Optimized production and characterization of endo-ß-mannanase by Aspergillus niger for generation of prebiotic mannooligosaccharides from guar gum.

Optimized production of Aspergillus niger ATCC 26011 endo-ß-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.

Heterologous expression and characterization of mutant cellulase from indigenous strain of Aspergillus niger.

The purpose of current research work was to investigate the effect of mutagenesis on endoglucanase B activity of indigenous strain of Aspergillus niger and its heterologous expression studies in the pET28a+ vector. The physical and chemical mutagens were employed to incorporate mutations in A. niger. For determination of mutations, mRNA was isolated followed by cDNA synthesis and cellulase gene was amplified, purified and sequenced both from native and mutant A. niger. On comparison of gene sequences, it was observed that 5 nucleotide base pairs have been replaced in the mutant cellulase. The mutant recombinant enzyme showed 4.5 times higher activity (428.5 µmol/mL/min) as compared to activity of native enzyme (94 µmol/mL/min). The mutant gene was further investigated using Phyre2 and I-Tesser tools which exhibited 71% structural homology with Endoglucanase B of Thermoascus aurantiacus. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), solvent accessible surface area (SASA), radius of gyration (Rg) and hydrogen bonds analysis were carried at 35°C and 50°C to explore the integrity of structure of recombinant mutant endoglucanase B which corresponded to its optimal temperature. Hydrogen bonds analysis showed more stability of recombinant mutant endoglucanase B as compared to native enzyme. Both native and mutant endoglucanase B genes were expressed in pET 28a+ and purified with nickel affinity chromatography. Theoretical masses determined through ExPaSy Protparam were found 38.7 and 38.5 kDa for native and mutant enzymes, respectively. The optimal pH and temperature values for the mutant were 5.0 and 50°C while for native these were found 4.0 and 35°C, respectively. On reacting with carboxy methyl cellulose (CMC) as substrate, the mutant enzyme exhibited less Km (0.452 mg/mL) and more Vmax (50.25 µmol/ml/min) as compared to native having 0.534 mg/mL as Km and 38.76 µmol/ml/min as Vmax. Among metal ions, Mg2+ showed maximum inducing effect (200%) on cellulase activity at 50 mM concentration followed by Ca2+ (140%) at 100 mM concentration. Hence, expression of a recombinant mutant cellulase from A. niger significantly enhanced its cellulytic potential which could be employed for further industrial applications at pilot scale.

Low frequency magnetic field assisted production of acidic protease by Aspergillus niger.

Acid protease is widely used in industries such as food processing and feed additives. In the study, low frequency magnetic field (LF-MF) as an aid enhances acid protease production by Aspergillus niger (A. niger). The study assessed mycelial biomass, the enzymic activity of the acidic protease and underlying mechanism. At low intensities, alternating magnetic field (AMF) is more effective than static magnetic fields (SMF). Under optimal magnetic field conditions, acid protease activity and biomass increased by 91.44% and 16.31%, as compared with the control, respectively. Maximum 19.87% increase in enzyme activity after magnetic field treatment of crude enzyme solution in control group. Transcriptomics analyses showed that low frequency alternating magnetic field (LF-AMF) treatment significantly upregulated genes related to hydrolases and cell growth. Our results showed that low-frequency magnetic fields can enhance the acid protease production ability of A. niger, and the effect of AMF is better at low intensities. The results revealed that the effect of magnetic field on the metabolic mechanism of A. niger and provided a reference for magnetic field-assisted fermentation of A. niger.

Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

One-step high efficiency separation of prolyl endopeptidase from Aspergillus niger and its application.

Prolyl endopeptidase from Aspergillus niger (An-PEP) is an enzyme that recognizes C-terminal peptide bonds of amino acid chains and cleaves them by hydrolysis. An aqueous two-phase system (ATPS) was used to separate An-PEP from fermentation broth. Through single factor experiments, the ATPS containing 16 % (w/w) PEG2000 and 15 % (w/w) (NH4)2SO4 at pH 6.0 obtained the recovery of 79.74 ± 0.16 % and the purification coefficient of 7.64 ± 0.08. It was then used to produce soy protein isolate peptide (SPIP) by hydrolysis of soy protein isolate (SPI), and SPIP-Ferrous chelate (SPIP-Fe) was prepared with SPIP and Fe2+. The chelation conditions were optimized by RSM, as the chelation time was 30 min, chelation temperature was 25 °C, SPIP mass to VC mass was two to one and pH was 6.0. The obtained chelation rate was 82.56 ± 2.30 %. The change in the structures and functional features of SPIP before and after chelation were investigated. The FTIR and UV-Vis results indicated that the chelation of Fe2+ and SPIP depended mainly on the formation of amide bonds. The fluorescence, SEM and amino acid composition analysis results indicated that Fe2+ could induce and stabilize the surface conformation and change the amino acid distribution on the surfaces of SPIP. The chelation of SPIP and Fe2+ resulted in the enhancement of radical scavenging activities and ACE inhibitory activities. This work provided a new perspective for the further development of peptide-Fe chelates for iron supplement.

Enhancing Acid Resistance of Aspergillus niger Pectin Lyase through Surface Charge Design for Improved Application in Juice Clarification.

Pectin lyases (PNLs) can enhance juice clarity and flavor by degrading pectin in highly esterified fruits, but their inadequate acid resistance leads to rapid activity loss in juice. This study aimed to improve the acid resistance of Aspergillus niger PNL pelA through surface charge design. A modification platform was established by fusing pelA with a protein tag and expressing the fusion enzyme in Escherichia coli. Four single-point mutants were identified to increase the surface charge using computational tools. Moreover, the combined mutant M6 (S514D/S538E) exhibited 99.8% residual activity at pH 3.0. The M6 gene was then integrated into the A. niger genome using a multigene integration system to obtain the recombinant PNL AM6. Notably, AM6 improved the light transmittance of orange juice to 45.3%, which was 8.39 times higher than that of pelA. In conclusion, AM6 demonstrated the best-reported acid resistance, making it a promising candidate for industrial juice clarification.

A catalytic membrane approach as a way to obtain sweet and unsweet lactose-free milk.

The growing need in the current market for innovative solutions to obtain lactose-free (L-F) milk is caused by the annual increase in the prevalence of lactose intolerance inside as well as the newborn, children, and adults. Various configurations of enzymes can yield two distinct L-F products: sweet (ß-galactosidase) and unsweet (ß-galactosidase and glucose oxidase) L-F milk. In addition, the reduction of sweetness through glucose decomposition should be performed in a one-pot mode with catalase to eliminate product inhibition caused by H2O2. Both L-F products enjoy popularity among a rapidly expanding group of consumers. Although enzyme immobilization techniques are well known in industrial processes, new carriers and economic strategies are still being searched. Polymeric carriers, due to the variety of functional groups and non-toxicity, are attractive propositions for individual and co-immobilization of food enzymes. In the presented work, two strategies (with free and immobilized enzymes; ß-galactosidase NOLA, glucose oxidase from Aspergillus niger, and catalase from Serratia sp.) for obtaining sweet and unsweet L-F milk under low-temperature conditions were proposed. For free enzymes, achieving the critical assumption, lactose hydrolysis and glucose decomposition occurred after 1 and 4.3 h, respectively. The tested catalytic membranes were created on regenerated cellulose and polyamide. In both cases, the time required for lactose and glucose bioconversion was extended compared to free enzymes. However, these preparations could be reused for up to five (ß-galactosidase) and ten cycles (glucose oxidase with catalase).

Simple one-step treatment for saccharification of mango peels using an optimized enzyme cocktail of Aspergillus niger ATCC 9642.

Developing efficient microbiological methods to convert polysaccharide-rich materials into fermentable sugars, particularly monosaccharides, is vital for advancing the bioeconomy and producing renewable chemicals and energy sources. This study focused on optimizing the production conditions of an enzyme cocktail from Aspergillus niger ATCC 9642 using solid-state fermentation (SSF) and assessing its effectiveness in saccharifying mango peels through a simple, rapid, and efficient one-step process. A rotatable central composite design was employed to determine optimal conditions of moisture, time, and pH for enzyme production in SSF medium. The optimized enzyme cocktail exhibited cellulase activity (CMCase) at 6.28 U/g, filter paper activity (FPase) at 3.29 U/g, and pectinase activity at 117.02 U/g. These optimal activities were achieved with an SSF duration of 81 h, pH of 4.66, and a moisture content of 59%. The optimized enzyme cocktail effectively saccharified the mango peels without the need for chemical agents. The maximum saccharification yield reached approximately 81%, indicating efficient conversion of mango peels into sugars. The enzyme cocktail displayed consistent thermal stability within the tested temperature range of 30-60°C. Notably, the highest sugar release occurred within 36 h, with glucose, arabinose, galactose, and xylose being the primary monosaccharides released during saccharification. This study highlights the potential application of Aspergillus niger ATCC 9642 and SSF for enzymatic production, offering a simple and high-performance process for monosaccharide production. The optimized enzyme cocktail obtained through solid-state fermentation demonstrated efficient saccharification of mango peels, suggesting its suitability for industrial-scale applications.

Crystal structure and substrate recognition mechanism of the prolyl endoprotease PEP from Aspergillus niger.

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates.

Purification, Identification, and Characterization of a Glycoside Hydrolase Family 11-Xylanase with High Activity from Aspergillus niger VTCC 017.

Xylanases (EC 3.2.1.8) have been considered as a potential green solution for the sustainable development of a wide range of industries including pulp and paper, food and beverages, animal feed, pharmaceuticals, and biofuels because they are the key enzymes that degrade the xylosidic linkages of xylan, the major component of the second most abundant raw material worldwide. Therefore, there is a critical need for the industrialized xylanases which must have high specific activity, be tolerant to organic solvent or detergent and be active during a wide range of conditions, such as high temperature and pH. In this study, an extracellular xylanase was purified from the culture broth of Aspergillus niger VTCC 017 for primary structure determination and properties characterization. The successive steps of purification comprised centrifugation, Sephadex G-100 filtration, and DEAE-Sephadex chromatography. The purified xylanase (specific activity reached 6596.79 UI/mg protein) was a monomer with a molecular weight of 37 kDa estimating from SDS electrophoresis. The results of LC/MS suggested that the purified protein is indeed an endo-1,4-ß-D-xylanase. The purified xylanase showed the optimal temperature of 55 °C, and pH 6.5 with a stable xylanolytic activity within the temperature range of 45-50 °C, and within the pH range of 5.0-8.0. Most divalent metal cations including Zn2+, Fe2+, Mg2+, Cu2+, Mn2+ showed some inhibition of xylanase activity while the monovalent metal cations such as K+ and Ag+ exhibited slight stimulating effects on the enzyme activity. The introduction of 10-30% different organic solvents (n-butanol, acetone, isopropanol) and several detergents (Triton X-100, Tween 20, and SDS) slightly reduced the enzyme activity. Moreover, the purified xylanase seemed to be tolerant to methanol and ethanol and was even stimulated by Tween 80. Overall, with these distinctive properties, the putative xylanase could be a successful candidate for numerous industrial uses.

Biocatalytic one pot three component approach: Facile synthesis, characterization, molecular modelling and hypoglycemic studies of new thiazolidinedione festooned quinoline analogues catalyzed by alkaline protease from Aspergillus niger.

A novel ANAP (Aspergillus niger from alkaline protease) catalyzed one pot three component approach in the synthesis of new thiazolidinedione festooned quinoline analogues via Knoevenagel condensation and N-alkylation have been reported. The catalytic effect of enzyme was monitored and optimized by adjusting various parameters including catalyst concentration, choice of solvent and temperature. The isolated alkaline protease exhibits favorable features for the reaction response such as the shorter reaction time, simple work-up procedure, clean reaction profiles and excellent product yields through reusability of the catalyst upto five cycles. In silico molecular docking simulations were carried out to find out the effective binding affinity of the synthesized quinoline analogues 4(a-i) towards PPARγ protein (Id-2XKW). In vitro α-amylase and α-glucosidase assays were performed for hypoglycemic activity evaluation. In vivo hypoglycemic studies carried out on streptozotocin (SZT) induced diabetic male albino rats have shown that compounds 4e and 4f significantly reduced blood glucose levels with percentage reduction of 43.7 ± 0.91 and 45.6 ± 0.28 at a concentration of 50 mg/kg body wt. The results obtained from molecular docking simulations and in vitro enzyme assays are in consistent with in-vivo studies which clearly demonstrated that out of the synthesized quinoline analogues, compounds 4e and 4f possess promising hypoglycemic activity which was on par to that of standards pioglitazone and rosiglitazone respectively.

Revealing methyl-esterification patterns of pectins by enzymatic fingerprinting: Beyond the degree of blockiness.

Citrus pectins were studied by enzymatic fingerprinting using a simultaneous enzyme treatment with endo-polygalacturonase (endo-PG) from Kluyveromyces fragilis and pectin lyase (PL) from Aspergillus niger to reveal the methyl-ester distribution patterns over the pectin backbone. Using HILIC-MS combined with HPAEC enabled the separation and identification of the diagnostic oligomers released. Structural information on the pectins was provided by using novel descriptive parameters such as degree of blockiness of methyl-esterified oligomers by PG (DBPGme) and degree of blockiness of methyl-esterified oligomers by PL (DBPLme). This approach enabled us to clearly differentiate citrus pectins with various methyl-esterification patterns. The simultaneous use of PG and PL showed additional information, which is not revealed in digests using PG or PL alone. This approach can be valuable to differentiate pectins having the same DM and to get specific structural information on pectins and therefore to be able to better predict their physical and biochemical functionalities.

Machine learning prediction of novel pectinolytic enzymes in Aspergillus niger through integrating heterogeneous (post-) genomics data.

Pectinolytic enzymes are a variety of enzymes involved in breaking down pectin, a complex and abundant plant cell-wall polysaccharide. In nature, pectinolytic enzymes play an essential role in allowing bacteria and fungi to depolymerize and utilize pectin. In addition, pectinases have been widely applied in various industries, such as the food, wine, textile, paper and pulp industries. Due to their important biological function and increasing industrial potential, discovery of novel pectinolytic enzymes has received global interest. However, traditional enzyme characterization relies heavily on biochemical experiments, which are time consuming, laborious and expensive. To accelerate identification of novel pectinolytic enzymes, an automatic approach is needed. We developed a machine learning (ML) approach for predicting pectinases in the industrial workhorse fungus, Aspergillus niger. The prediction integrated a diverse range of features, including evolutionary profile, gene expression, transcriptional regulation and biochemical characteristics. Results on both the training and the independent testing dataset showed that our method achieved over 90 % accuracy, and recalled over 60 % of pectinolytic genes. Application of the ML model on the A. niger genome led to the identification of 83 pectinases, covering both previously described pectinases and novel pectinases that do not belong to any known pectinolytic enzyme family. Our study demonstrated the tremendous potential of ML in discovery of new industrial enzymes through integrating heterogeneous (post-) genomimcs data.

An efficient method based on an inhibitor-enzyme complex to screen an active compound against lipase from Toona sinensis.

As a popular vegetable, Toona sinensis has a wide range of bioactivities including lipase inhibitory activity. In the present study, an efficient and rapid method using a ligand-enzyme complex was established for screening of an active compound against lipase from Toona sinensis. The ethyl acetate extract of Toona sinensis showed good lipase inhibitory activity. After incubation with lipase, one of the compounds in the extract decreased significantly while comparing the HPLC chromatograms before and after incubation, which indicated that it may be the active compound bound to lipase. Then, the compound was isolated using a Sephadex LH-20 column and identified as 1,2,3,4,6-penta-O-galloyl-ß-D-glucose. The in vitro activity test showed that the compound had good inhibitory activity against lipase, and its IC50 value was 118.8 ± 1.53 µg mL-1. The kinetic experiments indicated that 1,2,3,4,6-penta-O-galloyl-ß-D-glucose inhibited lipase through mixed competitive and non-competitive inhibitions. Further docking results showed that the target compound could bind to the active site of lipase stably through seven hydrogen bonds, resulting in a docking energy of -8.31 kcal mol-1. The proposed method can not only screen the lipase inhibitors from Toona sinensis quickly and effectively, but also provide an effective way for the rapid screening of active substances in natural food and plants.

Production and immobilization of ß-glucanase from Aspergillus niger with its applications in bioethanol production and biocontrol of phytopathogenic fungi.

ß-Glucanase has received great attention in recent years regarding their potential biotechnological applications and antifungal activities. Herein, the specific objectives of the present study were to purify, characterize and immobilize ß-glucanase from Aspergillus niger using covalent binding and cross linking techniques. The evaluation of ß-glucanase in hydrolysis of different lignocellulosic wastes with subsequent bioethanol production and its capability in biocontrol of pathogenic fungi was investigated. Upon nutritional bioprocessing, ß-glucanase production from A. niger EG-RE (MW390925.1) preferred ammonium nitrate and CMC as the best nitrogen and carbon sources, respectively. The soluble enzyme was purified by (NH4)2SO4, DEAE-Cellulose and Sephadex G200 with 10.33-fold and specific activity of 379.1 U/mg protein. Tyrosyl, sulfhydryl, tryptophanyl and arginyl were essential residues for enzyme catalysis. The purified ß-glucanase was immobilized on carrageenan and chitosan with appreciable yield. However, the cross-linked enzyme exhibited superior activity along with remarkable improved thermostability and operational stability. Remarkably, the application of the above biocatalyst proved to be a promising candidate in liberating the associate lignocellulosic reducing sugars, which was utilized for ethanol production by Saccharomyces cerevisiae. The purified ß-glucanase revealed an inhibitory effect on the growth of two tested phytopathogens Fusarium oxysporum and Penicillium digitatum.

Biochemical characterization of xylanase GH11 isolated from Aspergillus niger BCC14405 (XylB) and its application in xylooligosaccharide production.

OBJECTIVE: To develop an endo-ß-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process. RESULTS: The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 U mg-1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel Ò PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45 °C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708 mg g-1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis. CONCLUSION: The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass.

Application and characterization of crude fungal lipases used to degrade fat and oil wastes.

Aspergillus niger MH078571.1 and A. niger MH079049.1 were identified previously as the two highest Aspergillus niger strains producing lipase. Biochemical characterizations of lipase activity and stability for these two strains were examined and revealed that the optimal temperature is 45 °C at pH 8for A. niger MH078571.1 and 55 °C for MH079049.1. The lipase production of both strains was studied on medium contains waste oil, as a cheap source to reduce the industrial cost, showed that the optimal incubation period for the enzyme production is 3 days. Moreover, an experiment on lipase activates in organic solvents demonstrated that 50% of acetone is the best solvent for the two strains. In the presence of surfactants, 0.1% of tween 80 surfactant showed the best lipase activities. Furthermore, Mg2+ and Zn2+ ions enhanced the lipase activity of A. niger MH078571.1, while Na2+ and Cu2+ enhanced the enzyme activity of A. niger MH079049.1. Lipase activity was also tested for industrial applications such as integrating it with different detergents. Maximum lipase activity was obtained with 1% of Omo as a powder detergent for both strains. In liquid detergent, 0.1% of Fairy showed maximum lipase activity in A. niger MH078571.1, while the lipase in A. niger MH079049.1 was more effective in 1% of Lux. Moreover, the degradation of natural animal fat with crude enzyme was tested using chicken and sheep fats. The results showed that more than 90% of fats degraded after 5 days of the incubation period.

A new carboxypeptidase from Aspergillus niger with good thermostability, pH stability and broad substrate specificity.

A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg-1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40-50 °C or at pH 4.0-8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1-10 mmol L-1 Mg2+ enhanced the activity of CapA, whereas 1-10 mmol L-1 Cu2+, Fe2+, or Co2+, 10 mmol L-1 Mn2+, or 1-10 mmol L-1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-L-phenylalanyl-L-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited Km 0.063 mmol L-1 and kcat/Km 186.35 mmol L-1 s-1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

In silico and in vitro analysis of an Aspergillus niger chitin deacetylase to decipher its subsite sugar preferences.

Chitin deacetylases (CDAs) are found in many different organisms ranging from marine bacteria to fungi and insects. These enzymes catalyze the removal of acetyl groups from chitinous substrates generating various chitosans, linear copolymers consisting of N-acetylglucosamine (GlcNAc) and glucosamine. CDAs influence the degree of acetylation of chitosans as well as their pattern of acetylation, a parameter that was recently shown to influence the physicochemical properties and biological activities of chitosans. The binding site of CDAs typically consists of around four subsites, each accommodating a single sugar unit of the substrate. It has been hypothesized that the subsite preferences for GlcNAc or glucosamine units play a crucial role in the acetylation pattern they generate, but so far, this characteristic was largely ignored and still lacks structural data on the involved residues. Here, we determined the crystal structure of an Aspergillus niger CDA. Then, we used molecular dynamics simulations, backed up with a variety of in vitro activity assays using different well-defined polymeric and oligomeric substrates, to study this CDA in detail. We found that Aspergillus niger CDA strongly prefers a GlcNAc sugar unit at its -1 subsite and shows a weak GlcNAc preference at the other noncatalytic subsites, which was apparent both when deacetylating and N-acetylating oligomeric substrates. Overall, our results show that the combination of in vitro and in silico methods used here enables the detailed analysis of CDAs, including their subsite preferences, which could influence their substrate targets and the characteristics of chitosans produced by these species.

Vanillic acid and methoxyhydroquinone production from guaiacyl units and related aromatic compounds using Aspergillus niger cell factories.

BACKGROUND: The aromatic compounds vanillin and vanillic acid are important fragrances used in the food, beverage, cosmetic and pharmaceutical industries. Currently, most aromatic compounds used in products are chemically synthesized, while only a small percentage is extracted from natural sources. The metabolism of vanillin and vanillic acid has been studied for decades in microorganisms and many studies have been conducted that showed that both can be produced from ferulic acid using bacteria. In contrast, the degradation of vanillin and vanillic acid by fungi is poorly studied and no genes involved in this metabolic pathway have been identified. In this study, we aimed to clarify this metabolic pathway in Aspergillus niger and identify the genes involved. RESULTS: Using whole-genome transcriptome data, four genes involved in vanillin and vanillic acid metabolism were identified. These include vanillin dehydrogenase (vdhA), vanillic acid hydroxylase (vhyA), and two genes encoding novel enzymes, which function as methoxyhydroquinone 1,2-dioxygenase (mhdA) and 4-oxo-monomethyl adipate esterase (omeA). Deletion of these genes in A. niger confirmed their role in aromatic metabolism and the enzymatic activities of these enzymes were verified. In addition, we demonstrated that mhdA and vhyA deletion mutants can be used as fungal cell factories for the accumulation of vanillic acid and methoxyhydroquinone from guaiacyl lignin units and related aromatic compounds. CONCLUSIONS: This study provides new insights into the fungal aromatic metabolic pathways involved in the degradation of guaiacyl units and related aromatic compounds. The identification of the involved genes unlocks new potential for engineering aromatic compound-producing fungal cell factories.