Fabiano Jares Contesini

Enantioselective behavior of lipases from Aspergillus niger immobilized in different supports

Considering the extraordinary microbial diversity and importance of fungi as enzyme producers, the search for new biocatalysts with special characteristics and possible applications in biocatalysis is of great interest. Here, we report the performance in the resolution of racemic ibuprofen of a native enantioselective lipase from Aspergillus niger, free and immobilized in five types of support (Accurel EP-100, Amberlite MB-1, Celite, Montmorillonite K10 and Silica gel). Amberlite MB-1 was found to be the best support, with a conversion of 38.2%, enantiomeric excess of 50.7% and enantiomeric ratio (E value) of 19 in 72xa0h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (Exa0=xa023) in a shorter reaction period (48xa0h) at 35°C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8xa0months of storage at 4°C and could be reused at least six times.

Response surface analysis for the production of an enantioselective lipase from Aspergillus niger by solid-state fermentation

The lipase produced by the Aspergillus niger strain AC-54 has been widely studied due to its enantioselectivity for racemic mixtures. This study aimed to optimize the production of this enzyme using statistical methodology. Initially a Plackett-Burman (PB) design was used to evaluate the effects of the culture medium components and the culture conditions. Twelve factors were screened: water content, glucose, yeast extract, peptone, olive oil, temperature, NaH2P04, KH2P04, MgS04-7H20, CaCl2, NaCI, and MnS04. The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH2P04, and KH2P04, which were optimized using response surface methodology (RSM) and a mathematical model obtained to explain the behavioral process. The best lipase activity was attained using the following conditions: water content (20%), glucose (4.8%), yeast extract (4.0%), and NaH2P04 (4.0%). The predicted lipase activity was 33.03 U/ml and the experimental data confirmed the validity of the model. The enzymatic activity was expressed as µmoles of oleic acid released per minute of reaction (µmol/min).

An overview of Bacillus proteases: from production to application

Abstract Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.

Immobilization of glucosyltransferase from Erwinia sp. using two different techniques

Two different techniques of glucosyltransferase immobilization were studied for the conversion of sucrose into isomaltulose. The optimum conditions for immobilization of Erwinia sp. glucosyltransferase onto Celite 545, determined using response surface methodology, was pH 4.0 and 170 U of glucosyltransferase/g of Celite 545. Using this conditions more than 60% conversion of sucrose into isomaltulose can be obtained. The immobilization of glucosyltransferase was also studied by its entrapment in microcapsules of low-methoxyl pectin and fat (butter and oleic acid). The non-lyophilized microcapsules of pectin, containing the enzyme and fat, showed higher glucosyltransferase activity, compared with lyophilized microcapsules containing enzyme plus fat, and also lyophilized microcapsules containing enzyme without fat addition. The non-lyophilized microcapsules of pectin containing the glucosyltransferase and fat, converted 30% of sucrose into isomaltulose in the first batch. However the conversion decreased to 5% at the 10th batch, indicating inactivation of the enzyme.

Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase

The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4 °C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid + docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36 h, at 40 °C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids.

Xyloglucan breakdown by endo-xyloglucanase family 74 from Aspergillus fumigatus

Xyloglucan is the most abundant hemicellulose in primary walls of spermatophytes except for grasses. Xyloglucan-degrading enzymes are important in lignocellulosic biomass hydrolysis because they remove xyloglucan, which is abundant in monocot-derived biomass. Fungal genomes encode numerous xyloglucanase genes, belonging to at least six glycoside hydrolase (GH) families. GH74 endo-xyloglucanases cleave xyloglucan backbones with unsubstituted glucose at the −1 subsite or prefer xylosyl-substituted residues in the −1 subsite. In this work, 137 GH74-related genes were detected by examining 293 Eurotiomycete genomes and Ascomycete fungi contained one or no GH74 xyloglucanase gene per genome. Another interesting feature is that the triad of tryptophan residues along the catalytic cleft was found to be widely conserved among Ascomycetes. The GH74 from Aspergillus fumigatus (AfXEG74) was chosen as an example to conduct comprehensive biochemical studies to determine the catalytic mechanism. AfXEG74 has no CBM and cleaves the xyloglucan backbone between the unsubstituted glucose and xylose-substituted glucose at specific positions, along the XX motif when linked to regions deprived of galactosyl branches. It resembles an endo-processive activity, which after initial random hydrolysis releases xyloglucan-oligosaccharides as major reaction products. This work provides insights on phylogenetic diversity and catalytic mechanism of GH74 xyloglucanases from Ascomycete fungi.

β-Glucosidase From Aspergillus

Abstract Different species from the genus Aspergillus, such as A. niger and A. oryzae, are able to produce a wide range of carbohydrate-active enzymes (CAZymes), including β-glucosidase, that present several industrial applications. This glycosyl hydrolase hydrolyzes β-linked O-glycosyl bond of various oligomeric saccharides or glycosides from cellulose. Hence, it is a promising biocatalyst for the synthesis of stereo- and regiospecific glycosides or oligosaccharides, molecules potentially useful as functional materials, nutraceuticals, or pharmaceuticals. In addition, this enzyme can be used in combination with other enzymes in the degradation of lignocellulosic materials for the obtainment of second-generation ethanol. In this perspective, this chapter highlights the production, purification, characterization, applications, and immobilization of β-glucosidases from different Aspergillus species.

Protein profile in Aspergillus nidulans recombinant strains overproducing heterologous enzymes

Filamentous fungi are robust cell factories and have been used for the production of large quantities of industrially relevant enzymes. However, the production levels of heterologous proteins still need to be improved. Therefore, this article aimed to investigate the global proteome profiling of Aspergillus nidulans recombinant strains in order to understand the bottlenecks of heterologous enzymes production. About 250, 441 and 424 intracellular proteins were identified in the control strain Anid_pEXPYR and in the recombinant strains Anid_AbfA and Anid_Cbhl respectively. In this context, the most enriched processes in recombinant strains were energy pathway, amino acid metabolism, ribosome biogenesis, translation, endoplasmic reticulum and oxidative stress, and repression under secretion stress (RESS). The global protein profile of the recombinant strains Anid_AbfA and Anid_Cbhl was similar, although the latter strain secreted more recombinant enzyme than the former. These findings provide insights into the bottlenecks involved in the secretion of recombinant proteins in A. nidulans, as well as in regard to the rational manipulation of target genes for engineering fungal strains as microbial cell factories.

Agro-Industrial Residues and Microbial Enzymes

Abstract Bioconversion of renewable lignocellulosic biomass to biofuels and high value-added products is nowadays a field of much attention and promise. Furthermore, lignin can be used for the smooth generation of polymers using laccase or a laccase-mediator system. Food industries provide other important uses of residues, such as underutilized fish, and by-products from the fishing industries for the production of bioactive peptides using microbial proteases. On the other hand, fat wastes, such as waste from cooking oil, are very interesting substrates for the production of industrially relevant compounds, mainly biodiesel, using lipases from different microorganisms. Again, phenolic compounds are very important because of their biological activities, presenting impressive antioxidant activity. More interestingly, they can be obtained using enzymes from different microorganisms, which are capable of producing antioxidative phenolics from different wastes. Although microbial enzymes are highly effective tools for modifying agro-industrial residues in generating high value-added products, the use of native enzymes are frequently infeasible in large scales. Therefore, different techniques of molecular biology are necessary to surpass these limitations. These techniques include the use of expression models that are more feasible for the industrial production of enzymes, and genetic and protein engineering focusing on the overexpression of the enzymes to have the desired enzymes with improved characteristics, such as better enzymatic activity, stability, and selectivity.

High value added lipids produced by microorganisms: a potential use of sugarcane vinasse

Abstract This review aims to present an innovative concept of high value added lipids produced by heterotrophic microorganisms, bacteria and fungi, using carbon sources, such as sugars, acids and alcohols that could come from sugarcane vinasse, which is the main byproduct from ethanol production that is released in the distillation step. Vinasse is a rich carbon source and low-cost feedstock produced in large amounts from ethanol production. In 2019, the Brazilian Ministry of Agriculture, Livestock and Food Supply estimates that growth of ethanol domestic consumption will be 58.8 billion liters, more than double the amount in 2008. This represents the annual production of more than 588 billion liters of vinasse, which is currently used as a fertilizer in the sugarcane crop, due to its high concentration of minerals, mainly potassium. However, studies indicate some disadvantages such as the generation of Greenhouse Gas emission during vinasse distribution in the crop, as well as the possibility of contaminating the groundwater and soil. Therefore, the development of programs for sustainable use of vinasse is a priority. One profitable alternative is the fermentation of vinasse, followed by an anaerobic digester, in order to obtain biomaterials such as lipids, other byproducts, and methane. Promising high value added lipids, for instance carotenoids and polyunsaturated fatty acids (PUFAS), with a predicted market of millions of US