Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- biokatalýza * MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- Pichia genetika metabolismus MeSH
- průmyslová mikrobiologie metody MeSH
- quercetin chemie metabolismus MeSH
- rutin chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.
- MeSH
- alfa-N-acetylgalaktosaminidasa biosyntéza chemie genetika MeSH
- Aspergillus niger enzymologie genetika MeSH
- buněčné kultury MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- elektronová mikroskopie MeSH
- gelová chromatografie MeSH
- glykosylace MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- molekulární sekvence - údaje MeSH
- molekulová hmotnost MeSH
- rekombinantní proteiny biosyntéza chemie genetika MeSH
- Saccharomyces cerevisiae enzymologie genetika MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
β-Mannosidase (EC 3.2.1.25) is an exoglycosidase specific for the hydrolysis of terminal β-linked mannoside in various sugar chains. cDNA corresponding to the β-mannosidase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Pichia pastoris. The β-mannosidase gene contains an open reading frame which encodes the protein with 933 amino acid residues. The wild type and recombinant proteins were purified to apparent homogeneity and biochemically characterized (K(M) 0.28 and 0.44 mmol/l for p-nitrophenyl β-d-mannopyranoside, pI 4.2 and 4.0, and their pH optima were at pH 4.5 and 5.5 and 65°C, respectively).
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- beta-mannosidasa biosyntéza chemie genetika izolace a purifikace MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fungální proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- klonování DNA MeSH
- koncentrace vodíkových iontů MeSH
- kultivační média MeSH
- Pichia enzymologie genetika MeSH
- rekombinantní proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- stabilita enzymů MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE OF WORK: our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles. The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l(-1) culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l(-1) culture, respectively, with phenylacetonitrile as substrate.
- MeSH
- aminohydrolasy genetika metabolismus MeSH
- Aspergillus niger enzymologie genetika MeSH
- Escherichia coli genetika MeSH
- exprese genu MeSH
- fungální proteiny genetika metabolismus MeSH
- genom fungální MeSH
- Gibberella enzymologie genetika MeSH
- klonování DNA MeSH
- Neurospora crassa enzymologie genetika MeSH
- nitrily metabolismus MeSH
- organické látky metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We performed a laboratory evolution study with the epoxide hydrolase from Aspergillus niger M200. This enzyme exhibits no enantioconvergence with the substrates styrene oxide or para-chlorostyrene oxide, i.e. racemic vicinal diols are produced from the racemic substrates. After saturation mutagenesis, screening by chiral gas chromatography revealed enzyme variants with improved enantioconvergence as manifested by an increased enantiomeric excess of the diol product. Nine amino acid exchanges accumulated in the active site and the substrate access tunnel over the course of 5 productive rounds of iterative saturation mutagenesis, resulting in an enantioconvergent epoxide hydrolase variant. The final mutant enzyme transformed racemic styrene oxide and para-chlorostyrene oxide to (R)-diol enantiomers with enantiomeric excesses of 70%. Sequential bi-enzymatic reactions using the wild-type EH and/or its evolved variants enabled preparation of the chiral building blocks (R)-phenyl-1,2-ethanediol and (R)-para-chlorophenyl-1,2-ethanediol from inexpensive racemic epoxides with enantiomeric excesses of 91% and 88%, respectively.
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- epoxid hydrolasy chemie genetika metabolismus MeSH
- epoxidové sloučeniny chemie metabolismus MeSH
- katalytická doména MeSH
- katalýza MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- stereoizomerie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.
- MeSH
- aminohydrolasy biosyntéza genetika izolace a purifikace metabolismus MeSH
- Aspergillus niger enzymologie genetika MeSH
- bakteriální proteiny genetika izolace a purifikace metabolismus MeSH
- klonování DNA metody MeSH
- komplementární DNA MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce MeSH
- radiační rozptyl MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza DNA MeSH
- sekvenční seřazení MeSH
- simulace molekulární dynamiky MeSH
- stabilita enzymů MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- publikace stažené z tisku MeSH
Two genes in the genome of Aspergillus niger, aglA and aglB, have been assigned to encode for α-d-galactosidases variant A and B. However, analyses of primary and 3D structures based on structural models of these two enzymes revealed significant differences in their active centers suggesting important differences in their specificity for the hydrolyzed carbohydrates. To test this unexpected finding, a large screening of libraries from 42 strains of filamentous fungi succeeded in identifying an enzyme from A. niger CCIM K2 that exhibited both α-galactosidase and α-N-acetylgalactosaminidase activities, with the latter activity predominating. The enzyme protein was sequenced, and its amino acid sequence could be unequivocally assigned to the enzyme encoded the aglA gene. Enzyme activity measurements and substrate docking clearly demonstrated the preference of the identified enzyme for α-N-acetyl-d-galactosaminide over α-d-galactoside. Thus, we provide evidence that the α-galactosidase type A gene aglA from A. niger in fact encodes a fully functional α-N-acetylgalactosaminidase using a retaining mechanism.
- MeSH
- alfa-galaktosidasa genetika MeSH
- alfa-N-acetylgalaktosaminidasa chemie genetika MeSH
- Aspergillus niger enzymologie genetika MeSH
- geny hub MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The gene encoding an epoxide hydrolase from Aspergillus niger M200 has been cloned and its sequence determined. The gene is interrupted by seven introns, one exon being only nine nucleotides long. The non-coding 5'- and 3'-regions of the mRNA are composed of 47 and 76 nucleotides, respectively. Overexpression of the fungal epoxide hydrolase in E. coli TOP10 has led to a 15-fold increase in specific activity (compared to the wild-type strain). Saturation mutagenesis at codon 217 resulted in the discovery of nine enzyme variants showing in several cases profound differences in activity and enantioselectivity towards various epoxides when compared to the data of the wild-type enzyme. The site 217 is located at the entrance of the tunnel that provides the substrate with access to the active site. The exchange of Ala at this position for Cys has led to a doubled enantioselectivity (E-value of 5.0) towards benzyl glycidyl ether. The same substitution resulted in a threefold-enhanced activity of the enzyme towards allyl glycidyl ether and styrene oxide without affecting enantioselectivity. The variant A217L showed an enhanced enantioselectivity towards tert-butyl glycidyl ether reaching an E-value of 100 (from 60 for the wild-type enzyme). Replacement of A217 by Val has led to higher activity towards allyl glycidyl ether by a factor of six. The substitutions Ala-->Glu and Ala-->Gln increased the enantioselectivity towards allyl glycidyl ether and styrene oxide by over 50% to E-values of 10 and 16, respectively. The study underlines that single amino acid exchanges in the substrate tunnel region can lead to significant improvements in enantioselectivity and activity of the epoxide hydrolase from A. niger M200.
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- biotechnologie MeSH
- DNA fungální genetika MeSH
- epoxid hydrolasy genetika chemie metabolismus MeSH
- Escherichia coli genetika MeSH
- financování organizované MeSH
- fylogeneze MeSH
- geny hub MeSH
- katalytická doména MeSH
- kinetika MeSH
- klonování DNA MeSH
- komplementární DNA genetika MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- proteinové inženýrství MeSH
- rekombinantní proteiny genetika chemie metabolismus MeSH
- sekvence nukleotidů MeSH
- stereoizomerie MeSH
- substituce aminokyselin MeSH
- substrátová specifita MeSH
