activity to be obtained. Indeed, it was recorded at acidic pH, but at 150 mM, where evaluation of the recovered activity revealed hyperactivation of the enzyme. These findings were supported by adsorption isotherms performed under different conditions. Based on zeta potential measurements, electrostatic interactions contributed differently to protein-support binding under the conditions tested, showing a strong correlation with experimentally determined immobilization parameters. Raman spectra revealed an increase in hydrophobicity around tryptophan residues, whereas the enzyme immobilization significantly reduced the phenylalanine signal in CRL. This suggests that this residue was involved in the interaction with Ca2Fe2O2 and molecular docking analysis confirmed these findings. Fluorescence spectroscopy showed distinct behaviors in the CRL emission patterns with the addition of Ca2Fe2O5 at pH 4 and 7. The calculated thermodynamic parameters indicated that the contact would be mediated by hydrophobic interactions at both pHs, as well as by ionic ones at pH 4. In this approach, this work adds to our understanding of the design of biocatalysts immobilized in iron oxide NPs.

Tuning surface interactions on MgFe2O4 nanoparticles to induce interfacial hyperactivation in Candida rugosa lipase immobilization.

Lipase adsorption on solid supports can be mediated by a precise balance of electrostatic and hydrophobic interactions. A suitable fine-tuning could allow the immobilized enzyme to display high catalytic activity. The objective of this work was to investigate how pH and ionic strength fluctuations affected protein-support interactions during immobilization via physical adsorption of a Candida rugosa lipase (CRL) on MgFe2O5. The highest amount of immobilized protein (IP) was measured at pH 4, and an ionic strength of 90 mM. However, these immobilization conditions did not register the highest hydrolytic activity (HA) in the biocatalyst (CRLa@MgFe2O4), finding the best values also at acidic pH but with a slight shift towards higher values of ionic strength around 110 mM. These findings were confirmed when the adsorption isotherms were examined under different immobilization conditions so that the maximum measurements of IP did not coincide with that of HA. Furthermore, when the recovered activity was examined, a strong interfacial hyperactivation of the lipase was detected towards acidic pH and highly charged surrounding environments. Spectroscopic studies, as well as in silico molecular docking analyses, revealed a considerable involvement of surface hydrophobic protein-carrier interactions, with aromatic aminoacids, especially phenylalanine residues, playing an important role. In light of these findings, this study significantly contributes to the body of knowledge and a better understanding of the factors that influence the lipase immobilization process on magnetic inorganic oxide nanoparticle surfaces.

Genome sequencing and genomic analysis of Amycolatopsis tucumanensis DSM 45259 applicable in gray, red, and nano-biotechnology.

Through the years, the genus Amycolatopsis has demonstrated its biotechnological potential. The need to clean up the environment and produce new antimicrobial molecules led to exploit promising bacterial genera such as Amycolatopsis. In this present work, we analyze the genome of the strain Amycolatopsis tucumanensis AB0 previously isolated from copper-polluted sediments. Phylogenomic and comparative analysis with the closest phylogenetic neighbor was performed. Our analysis showed the genetic potential of the strain to deal with heavy metals such as copper and mitigate oxidative stress. In addition, the ability to produce copper oxide nanoparticles and the presence of genes potentially involved in the synthesis of secondary metabolites suggest that A. tucumanensis may find utility in gray, red, and nano-biotechnology. To our knowledge, this is the first genomic analysis of an Amycolatopsis strain with potential for different biotechnological fields.

Silver nanoparticles synthesized by the heavy metal resistant strain Amycolatopsis tucumanensis and its application in controlling red strip disease in sugarcane.

The production of bioethanol and sugar from sugarcane is an important economic activity in several countries. Sugarcane is susceptible to different phytopathogens. Over the last years, the red stripe disease caused by the bacterium Acidovorax avenae subsp. avenae produced significant losses in sugarcane crops. Bio-nanotechnology emerged as an eco-friendly alternative to the biosynthesis of antimicrobial molecules. The aims of this study were to (a) produce extracellular silver nanoparticles using the heavy metal resistant strain Amycolatopsis tucumanensis, (b) evaluate their antibacterial in vitro effect and (c) determine the potential of silver nanoparticles to protect sugarcane against red stripe disease. Amycolatopsis tucumanensis synthesized spherical silver nanoparticles with an average size of 35 nm. Nanoparticles were able to control the growth of A. avenae subsp. avenae in in vitro assays. In addition, in vivo assays in sugarcane showed a control upon the red stripe disease when silver nanoparticles were applied as preventive treatment. The Disease Severity Index was 28.94% when silver nanoparticles were applied 3 days before inoculation with A. avenae subsp. a venae. To our knowledge, this is the first report of silver nanoparticles extracellularly synthesized by an Amycolatopsis strain that were able to inhibited the growth of A. avenae subsp. avenae and control the red stripe disease in sugarcane.

Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-ß-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

Control of microbial biofilm formation as an approach for biomaterials synthesis.

The search for new biomaterials with superior mechanical properties is the focus in the area of materials science. A promising pathway is drawing inspiration from nature to design and develop materials with enhanced properties. In this work, a novel strategy to produce functionalized supramolecular bionanomaterials from the microbial biofilm is reported. Tuneable biofilms with specific characteristics were obtained by controlling the culture condition of the microorganism. When the exopolysaccharide (EPS) production was desired the tryptone was the best nutritional component for the EPS production into the biofilm. However, for the expression of a high amount of amyloid protein the combination of peptone and glucose was the best nutritional choice. Each biofilm obtained showed its owner rheology properties. These properties were altered by the addition of extracellular DNA, which increased the viscosity of the biofilm and induced a viscoelastic hydrogel behavior. Besides, as a proof of concept of bionanomaterial, a novel supramolecular polymeric hybrid EPS-Amyloid protein (EPAP) was obtained from the biofilm and it was tested as a new natural functionalized support for enzyme immobilization. The results suggest that this technology could be used as a new concept to obtain biomaterials from biofilms by controlling the nutritional conditions of a microorganism. Understanding environmental factors affecting biofilm formation will help the development of methods for controlling biofilm production and therefore obtaining new biomaterials.

Raw sugarcane bagasse as carbon source for xylanase production by Paenibacillus species: a potential degrader of agricultural wastes.

Paenibacillus species isolated from a variety of natural sources have shown to be important glycoside hydrolases producers. These enzymes play a key role in bio-refining applications, as they are central biocatalysts for the processing of different types of polymers from vegetal biomass. Xylanase production by three native isolates belonging to the genus Paenibacillus was approached by utilizing mineral-based medium and agricultural by-products as a convenient source to produce biocatalysts suitable for their degradation. While varieties of alkali pretreated sugarcane bagasse were useful substrates for the strains from Paenibacillus genus evaluated, raw sugarcane bagasse was the most effective substrate for endoxylanase production by Paenibacillus sp. AR247. This strain was then selected to further improvement of its enzyme production by means of a two-step statistical approach. It was determined that the carbon source, provided as an inexpensive agro-waste, as well as phosphate and magnesium were the culture media components that most influenced the enzyme production, which was improved three times compared to the screening results.

Endoglucanase and xylanase production by Bacillus sp. AR03 in co-culture.

The behavior of three isolates retrieved from different cellulolytic consortia, Bacillus sp. AR03, Paenibacillus sp. AR247 and Achromobacter sp. AR476-2, were examined individually and as co-cultures in order to evaluate their ability to produce extracellular cellulases and xylanases. Utilizing a peptone-based medium supplemented with carboxymethyl cellulose (CMC), an increase estimation of 1.30 and 1.50 times was obtained by the co-culture containing the strains AR03 and AR247, with respect to enzyme titles registered by their individual cultivation. On the contrary, the extracellular enzymatic production decreased during the co-cultivation of strain AR03 with the non-cellulolytic Achromobacter sp. AR476-2. The synergistic behavior observed through the combined cultivation of the strains AR03 and AR247 might be a consequence of the consumption by Paenibacillus sp. AR247 of the products of the CMC hydrolysis (i.e., cellobiose and/or cello-oligosaccharides), which were mostly generated by the cellulase producer Bacillus sp. AR03. The effect observed could be driven by the requirement to fulfill the nutritional supply from both strains on the substrate evaluated. These results would contribute to a better description of the degradation of the cellulose fraction of the plant cell walls in nature, expected to an efficient utilization of renewable sources.

Draft Genome Sequence of Achromobacter sp. Strain AR476-2, Isolated from a Cellulolytic Consortium.

Achromobacter sp. AR476-2 is a noncellulolytic strain previously isolated from a cellulolytic consortium selected from samples of insect gut. Its genome sequence could contribute to the unraveling of the complex interaction of microorganisms and enzymes involved in the biodegradation of lignocellulosic biomass in nature.

Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil.

The intracellular lipase production by Mucor circinelloides URM 4182 was investigated through a step-by-step strategy to attain immobilized whole-cells with high lipase activity. Physicochemical parameters, such as carbon and nitrogen sources, inoculum size and aeration, were studied to determine the optimum conditions for both lipase production and immobilization in polyurethane support. Olive oil and soybean peptone were found to be the best carbon and nitrogen sources, respectively, to enhance the intracellular lipase activity. Low inoculum level and poor aeration rate also provided suitable conditions to attain high lipase activity (64.8 ± 0.8 U g(-1)). The transesterification activity of the immobilized whole- cells was assayed and optimal reaction conditions for the ethanolysis of babassu oil were determined by experimental design. Statistical analysis showed that M. circinelloides whole-cells were able to produce ethyl esters at all tested conditions, with the highest yield attained (98.1 %) at 35 °C using an 1:6 oil-to-ethanol molar ratio. The biocatalyst operational stability was also assayed in a continuous packed bed reactor (PBR) charged with glutaraldehyde (GA) and Aliquat-treated cells revealing half-life of 43.0 ± 0.5 and 20.0 ± 0.8 days, respectively. These results indicate the potential of immobilized M. circinelloides URM 4182 whole-cells as a low-cost alternative to conventional biocatalysts in the production of ethyl esters from babassu oil.

Catalytic properties of mycelium-bound lipases from Aspergillus niger MYA 135.

A constitutive level of a mycelium-bound lipolytic activity from Aspergillus niger MYA 135 was strongly increased by 97% in medium supplemented with 2% olive oil. The constitutive lipase showed an optimal activity in the pH range of 3.0-6.5, while the mycelium-bound lipase activity produced in the presence of olive oil had two pH optima at pH 4 and 7. Interestingly, both lipolytic sources were cold-active showing high catalytic activities in the temperature range of 4-8 degrees C. These mycelium-bound lipase activities were also very stable in reaction mixtures containing methanol and ethanol. In fact, the constitutive lipase maintained almost 100% of its activity after exposure by 1 h at 37 degrees C in ethanol. A simple methodology to evaluate suitable transesterification activities in organic solvents was also reported.