scholarly journals Regioselective chemo-enzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

2020 ◽  
Author(s):  
Olga Gherbovet ◽  
Fernando Ferreira ◽  
Apolline Clément ◽  
Mélanie Ragon ◽  
Julien Durand ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Feruloyl Esterase ◽  
Thermomyces Lanuginosus ◽  
Hydroxyl Group ◽  
Chromogenic Substrates ◽  
Carbohydrate Active Enzymes ◽  
Experimental Application ◽  
Synthetic Routes ◽  
Feruloyl Esterases ◽  
Regioselective Functionalization

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. In the case of feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl O-5-feruloyl-α-l-arabinofuranosides 1a and 1b were significantly shortened (7-8 steps reduced to 4-6) and transesterification yields enhanced (from 46 to 73% for 1a and 47 to 86 % for 1b). This was achieved using enzymatic (immobilized Lipolase 100T from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxyl group of α‐l‐arabinofuranosides. Moreover, a novel feruloylated-butanetriol 4-nitrocatechol-1-yl analog 12, containing a cleavable hydroxylated linker was also synthesized in 29% overall yield in 3 steps (convergent synthesis). The latter route combined regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of 12 as a substrate to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.

scholarly journals Regioselective chemoenzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases

2021 ◽  
Vol 17 ◽  
pp. 325-333
Author(s):  
Olga Gherbovet ◽  
Fernando Ferreira ◽  
Apolline Clément ◽  
Mélanie Ragon ◽  
Julien Durand ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Feruloyl Esterase ◽  
Thermomyces Lanuginosus ◽  
Chromogenic Substrates ◽  
Carbohydrate Active Enzymes ◽  
Experimental Application ◽  
Primary Hydroxy Group ◽  
Synthetic Routes ◽  
Feruloyl Esterases ◽  
Regioselective Functionalization

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. For feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl 5-O-feruloyl-α-ʟ-arabinofuranosides were significantly shortened (from 7 or 8 to 4–6 steps), and the transesterification yields were enhanced (from 46 to 73% and from 47 to 86%, respectively). This was achieved using enzymatic (immobilized Lipozyme® TL IM from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxy group of α‐ʟ‐arabinofuranosides. Moreover, a novel feruloylated 4-nitrocatechol-1-yl-substituted butanetriol analog, containing a cleavable hydroxylated linker, was also synthesized in 32% overall yield in 3 steps (convergent synthesis). The latter route combined the regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of this strategy to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.

Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger

FEBS Journal ◽  
2005 ◽  
Vol 272 (17) ◽  
pp. 4362-4371 ◽  
Author(s):  
Craig B. Faulds ◽  
Rafael Molina ◽  
Ramon Gonzalez ◽  
Fiona Husband ◽  
Nathalie Juge ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Substrate Specificity ◽  
Feruloyl Esterase

scholarly journals Regulation of the Feruloyl Esterase (faeA) Gene from Aspergillus niger

1999 ◽  
Vol 65 (12) ◽  
pp. 5500-5503 ◽  
Author(s):  
Ronald P. de Vries ◽  
Jaap Visser
Keyword(s):  
Cell Wall ◽  
Aspergillus Niger ◽  
Ferulic Acid ◽  
Methoxy Group ◽  
Plant Cell Wall ◽  
Ring Structure ◽  
Feruloyl Esterase ◽  
Cell Wall Polysaccharides ◽  
Aromatic Residues ◽  
Encoding Gene

ABSTRACT Feruloyl esterases can remove aromatic residues (e.g., ferulic acid) from plant cell wall polysaccharides (xylan, pectin) and are essential for complete degradation of these polysaccharides. Expression of the feruloyl esterase-encoding gene (faeA) fromAspergillus niger depends on d-xylose (expression is mediated by XlnR, the xylanolytic transcriptional activator) and on a second system that responds to aromatic compounds with a defined ring structure, such as ferulic acid and vanillic acid. Several compounds were tested, and all of the inducing compounds contained a benzene ring which had a methoxy group at C-3 and a hydroxy group at C-4 but was not substituted at C-5. Various aliphatic groups occurred at C-1. faeA expression in the presence of xylose or ferulic acid was repressed by glucose. faeA expression in the presence of ferulic acid and xylose was greater thanfaeA expression in the presence of either compound alone. The various inducing systems allow A. niger to produce feruloyl esterase not only during growth on xylan but also during growth on other ferulic acid-containing cell wall polysaccharides, such as pectin.

Exploration of members ofAspergillussectionsNigri,Flavi, andTerreifor feruloyl esterase production

2006 ◽  
Vol 52 (9) ◽  
pp. 886-892 ◽  
Author(s):  
Ourdia Bouzid ◽  
Eric Record ◽  
Michèle Asther ◽  
Mireille Haon ◽  
David Navarro ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Methyl Esters ◽  
Type A ◽  
Sugar Beet Pulp ◽  
Feruloyl Esterase ◽  
Type B ◽  
Original Activity ◽  
Beet Pulp ◽  
Feruloyl Esterases ◽  
Oat Spelt

The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat–spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat–spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat–spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.Key words: Aspergillus, Nigri, Flavi, Terrei, feruloyl esterase.

Production of a chimeric enzyme tool associating the Trichoderma reesei swollenin with the Aspergillus niger feruloyl esterase A for release of ferulic acid

2006 ◽  
Vol 73 (4) ◽  
pp. 872-880 ◽  
Author(s):  
Anthony Levasseur ◽  
Markku Saloheimo ◽  
David Navarro ◽  
Martina Andberg ◽  
Frédéric Monot ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Ferulic Acid ◽  
Trichoderma Reesei ◽  
Feruloyl Esterase ◽  
Chimeric Enzyme

Synthesis of des-A-B-secocholestanes

1984 ◽  
Vol 62 (6) ◽  
pp. 1081-1084 ◽  
Author(s):  
Wojciech J. Szczepek ◽  
Jacek W. Morzycki ◽  
Zbigniew Bończa-Tomaszewski ◽  
Michał Chodyński ◽  
Władysław J. Rodewald
Keyword(s):  
Hydroxyl Group ◽  
Dioic Acid ◽  
Ruthenium Tetroxide ◽  
Enol Acetate ◽  
Synthetic Routes ◽  
Hydrolysis Of

Two independent synthetic routes to 10-oxo-5, 10-seco-des-A-cholestan-5-oic acid (5) are described. The route including the Baeyer–Villiger rearrangement of (10R)-des-A-cholestan-5-one (2a), followed by hydrolysis of the lactone 3a obtained and oxidation of the C-10 hydroxyl group, seems to be superior to ozonolysis of the 5(10)-enol acetate 6 of des-A-ketone which resulted in the formation of 5,10-seco-des-A-19-norcholestan-5,10-dioic acid (7), in addition to the compound 5. Product 7 and its 6-nor analog 12 have also been obtained by ruthenium tetroxide oxidation of the respective A-ring phenols 8, 9, and 11.

scholarly journals Cloning, characterization and directed evolution of a xylosidase from Aspergillus niger

2016 ◽  
Author(s):  
◽  
Bibi Khadija Khan
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Random Mutagenesis ◽  
Thermomyces Lanuginosus ◽  
Major Protein ◽  
Bioethanol Production ◽  
Xylanase Production ◽  
Fluorescent Band ◽  
Gap Promoter ◽  
Major Protein Band

β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant.

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