Delineation of the CYP505E subfamily of fungal self-sufficient in-chain hydroxylating cytochrome P450 monooxygenases.

Cytochrome P450 monooxygenases (CYP450s) are abundant in eukaryotes, specifically in plants and fungi where they play important roles in the synthesis and degradation of secondary metabolites. In eukaryotes, the best studied "self-sufficient" CYP450s, with a fused redox partner, belong to the CYP505 family. Members of the CYP505 family are generally considered sub-terminal fatty acid hydroxylases. CYP505E3 from Aspergillus terreus, however, gives remarkable in-chain hydroxylation at the ω-7 position of C10 to C16 alkanes and C12 and C14 fatty alcohols. Because CYP505E3 is a promising catalyst for the synthesis of δ-dodecalactone, we set out to delineate the unique ω-7 hydroxylase activity of CYP505E3. CYP505E3 and six additional CYP505Es as well as four closely related CYP505s from four different subfamilies were expressed in Pichia pastoris. Only the CYP505Es, sharing more than 70% amino acid identity, displayed significant ω-7 hydroxylase activity toward 1-dodecanol, dodecanoic acid, and tetradecanoic acid giving products that can readily be converted to δ-dodecalactone. Concentrations of δ-dodecalactone, directly extracted from dodecanoic acid biotransformations, were higher than previously obtained with E. coli. Searches of the UniProt and NCBI databases yielded a total of only 23 unique CYP505Es, all from the Aspergillaceae. Given that CYP505Es with this remarkable activity occur in only a few Aspergillus and Penicillium spp., we further explored the genetic environments in which they occur. These were found to be very distinct environments which include a specific ABC transporter but could not be linked to apparent secondary metabolite gene clusters. KEY POINTS: • Identified CYP505Es share > 70% amino acid identity. • CYP505Es hydroxylate 1-dodecanol, dodecanoic, and tetradecanoic acid at ω-7 position. • CYP505E genes occur in Aspergillus and Penicillium spp. near an ABC transporter.

Identification and Characterization of a Cryptic Bifunctional Type I Diterpene Synthase Involved in Talaronoid Biosynthesis from a Marine-Derived Fungus.

We report the identification of the tnd biosynthetic cluster from the marine-derived fungus Aspergillus flavipes and the in vivo characterization of a cryptic type I diterpene synthase. The heterologous expression of the bifunctional terpene synthase led to the discovery of a diterpene backbone, talarodiene, harboring a benzo[a]cyclopenta[d]cyclooctane tricyclic fused ring system. The conversion of geranylgeranyl diphosphate to talarodiene was investigated using 13C-labeling studies, and stable isotope tracer experiments showed the biotransformation of talarodiene into talaronoid C.

A time-dependently regulated gene network reveals that Aspergillus protease affects mitochondrial metabolism and airway epithelial cell barrier function via mitochondrial oxidants.

The airway epithelium maintains tight barrier integrity to prevent penetration of pathogens; thus, impairment of the barrier function is an important and common histological feature in asthmatic patients. Proteolytic allergens from fungi, pollen, and house dust mites can disrupt epithelial barrier integrity, but the mechanism remains unclear. Aspergillus oryzae protease (AP)-induced mitochondrial reactive oxygen species (ROS) contribute to the epithelial inflammatory response. However, as mitochondrial ROS affect various cellular functions, such as metabolism, cell death, cell proliferation, and redox homeostasis through signal transduction, it is difficult to understand the detailed action mechanism of AP by measuring changes in a single gene or protein of a specific signaling pathway. Moreover, mitochondrial ROS can directly oxidize DNA to activate transcription, thereby affecting the expression of various genes at the transcriptional level. Therefore, we conducted whole-genome analysis and used a network-based approach to understand the effect of AP and AP-induced mitochondrial ROS in human primary airway epithelial cells and to evaluate the mechanistic basis for AP-mediated epithelial barrier dysfunction. Our results indicate that production of mitochondrial ROS following AP exposure induce mitochondrial dysfunction at an early stage. Over time, changes in genome expression were further expanded without remaining mitochondrial ROS. Specifically, genes involved in the apoptotic functions and intercellular junctions were affected, consequently impairing the cellular barrier integrity. This change was recovered by scavenging mitochondrial ROS at an early point after exposure to AP. In conclusion, our findings indicate that instantly increased mitochondrial ROS at the time of exposure to allergenic proteases consequently induces epithelial barrier dysfunction at a later time point, resulting in pathological changes. These data suggest that antioxidant therapy administered immediately after exposure to proteolytic antigens may be effective in maintaining epithelial barrier function.

Molecular insights into the inhibition of glutamate dehydrogenase by the dicarboxylic acid metabolites.

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+ - or NADP+ -dependent reversible conversion of α-ketoglutarate (AKG) to l-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP+ -dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate, and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

Inhibition analysis of aflatoxin by in silico targeting the thioesterase domain of polyketide synthase enzyme in Aspergillus ssp.

The spread of fungal growth causes enormous economic, agricultural, and health problems for humans, such as Aspergillus sp., which produce aflatoxins. Thus, the inhibition of aflatoxin production became a precious target. In this research, the thioesterase (TE) domain from Polyketide synthase enzyme was selected to employ the in silico docking, using AutoDock Vina, against 623 natural compounds from the South African natural compound database (SANCDB), to identify potential inhibitors that can selectively inhibit thioesterase domain. The top ten inhibitors components were pinocembrin, typhaphthalide, p-coumaroylputrescine, dilemmaone A, 9-angelylplatynecine, 2,4,6-octatrienal, 4,8-dichloro-3,7-dimethyl-, (2e,4z,6e)-, lilacinobiose, 1,3,7-octatriene, 5,6-dichloro-2-(dichloromethyl)-6-methyl-, [r*,s*-(e)]-(-)- (9ci), lilacinobiose, 1,3,7-octatriene, 5,6-dichloro-2-(dichloromethyl)-6-methyl-, [r*,s*-(e)]-(-)- (9ci), 1,3,7-octatriene, 1,5,6-trichloro-2-(dichloromethyl)-6-methyl-, [r*,s*-(z,e)] and 9-angelylhastanecine and that depending on the lowest binding energy, the best chemical interactions and the best drug-likeness. The results of those components gave successful inhibition with the thioesterase domain. So, they can be used for inhibition and controlling aflatoxin contamination of agriculture crop yields, specially, pinocembrin which gave promising results.Communicated by Ramaswamy H. Sarma.

Purification and characterization of a highly-stable fungal xylanase from Aspergillus tubingensis cultivated on palm wastes through combined solid-state and submerged fermentation.

Fungal xylanase was produced from lignocellulosic palm wastes through combined solid-state fermentation (SSF) and submerged fermentation (SmF) by Aspergillus tubingensis TSIP9 in a helical-impeller equipped bioreactor. The combined SSF-SmF promoted the xylanase production by 15 and 70% higher than SSF and SmF, respectively. Sequential purification yielded 7.4-fold purified xylanase with 9.07% recovery. The maximum activities of crude and purified xylanase were observed at the same pH of 5.0 and the same temperature of 50 °C while purified xylanase is more active and highly stable at a wider pH range of 3-8 and temperature of 30-60 °C. The half-life of purified xylanase at various temperatures was also much improved by 2-8 folds compared to crude xylanase. Michaelis-Menten constants, Vmax and Km, for purified xylanase are 2,602.8 U/mg and 32.4 mg/mL, respectively. Purified xylanase activity was most enhanced with Ca2+ followed by Zn2+ and Fe2+ at 10 mM while significantly inhibited by Co2+, Cu2+, Pb2+, and Ag+. This study has shown the effectiveness of combined SSF-SmF for xylanase production and superior properties of purified xylanase for industrial processes.

Protease Produced by Endophytic Fungi: A Systematic Review.

The purpose of this systematic review was to identify the available literature of production, purification, and characterization of proteases by endophytic fungi. There are few complete studies that entirely exhibit the production, characterization, and purification of proteases from endophytic fungi. This study followed the PRISMA, and the search was conducted on five databases: PubMed, PMC, Science Direct, Scopus Articles, and Web of Science up until 18 May 2021, with no time or language restrictions. The methodology of the selected studies was evaluated using GRADE. Protease production, optimization, purification, and characterization were the main evaluated outcomes. Of the 5540 initially gathered studies, 15 met the inclusion criteria after a two-step selection process. Only two studies optimized the protease production using statistical design and two reported enzyme purification and characterization. The genus Penicillium and Aspergillus were the most cited among the eleven different genera of endophytic fungi evaluated in the selected articles. Six studies proved the ability of some endophytic fungi to produce fibrinolytic proteases, demonstrating that endophytic fungi can be exploited for the further production of agents used in thrombolytic therapy. However, further characterization and physicochemical studies are required to evaluate the real potential of endophytic fungi as sources of industrial enzymes.

Mycofabrication of AgONPs derived from Aspergillus terreus FC36AY1 and its potent antimicrobial, antioxidant, and anti-angiogenesis activities.

BACKGROUND: There is an emergency need for the natural therapeutic agents to treat arious life threatening diseases such as cardio- vascular disease, Rheumatoid arthritis and cancer. Among these diseases, cancer is found to be the second life threatening disease; in this view the present study focused to synthesize the silver oxide nanoparticles (AgONPs) from endophytic fungus. METHODS: The endophytic fungus was isolated from a medicinal tree Aegle marmelos (Vilva tree) and the potential strain was screened through antagonistic activity. The endophytic fungus was identified through microscopic (Lactophenol cotton blue staining and spore morphology in culture media) and Internal Transcribed Spacer (ITS) 1, ITS 4 and 18S rRNA amplification. The endophyte was cultured for the synthesis of AgONPs and the synthesized NPs were characterized through UV- Vis, FT- IR, EDX, XRD and SEM. The synthesized AgONPs were determined for antimicrobial, antioxidant and anti- angiogenic activity. RESULTS: About 35 pigmented endophytic fungi were isolated, screened for antagonistic activity against 12 pathogens and antioxidant activity through DPPH radical scavenging assay; among the isolates, FC36AY1 explored the highest activity and the strain FC36AY1 was identified as Aspergillus terreus. The AgONPs were synthesized from the strain FC36AY1 and characterized for its confirmation, functional groups, nanostructures with unit cell dimensions, size and shape, presence of elements through UV-Vis spectrophotometry, FT-IR, XRD, SEM with EDX analysis. The myco-generated AgONPs manifested their antimicrobial and antioxidant properties with maximum activity at minimum concentration. Moreover, the inhibition of angiogenesis by the AgONPs in Hen's Egg Test on the Chorio-Allantoic Membrane analysis were tested on the eggs of Chittagong breed evinced at significant bioactivity least concentration at 0.1 µg/mL. CONCLUSIONS: Thus, the results of this study revealed that the fungal mediated AgONPs can be exploited as potential in biomedical applications.

Insights into carbon nanotube-assisted electro-oxidation of polycyclic aromatic hydrocarbons for mediated bioelectrocatalysis.

A series of polycyclic aromatics, naphthalene, phenanthrene, perylene, pyrene, 1-pyrenebutyric acid N-hydroxysuccinimide ester (pyrene NHS) and coronene, were immobilized via π stacking on carbon nanotube (CNT) electrodes and electro-oxidized in aqueous solutions. The obtained quinones were characterized and evaluated for the mediated electron transfer with FAD dependent glucose dehydrogenase during catalytic glucose oxidation.

A Machine Learning Study on the Thermostability Prediction of (R)-ω-Selective Amine Transaminase from Aspergillus terreus.

Artificial intelligence technologies such as machine learning have been applied to protein engineering, with unique advantages in protein structure, function prediction, catalytic activity, and other issues in recent years. Screening better mutants is still a bottleneck in protein engineering. In this paper, a new sequence-activity relationship method was analyzed for its application in improving the thermal stability of Aspergillus terreus (R)-ω-selective amine transaminase. The experimental data from 6 single-point mutated enzymes were used as a learning dataset to build models and predict the thermostability of 26 mutants. Based on digital signal processing (DSP), this method digitized the amino acid sequence of proteins by fast Fourier transform (FFT) and then established the best model applying partial least squares regression (PLSR) to screen out all possible mutants, especially those with high performance. In protein engineering, the innovative sequence activity relationship (ISAR) method can make a reasonable prediction using limited experimental data and significantly reduce the experimental cost. The half-life (T 1/2) of (R)-ω-transaminase was fitted with the amino acid sequence by the ISAR algorithm, resulting in an R 2 of 0.8929 and a cvRMSE of 4.89. At the same time, the mutants with higher T 1/2 than the existing ones were predicted, laying the groundwork for better (R)-ω-transaminase in the later stage. The ISAR algorithm is expected to provide a new technique for protein evolution and screening.

Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus luchuensis.

Certain Aspergillus and Penicillium spp. produce the fungal cell wall component nigeran, an unbranched d-glucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation. The mechanism underlying nigeran biosynthesis and the physiological role of nigeran in fungal survival are not clear. We used RNA sequencing (RNA-seq) to identify genes involved in nigeran synthesis in the filamentous fungus Aspergillus luchuensis when grown under nitrogen-free conditions. agsB, which encodes a putative α-1,3-glucan synthase, and two adjacent genes (agtC and gnsA) were upregulated under conditions of nitrogen starvation. Disruption of agsB in A. luchuensis (ΔagsB) resulted in the complete loss of nigeran synthesis. Furthermore, the overexpression of agsB in an Aspergillus oryzae strain that cannot produce nigeran resulted in nigeran synthesis. These results indicated that agsB encodes a nigeran synthase. Therefore, we have renamed the A. luchuensis agsB gene the nigeran synthase gene (nisA). Nigeran synthesis in an agtC mutant (ΔagtC) increased to 121%; conversely, those in the ΔgnsA and ΔagtC ΔgnsA strains decreased to 64% and 63%, respectively, compared to that in the wild-type strain. Our results revealed that AgtC and GnsA play an important role in regulating not only the quantity of nigeran but also its polymerization. Collectively, our results demonstrated that nisA (agsB) is essential for nigeran synthesis in A. luchuensis, whereas agtC and gnsA contribute to the regulation of nigeran synthesis and its polymerization. This research provides insights into fungal cell wall biosynthesis, specifically the molecular evolution of fungal α-glucan synthase genes and the potential utilization of nigeran as a novel biopolymer. IMPORTANCE The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3/1,4-glucosidic linkages. The mechanisms regulating the biosynthesis and function of nigeran are unknown. Here, we performed RNA sequencing of Aspergillus luchuensis cultured under nitrogen-free or low-nitrogen conditions. A putative α-1,3-glucan synthase gene, whose transcriptional level was upregulated under nitrogen-free conditions, was demonstrated to encode nigeran synthase. Furthermore, two genes encoding an α-glucanotransferase and a hypothetical protein were shown to be involved in controlling the nigeran content and molecular weight. This study reveals genes involved in the synthesis of nigeran, a potential biopolymer, and provides a deeper understanding of fungal cell wall biosynthesis.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Molecular insights into the endoperoxide formation by Fe(II)/α-KG-dependent oxygenase NvfI.

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.

Alternative splicing regulates the α-glucosidase synthesis in Aspergillus neoniger NCIM 1400.

Aspergillus neoniger NCIM 1400 whose cell-free fraction was earlier established for transglycosylation activity conferred by α-glucosidase gene (agdA), was subjected to sequence analysis. Preliminary results revealed certain dynamics in the intron splicing mechanism, and to ascertain these molecular events, a detailed study was carried. The electrophoresis results from the cDNA portion (B-fragment) of agdA showed multiple bands, indicating the amplification of one or more fragments. The sequence results of cDNA cloned vector revealed the retention type of alternative splicing in the agdA. The splicing mechanism of agdA in NCIM 1400 was compared to different A. niger strains, which harbours agdA orthologues, using PCR. It was observed that effective intron splicing leads to higher α-glucosidase activity from these selected Aspergillus spp. To explore the dynamics of intron retention in A. neoniger NCIM 1400, time-course analysis of intron retention, enzyme activity, and sugar consumption were carried over a period of 168 h of fungal growth. RT-qPCR results revealed that introns retention was not detected during the initial growth phase when the maltose and its hydrolysed product, glucose were consumed. Here we demonstrate that exhaustion of maltose causes increase in retention of introns in the mRNA transcripts of agdA gene, and this could be the possible mode of regulating this gene.

Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review.

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.

Identification and characterization of extracellular enzymes secreted by Aspergillus spp. involved in lipolysis and lipid-antioxidation during katsuobushi fermentation and ripening.

A mild-flavored soup stock made from katsuobushi is an important element of traditional Japanese cuisine and is the basic seasoning responsible for the taste. Fermented and ripened katsuobushi, known as karebushi, is manufactured by simmering skipjack tuna that is then smoke-dried, fermented, and ripened in a repeated molding process by five dominant Aspergillus species. Here, our aim was to characterize and identify the lipolytic enzymes secreted by the dominant Aspergillus species, especially A. chevalieri and A. pseudoglaucus, which are involved in hydrolyzing lipids during the molding process. The crude enzyme preparations from the five Aspergillus spp. cultivated on katsuobushi solid medium hydrolyzed triglycerides in fish oil, and more saturated and unsaturated fatty acids (C16:0, C16:1, C18:0, C18:1) were produced than major polyunsaturated fatty acids (C20:5, C22:6). On the basis of ion exchange chromatograms, the composition of the lipolytic enzymes was different in the five species. There was at least one active fraction with high hydrolytic activity toward fish oil in four of the Aspergillus spp., but not A. sydowii; the lipolytic enzyme secreted by A. sydowii had quite high activity toward the artificial substrate p-nitrophenyl butyrate, but low activity toward the natural oil. The lipolytic fractions from A. chevalieri and A. pseudoglaucus were further purified by hydrophobic interaction chromatography then gel-filtration chromatography; LC-MS-MS Mascot analysis identified a variety of lipolytic enzymes, including cutinase, esterase, phospholipase, and carboxyl esterase in the lipolytic fractions from these species. The identified enzymes had 30%-70% identity to previously reported or manually annotated lipases or esterases from taxa other than Aspergillus. The different lipolytic enzymes likely acted on triglycerides in the katsuobushi fish oil. Furthermore, catalase B and Cu/Zn superoxide dismutase, which limit oxidative damage of lipids, were also identified. These antioxidant enzymes may prevent lipid oxidation and rancidity as the lipolytic enzymes hydrolyze lipids during the long fermentation and ripening process. Umami and richness tastes tended to increase in extracts from culture of protease- and peptidase-producing A. sydowii. Our results will aid in the selection and application of desirable strains of Aspergillus species as starter cultures to improve the storage and quality of fermented and ripened karebushi.

Interspecific evolutionary relationships of alpha-glucuronidase in the genus Aspergillus.

The increased availability and production of lignocellulosic agroindustrial wastes has originated proposals for their use as raw material to obtain biofuels (ethanol and biodiesel) or derived products. However, for biomass generated from lignocellulosic residues to be successfully degraded, in most cases it requires a physical (thermal), chemical, or enzymatic pretreatment before the application of microbial or enzymatic fermentation technologies (biocatalysis). In the context of enzymatic technologies, fungi have demonstrated to produce enzymes capable of degrading polysaccharides like cellulose, hemicelluloses and pectin. Because of this ability for degrading lignocellulosic material, researchers are making efforts to isolate and identify fungal enzymes that could have a better activity for the degradation of plant cell walls and agroindustrial biomass. We performed an in silico analysis of alpha-glucoronidase in 82 accessions of the genus Aspergillus. The constructed dendrograms of amino acid sequences defined the formation of 6 groups (I, II, III, IV, V, and VI), which demonstrates the high diversity of the enzyme. Despite this ample divergence between enzyme groups, our 3D structure modeling showed both conservation and differences in amino acid residues participating in enzyme-substrate binding, which indicates the possibility that some enzymes are functionally specialized for the specific degradation of a substrate depending on the genetics of each species in the genus and the condition of the habitat where they evolved. The identification of alpha-glucuronidase isoenzymes would allow future use of genetic engineering and biocatalysis technologies aimed at specific production of the enzyme for its use in biotransformation.

Evaluation of nutraceutical application of xylooligosaccharide enzymatically produced from cauliflower stalk for its value addition through a sustainable approach.

There is increasing attention on the exploration of waste feedstocks as economically viable substrates for the production of prebiotic oligosaccharides, especially xylooligosaccharides, as excellent candidates for the maintenance and promotion of gut microbiota. XOS, an emerging prebiotic that has several functional attributes and beneficial health effects, is mainly produced by different processes, especially enzymatic hydrolysis through the valorisation of xylan enriched lignocellulosic materials. The present study deals with the enzymatic production of xylooligosaccharide (XOS) from xylan rich cauliflower stalk, a novel source. Delignification with alkali (NaOH) was found to be more efficient than acid and autohydrolysis, resulting in a higher extraction yield of xylan (18.42%). Alkaline extraction for 120 minutes at 1.25 M alkali concentration produced maximum xylan yield. FTIR analysis of xylan extracted from cauliflower stalk by an alkaline (NaOH) pretreatment method showed typical absorption bands at 1729 cm-1 that correspond to acetyl groups exhibiting the typical xylan specific band. Enzymatic hydrolysis was carried out with indigenously produced crude endoxylanase obtained from Aspergillus niger MTCC 9687 and the effects of substrate concentration, enzyme concentration, pH, time and temperature were investigated. High resolution MS analysis showed the presence of xylobiose as the major XOS. The major 1H spectral signals of XOS liberated from enzymatically hydrolysed alkali extracted cauliflower stalk xylan showed the presence of ß-anomeric protons in the spectral region of 4.0-4.7 ppm. Prebiotic efficacy of cauliflower stalk derived XOS alone and synbiotic combinations with known probiotic strains (Lactiplantibacillus plantarum, Bifidobacterium bifidum, Lactobacillus delbrueckii ssp. Helveticus) were evaluated. Butyrate was found to be the major short chain fatty acid produced by XOS supplemented fermentation media. All the synbiotic combinations showed significantly higher antioxidant and antimicrobial activities and reduced the viability of human bone cancer MG-63 cells. The individual profiles of antimicrobial components of XOS were identified as dihydroxy benzoic acid and aspartic acid by HPLC coupled to a photodiode array detector.

Modification of the transglucosylation properties of α-glucosidases from Aspergillus oryzae and Aspergillus sojae via a single critical amino acid replacement.

We constructed enzyme variants of the α-glucosidases from Aspergillus oryzae (AoryAgdS) and Aspergillus sojae (AsojAgdL) by mutating the amino acid residue at position 450. AoryAgdS_H450R acquired the ability to produce considerable amounts of α-1,6-transglucosylation products, whereas AsojAgdL_R450H changed to produce more α-1,3- and α-1,4-transglucosylation products than α-1,6-products. The 450th amino acid residue is critical for the transglucosylation of these α-glucosidases.