The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical ...industries. This challenge is highlighted by the estimate that 40–45% of drug candidates contain a chiral amine, fueling a demand for broadly applicable synthetic methods that deliver target structures in high yield and enantiomeric excess. Herein we describe the development and application of a “toolbox” of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant, which exhibits high activity and enantioselectivity toward substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemization reactions for the efficient asymmetric synthesis of the generic active pharmaceutical ingredients Solifenacin and Levocetirizine as well as the natural products (R)-coniine, (R)-eleagnine, and (R)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet–Spengler approach to the synthesis of (R)-harmicine.
Aspergillus niger YAT strain was obtained from Chinese brick tea (Collection number: CGMCC 10,568) and identified on the basis of morphological characteristics and internal transcribed spacer (ITS) ...sequence. The strain could degrade 54.83 % of β-cypermethrin (β-CY; 50 mg L⁻¹) in 7 days and 100 % of 3-phenoxybenzoic acid (3-PBA; 100 mg L⁻¹) in 22 h. The half-lives of β-CY and 3-PBA range from 3.573 to 11.748 days and from 5.635 to 12.160 h, respectively. The degradation of β-CY and 3-PBA was further described using first-order kinetic models. The pathway and mechanism of β-CY degraded by YAT were investigated by analyzing the degraded metabolites through high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Relevant enzymatic activities and substrate utilization were also investigated. β-CY degradation products were analyzed. Results indicated that YAT strain transformed β-CY into 3-PBA. 3-PBA was then gradually transformed into permethric acid, protocatechuic acid, 3-hydroxy-5-phenoxy benzoic acid, gallic acid, and phenol gradually. The YAT strain can also effectively degrade these metabolites. The results indicated that YAT strain has potential applications in bioremediation of pyrethroid insecticide (PI)-contaminated environments and fermented food.
Aspergillus section
Nigri is an important group of species for food and medical mycology, and biotechnology. The
Aspergillus niger ‘aggregate’ represents its most complicated taxonomic subgroup ...containing eight morphologically indistinguishable taxa:
A. niger,
Aspergillus tubingensis,
Aspergillus acidus,
Aspergillus brasiliensis,
Aspergillus costaricaensis,
Aspergillus lacticoffeatus,
Aspergillus piperis, and
Aspergillus vadensis.
Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of
A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins β-tubulin (
benA), calmodulin (
CaM), and the translation elongation factor-1 alpha (
TEF-1α) of a population of
A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population,
A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between
A. niger and the cryptic
A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B
2, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-γ-pyrones. In addition, sequence analysis of four putative
A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the
A. awamori phylospecies.
► We examined
A. awamori by polyphasic approach within
A. niger “aggregate” group. ► Multilocus phylogenetic analyses evidenced
A. awamori as a cryptic species. ► Morphological and extrolites data overlap between
A. niger and the cryptic
A. awamori. ► AFLP analysis of the full genome support
A. awamori as species. ► Both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B
2.
and its related species, known as
section
, are ubiquitously distributed across the globe and are often isolated from clinical specimens. In Japan,
section
is second most often isolated from clinical ...specimens following
We determined the species of
section
isolated in Japan by DNA sequencing of partial β-tubulin genes and investigated drug susceptibility by the CLSI M38-A2 method. The collection contained 20
, 59
, and 39
strains. Drug susceptibility testing revealed 30 to 55% of
, 6.8 to 18.6% of
, and 79.5 to 89.7% of
isolates to be less susceptible (so-called resistant) to itraconazole (ITC) and/or voriconazole (VRC) according to the epidemiologic cutoff values (ECVs) proposed for
previously. MIC distributions of ITC or VRC showed no remarkable differences between clinical and environmental isolates. When the
sequences were compared between susceptible and resistant strains, 18 amino acid mutations were specific for resistant isolates of
and
; however, none of them were confirmed to be associated with azole resistance. Three nonrelated
isolates possessed a partial deletion in
, likely attributable to being more susceptible to azoles than other isolates. One of five ITC-resistant
isolates showed higher expression of
than did susceptible strains. Our results show that
point mutations may have no association with azole resistance but that in some cases the overexpression of
may lead to the azole resistance in these species.
Phosphate solubilizing fungi (PSF) have huge potentials in enhancing release of phosphorus from fertilizer. Two PSF (NJDL-03 and NJDL-12) were isolated and identified as Penicillium oxalicum and ...Aspergillus niger respectively in this study. The quantification and identification of organic acids were performed by HPLC. Total concentrations of organic acids secreted by NJDL-03 and NJDL-12 are ~4000 and ~10,000 mg/L with pH values of 3.6 and 2.4 respectively after five-days culture. Oxalic acid dominates acidity in the medium due to its high concentration and high acidity constant. The two fungi were also cultured for five days with the initial pH values of the medium varied from 6.5 to 1.5. The biomass reached the maximum when the initial pH values are 4.5 for NJDL-03 and 2.5 for NJDL-12. The organic acids for NJDL-12 reach the maximum at the initial pH = 5.5. However, the acids by NJDL-03 continue to decrease and proliferation of the fungus terminates at pH = 2.5. The citric acid production increases significantly for NJDL-12 at acidic environment, whereas formic and oxalic acids decrease sharply for both two fungi. This study shows that NJDL-12 has higher ability in acid production and has stronger adaptability to acidic environment than NJDL-03.
Eukaryotic 1,4-β-endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applications because they effectively catalyze hydrolysis of cellulose, the main component of the plant ...cell wall. Here we expressed a glycoside hydrolase family (GH) 5 1,4-β-endoglucanase from Aspergillus niger (AnCel5A) in Pichia pastoris, which exhibits outstanding pH and heat stability. In order to further investigate the molecular mechanism of AnCel5A, apo-form and cellotetraose (CTT) complex enzyme crystal structures were solved to high resolution. AnCel5A folds into a typical (β/α)8-TIM barrel architecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to −4 – −1 subsites, and several polyethylene glycol molecules are found in positive subsites. In addition, several unique N-glycosylation motifs that may contribute to protein higher stability were observed from crystal structures. These results are of great importance for understanding the molecular mechanism of AnCel5A, and also provide guidance for further applications of the enzyme.
•An endoglucanase from Aspergillus niger, AnCel5A, was expressed in Pichia pastoris.•The recombinant AnCel5A shows outstanding pH and heat stability.•Crystal structures of AnCel5A in apo- and substrate-bound form were solved.
A variety of plant products have been recognized for their antifungal activity and recently have attracted food industry attention for their efficacy in controlling postharvest fungal decay of ...fruits. The antifungal activity of perillaldehyde (PAE) was evaluated against Aspergillus niger, a known cause of grape spoilage, and possible mechanisms were explored. PAE showed notable antifungal activity against A. niger, with a minimum inhibitory concentration (MIC) and a minimum fungicidal concentration (MFC) of 0.25 and 1μl/ml, respectively. The accumulation of mycelial biomass was also inhibited by PAE in a dose-dependent manner, completely inhibiting mycelial growth at 1μl/ml. In vivo data confirmed that the vapour treatment of grapes with various concentrations of PAE markedly improved control of A. niger and suppressed natural decay. Concentrations of PAE of 0.075μl/ml air showed the greatest inhibition of fungal growth compared to the controls. Further experiments indicated that PAE activated a membrane-active mechanism that inhibits ergosterol synthesis, increases membrane permeability (as evidenced by extracellular pH and conductivity measurements), and disrupts membrane integrity, leading to cell death. Our findings suggest that this membrane-active mechanism makes PAE a promising potential antifungal agent for postharvest control of grape spoilage.
•Perillaldehyde displayed notable in vitro antifungal activity against A. niger.•Practical efficacy of perillaldehyde was established as fumigant in grapes.•The membrane permeability of A. niger are changed by perillaldehyde.•The ergosterol synthesis and membrane integrity were affected by perillaldehyde.•Perillaldehyde could be used as a potential source of eco-friendly preservative.
The high cost of cellulases remains the most significant barrier to the economical production of bio‐ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and ...xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond OPF) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 106 spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.
The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a ...five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10–32 fold in the optimized medium condition (pH 6.5, temperature 30°C, 6% (v/v), inoculum size and 150rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.
Summary
Environmental microorganisms have been widely applied in heavy metal remediation. This study explored the mechanisms of lead tolerance of two typical filamentous fungi, Aspergillus niger and ...Penicillium oxalicum. It is shown that the mechanisms of reducing Pb toxicity by these two fungi have three major pathways. The secreted oxalic acid can react with Pb (II) to form insoluble Pb minerals, primarily lead oxalate. Then, the enhanced biosorption via forming new border of cell wall prevents the transportation of Pb (II) into hypha. In addition, the fungal activity could be maintained even at high Pb concentration due to the intracellular accumulation. It was confirmed that A. niger has the higher Pb tolerance (up to 1500 mg l−1 Pb level) compared with P. oxalicum (up to 1000 mg l−1). Meanwhile, Pb levels below 1000 mg l−1 partially stimulate the bioactivity of A. niger, which was confirmed by its elevated respiration (from 53 to 63 mg C l−1 medium h−1). This subsequently enhanced microbial functions of A. niger to resist Pb toxicity. A better understanding of Pb tolerance of these two fungi sheds a bright future of applying them to remediate lead‐contaminated environments.
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