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Background: Fermentation of sugars derived from plant biomass feedstock is crucial for sustainability. Hence, utilizing customized enzymatic cocktails to obtain oligosaccharides instead of monomers is an alternative fermentation strategy to produce prebiotics, cosmetics, and biofuels. This study developed an engineered strain of Aspergillus niger producing a tailored cellulolytic cocktail capable of partially degrading sugarcane straw to yield cellooligosaccharides., Results: The A. niger prtT∆ strain created resulted in a reduced extracellular protease production. The prtT∆ background was then used to create strains by deleting exoenzyme encoding genes involved in mono- or disaccharide formation. Consequently, we successfully generated a tailored prtT∆bglA∆ strain by eliminating a beta-glucosidase (bglA) gene and subsequently deleted two cellobiohydrolases and one beta-xylosidase encoding genes using a multiplex strategy, resulting in the Quintuple∆ strain (prtT∆; bglA∆; cbhA∆; cbhB∆; xlnD∆). When applied for sugarcane biomass degradation, the tailored secretomes produced by A. niger resulted in a higher ratio of cellobiose and cellotriose compared with glucose relative to the reference strain. Mass spectrometry revealed that the Quintuple∆ strain secreted alternative cellobiohydrolases and beta-glucosidases to compensate for the absence of major cellulases. Enzymes targeting minor polysaccharides in plant biomass were also upregulated in this tailored strain., Conclusion: Tailored secretome use increased COS/glucose ratio during sugarcane biomass degradation showing that deleting some enzymatic components is an effective approach for producing customized enzymatic cocktails. Our findings highlight the plasticity of fungal genomes as enzymes that target minor components of plant cell walls, and alternative cellulases were produced by the mutant strain. Despite deletion of important secretome components, fungal growth was maintained in plant biomass., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans, A. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories.

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes found in viruses, archaea, and bacteria as well as eukaryotes, such as fungi, algae and insects, actively contributing to the degradation of different polysaccharides. In Aspergillus nidulans, LPMOs from family AA9 ( An LPMO9s), along with an AA3 cellobiose dehydrogenase ( An CDH1), are cosecreted upon growth on crystalline cellulose and lignocellulosic substrates, indicating their role in the degradation of plant cell wall components. Functional analysis revealed that three target LPMO9s ( An LPMO9C, An LPMO9F and An LPMO9G) correspond to cellulose-active enzymes with distinct regioselectivity and activity on cellulose with different proportions of crystalline and amorphous regions. An LPMO9s deletion and overexpression studies corroborate functional data. The abundantly secreted An LPMO9F is a major component of the extracellular cellulolytic system, while An LPMO9G was less abundant and constantly secreted, and acts preferentially on crystalline regions of cellulose, uniquely displaying activity on highly crystalline algae cellulose. Single or double deletion of An LPMO9s resulted in about 25% reduction in fungal growth on sugarcane straw but not on Avicel, demonstrating the contribution of LPMO9s for the saprophytic fungal lifestyle relies on the degradation of complex lignocellulosic substrates. Although the deletion of An CDH1 slightly reduced the cellulolytic activity, it did not affect fungal growth indicating the existence of alternative electron donors to LPMOs. Additionally, double or triple knockouts of these enzymes had no accumulative deleterious effect on the cellulolytic activity nor on fungal growth, regardless of the deleted gene. Overexpression of An LPMO9s in a cellulose-induced secretome background confirmed the importance and applicability of An LPMO9G to improve lignocellulose saccharification. IMPORTANCE Fungal lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that boost plant biomass degradation in combination with glycoside hydrolases. Secretion of LPMO9s arsenal by Aspergillus nidulans is influenced by the substrate and time of induction. These findings along with the biochemical characterization of novel fungal LPMO9s have implications on our understanding of their concerted action, allowing rational engineering of fungal strains for biotechnological applications such as plant biomass degradation. Additionally, the role of oxidative players in fungal growth on plant biomass was evaluated by deletion and overexpression experiments using a model fungal system.

A chemical investigation of the filamentous fungus Aspergillus californicus led to the isolation of a polyketide-nonribosomal peptide hybrid, calipyridone A ( 1 ). A putative biosynthetic gene cluster cpd for production of 1 was next identified by genome mining. The role of the cpd cluster in the production of 1 was confirmed by multiple gene deletion experiments in the host strain as well as by heterologous expression of the hybrid gene cpdA in Aspergillus oryzae . Moreover, chemical analyses of the mutant strains allowed the biosynthesis of 1 to be elucidated. The results indicate that the generation of the 2-pyridone moiety of 1 via nucleophilic attack of the iminol nitrogen to the carbonyl carbon is different from the biosynthesis of other fungal 2-pyridone products through P450-catalyzed tetramic acid ring expansions. In addition, two biogenetic intermediates, calipyridones B and C, showed modest inhibition effects on the plaque-forming ability of SARS-CoV-2.

Filamentous fungi secrete protein with a very high efficiency, and this potential can be exploited advantageously to produce therapeutic proteins at low costs. A significant barrier to this goal is posed by the fact that fungal N-glycosylation varies substantially from that of humans. Inappropriate N-glycosylation of therapeutics results in reduced product quality, including poor efficacy, decreased serum half-life, and undesirable immune reactions. One solution to this problem is to reprogram the glycosylation pathway of filamentous fungi to decorate proteins with glycans that match, or can be remodeled into, those that are accepted by humans. In yeast, deletion of ALG3 leads to the accumulation of Man 5 GlcNAc 2 glycan structures that can act as a precursor for remodeling. However, in Aspergilli, deletion of the ALG3 homolog algC leads to an N-glycan pool where the majority of the structures contain more hexose residues than the Man 3-5 GlcNAc 2 species that can serve as substrates for humanized glycan structures. Hence, additional strain optimization is required. In this report, we have used gene deletions in combination with enzymatic and chemical glycan treatments to investigate N-glycosylation in the model fungus Aspergillus nidulans. In vitro analyses showed that only some of the N-glycan structures produced by a mutant A. nidulans strain, which is devoid of any of the known ER mannose transferases, can be trimmed into desirable Man 3 GlcNAc 2 glycan structures, as substantial amounts of glycan structures appear to be capped by glucose residues. In agreement with this view, deletion of the ALG6 homolog algF, which encodes the putative α-1,3- glucosyltransferase that adds the first glucose residue to the growing ER glycan structure, dramatically reduces the amounts of Hex 6-7 HexNAc 2 structures. Similarly, these structures are also sensitive to overexpression of the genes encoding the heterodimeric α-glucosidase II complex. Without the glucose caps, a new set of large N-glycan structures was formed. Formation of this set is mostly, perhaps entirely, due to mannosylation, as overexpression of the gene encoding mannosidase activity led to their elimination. Based on our new insights into the N-glycan processing in A. nidulans, an A. nidulans mutant strain was constructed in which more than 70% of the glycoforms appear to be Man 3-5 GlcNAc 2 species, which may serve as precursors for further engineering in order to create more complex human-like N-glycan structures., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Matricaria chamomilla L. contains antioxidant flavonoids that can have their bioactivity enhanced by enzymatic hydrolysis of specific glycosyl groups. This study implements an untargeted metabolomics approach based on ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry technique operating in MS E mode (UPLC-QTOF-MS E ) and spectrophotometric analysis of chamomile aqueous infusions, before and after hydrolysis by hesperidinase and β-galactosidase. Several phenolic compounds were altered in the enzymatically treated infusion, with the majority being flavonoid derivatives of apigenin, esculetin, and quercetin. Although enzymatically modifying the infusion only led to a small increase in antioxidant activity (DPPH • method), its inhibitory effect on pancreatic lipase was of particular interest. The enzymatically treated infusion exhibited a greater inhibitory effect (EC 50 of 35.6 µM) than unmodified infusion and kinetic analysis suggested mixed inhibition of pancreatic lipase. These results are of great relevance due to the potential of enzymatically treated functional foods in human health.

Filamentous fungi are important cell factories for large-scale enzyme production. However, production levels are often low, and this limitation has stimulated research focusing on the manipulation of genes with predicted function in the protein secretory pathway. This pathway is the major route for the delivery of proteins to the cell exterior, and a positive relationship between the production of recombinant enzymes and the unfolded protein response (UPR) pathway has been observed. In this study, Aspergillus nidulans was exposed to UPR-inducing chemicals and differentially expressed genes were identified by RNA-seq. Twelve target genes were deleted in A. nidulans recombinant strains producing homologous and heterologous GH10 xylanases. The knockout of pbnA (glycosyltransferase), ydjA (Hsp40 co-chaperone), trxA (thioredoxin) and cypA (cyclophilin) improved the production of the homologous xylanase by 78, 171, 105 and 125% respectively. Interestingly, these deletions decreased the overall protein secretion, suggesting that the production of the homologous xylanase was specifically altered. However, the production of the heterologous xylanase and the secretion of total proteins were not altered by deleting the same genes. Considering the results, this approach demonstrated the possibility of rationally increase the production of a homologous enzyme, indicating that trxA, cypA, ydjA and pbnA are involved in protein production by A. nidulans., (© 2020 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Both preventive and curative therapies have created a considerable demand for n-3 PUFAs (polyunsaturated fatty acids) from fish oil, such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, for human use. Bio-synthesized sardine oil (bioSO) concentrate containing an acylglycerols mixture with 50% n-3 PUFAs was obtained by Candida cylindracea lipase hydrolysis and subsequently used for in vivo tests in animals. Wistar rats received, by gavage, a dose of 0.2 g/kg/day of bioSO or unmodified sardine oil (unSO) or saline solution (control) for three consecutive days and the liver tissue was evaluated by a selective and sensitive lipidomic approach based on ultra-performance liquid chromatography-quadruple time-of-flight mass spectrometry (UPLC-QTOF-MS E ) and gas chromatography (GC). In addition, antioxidant parameters, response of oxidative stress marker and estimated fatty acid desaturase indexes were determined. The use of bioSO led to an increase in Cer d18:1/16:0, PE-Cer d14:2/18:0 and highly unsaturated phosphatylcholines (PC 38:4, PC 40:6 and PC 42:8) in the hepatic tissue membranes. There was also an increase in DHA incorporation in animals that received bioSO in comparison with the control animals. No differences in superoxide dismutase and catalase activity levels were observed between the groups, and malondialdehyde levels and delta 5-desaturase activity were higher in animals supplemented with bioSO. These results indicate that bioSO increase the hepatic incorporation of DHA, especially those esterified as PCs, and are probably absorbed and transported more effectively than the unSO. Enzymatically hydrolyzed compounds containing antioxidants may be a viable alternative for obtaining n-3 PUFA-enriched functional lipids., Competing Interests: Declarations of Competing Interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications., Results: In this study, BxlB-a highly secreted GH3 β-xylosidase from A. nidulans , presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N -glycans on BxlB secretion and function. The non-glycosylated mutant (BxlB non-glyc ) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlB wt ), while a partially glycosylated mutant (BxlB N1;5;7 ) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlB CC ), despite the high transcript levels. BxlB wt , BxlB non-glyc , and BxlB N1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlB N1;5;7 , to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlB N1;5 and BxlB N5;7 ) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlB non-glyc and BxlB N1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlB non-glyc and BxlB N1;5 ., Conclusions: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

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.

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., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Carbohydrate-Active Enzymes are key enzymes for biomass-to-bioproducts conversion. α-l-Arabinofuranosidases that belong to the Glycoside Hydrolase family 62 (GH62) have important applications in biofuel production from plant biomass by hydrolyzing arabinoxylans, found in both the primary and secondary cell walls of plants. In this work, we identified a GH62 α-l-arabinofuranosidase (AnAbf62A wt ) that was highly secreted when Aspergillus nidulans was cultivated on sugarcane bagasse. The gene AN7908 was cloned and transformed in A. nidulans for homologous production of AnAbf62A wt , and we confirmed that the enzyme is N-glycosylated at asparagine 83 by mass spectrometry analysis. The enzyme was also expressed in Escherichia coli and the studies of circular dichroism showed that the melting temperature and structural profile of AnAbf62A wt and the non-glycosylated enzyme from E. coli (AnAbf62A deglyc ) were highly similar. In addition, the designed glycomutant AnAbf62A N83Q presented similar patterns of secretion and activity to the AnAbf62A wt , indicating that the N-glycan does not influence the properties of this enzyme. The crystallographic structure of AnAbf62A deglyc was obtained and the 1.7Å resolution model showed a five-bladed β-propeller fold, which is conserved in family GH62. Mutants AnAbf62A Y312F and AnAbf62A Y312S showed that Y312 was an important substrate-binding residue. Molecular dynamics simulations indicated that the loop containing Y312 could access different conformations separated by moderately low energy barriers. One of these conformations, comprising a local minimum, is responsible for placing Y312 in the vicinity of the arabinose glycosidic bond, and thus, may be important for catalytic efficiency., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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$, could be produced using vinasse as carbon source, to guide an innovative concept for sustainable production. Example of lipids obtained from the fermentation of compounds present in vinasse are vitamin D, which comes from yeast sucrose fermentation and Omega 3, which can be obtained by bacteria and fungi fermentation. Additionally, several other compounds present in vinasse can be used for this purpose, including sucrose, ethanol, lactate, pyruvate, acetate and other carbon sources. Finally, this paper illustrates the potential market and microbial processes, using microorganisms, for lipid production.

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.

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 36h, 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., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Carbohydrases find a wide application in industrial processes and products, mainly in the food industry. With these enzymes, it is possible to obtain different types of sugar syrups (viz. glucose, fructose and inverted sugar syrups), prebiotics (viz. galactooligossacharides and fructooligossacharides) and isomaltulose, which is an interesting sweetener substitute for sucrose to improve the sensory properties of juices and wines and to reduce lactose in milk. The most important carbohydrases to accomplish these goals are of microbial origin and include amylases (α-amylases and glucoamylases), invertases, inulinases, galactosidases, glucosidases, fructosyltransferases, pectinases and glucosyltransferases. Yet, for all these processes to be cost-effective for industrial application, a very efficient, simple and cheap immobilization technique is required. Immobilization techniques can involve adsorption, entrapment or covalent bonding of the enzyme into an insoluble support, or carrier-free methods, usually based on the formation of cross-linked enzyme aggregates (CLEAs). They include a broad variety of supports, such as magnetic materials, gums, gels, synthetic polymers and ionic resins. All these techniques present advantages and disadvantages and several parameters must be considered. In this work, the most recent and important studies on the immobilization of carbohydrases with potential application in the food industry are reviewed.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Response surface methodology (RSM) based on a five-level, three-variable central composite design (CCD) was employed for modeling and optimizing the conversion yield of the enzymatic acylation of hesperidin with decanoic acid using immobilized Candida antarctica lipase B (CALB) in a two-phase system containing [bmim]BF(4). The three variables studied (molar ratio of hesperidin to decanoic acid, [bmim]BF(4)/acetone ratio and lipase concentration) significantly affected the conversion yield of acylated hesperidin derivative. Verification experiments confirmed the validity of the predicted model. The lipase showed higher conversion degree in a two-phase system using [bmim]BF(4) and acetone compared to that in pure acetone. Under the optimal reaction conditions carried out in a single-step biocatalytic process when the water content was kept lower than 200 ppm, the maximum acylation yield was 53.6%.

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, NaH(2)P0(4), KH(2)P0(4), MgS0(4)-7H(2)0, CaCl(2), NaCI, and MnS0(4). The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH(2)P0(4), and KH(2)P0(4), 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 micromoles of oleic acid released per minute of reaction (micromol/min).

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 72 h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (E = 23) in a shorter reaction period (48 h) at 35 degrees C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8 months of storage at 4 degrees C and could be reused at least six times.

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(-)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.