| 1. |
- Bonzom, Cyrielle, 1987, et al.
(author)
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Feruloyl esterases, effects of glycosylation on activity, stability and immobilization
- 2017
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Conference paper (other academic/artistic)abstract
- Feruloyl esterases (FAE; EC 3.1.1.73) are a subclass of carboxylic ester hydrolases. They catalyze the hydrolysis of ester linkages in plant cell walls materials releasing ferulic acid and other hydroxycinnamic acids and therefore have been classified in the CAZy database [1] in the carbohydrate esterase (CE) family. Currently, all FAEs in the CAZY database belong to the CE1 family. Immobilization is a powerful tool to allow enzyme reuse and therefore decrease their cost in the overall process. Immobilization on solid carriers can be achieved by different approaches, one of them is by physical adsorption. This technique relies mainly on the surface charges of the support and enzyme. Mesoporous silica particles (MPS) are porous supports made of silica which pore size can be tuned from 2 to 50 nm depending on the synthesis conditions [2]. Because they are negatively charged, silica particles allow for rapid enzyme immobilization by physical adsorption. Furthermore, the diameter of their pore size can be tuned to match the one of the enzyme used and greatly reduces leaching after immobilization.Depending on the production host of the enzyme, the glycosylation extent and/or pattern can vary from none in Escherichia coli to hyper-glycosylation in Pichia pastoris. Glycosylation has been shown to alter activity and stability of enzymes [3]. Our hypothesis was that glycosylation would influence the immobilization behavior of the studied FAE in MPS as well as its activity.In this study, the same enzyme MtFae1a from the fungus Myceliophtora thermophila has been expressed in three different host organisms: E. coli, M. thermophila and P. pastoris to achieve different degrees of glycosylation. The proteins were then purified and biochemically characterized. Different behaviors were observed depending on the production host of the enzymes, for instance in terms of specific activity and temperature optima. Furthermore, glycosylation affected the immobilization capacity of the enzymes on MPS, as well as the activity of immobilized FAEs.[1]B.L. Cantarel, P.M. Coutinho, C. Rancurel, T. Bernard, V. Lombard, B. Henrissat, The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics, Nucleic Acids Res. 37 (2009) D233–238. doi:10.1093/nar/gkn663.[2]D. Zhao, J. Feng, Q. Huo, N. Melosh, G.H. Fredrickson, B.F. Chmelka, G.D. Stucky, Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores, Science. 279 (1998) 548–552. doi:10.1126/science.279.5350.548.[3]A. Basso, P. Braiuca, S. Cantone, C. Ebert, P. Linda, P. Spizzo, P. Caimi, U. Hanefeld, G. Degrassi, L. Gardossi, In Silico Analysis of Enzyme Surface and Glycosylation Effect as a Tool for Efficient Covalent Immobilisation of CalB and PGA on Sepabeads®, Adv. Synth. Catal. 349 (2007) 877–886. doi:10.1002/adsc.200600337.
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| 2. |
- Bonzom, Cyrielle, 1987, et al.
(author)
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Feruloyl esterases immobilization in mesoporous silica particles
- 2016
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Conference paper (other academic/artistic)abstract
- Mesoporous silica materials (MPS) are an interesting choice as support to immobilize enzymes because MPS offer unique properties such as high enzyme loading and tunable pore size. They also provide the enzyme with a sheltered environment therefore reducing loss of function in industrial applications.Feruloyl esterases (FAEs) are naturally hydrolytic enzymes which are known for their action on lignocellulosic material and release ferulic acid (FA) which is bound to plant cell wall materials. Under specific conditions they are also able to perform synthetic reactions. In our work we focused on performing transesterification reactions with FAEs. The synthesis of butyl ferulate (BFA) from methyl ferulate (MFA) was chosen as a model reaction.Reduced water content of the reaction system, needed for the transesterification reaction to happen, can be achieved by replacing buffer with solvents. However, solvents can have a deleterious effect on the biocatalyst. Therefore, the use of ionic liquids instead of solvents was investigated. In addition, the enzymes were immobilized on MPS. In order to achieve a good immobilization yield and a good immobilized activity of the FAEs, several parameters were varied, and enzyme activity and selectivity were assessed. Since the reaction of interest was transesterification, the selectivity of the enzyme was quantified by determining the molar ratio between the product of transesterification reaction and the product of hydrolysis reaction: BFA/FA. Kinetic parameters, stability and reusability of the immobilized biocatalyst were also investigated. We found that the properties of enzyme themselves influence the immobilization process as well as the enzyme performance. Enzymes having a different isoelectric point or bearing different surface modifications such as glycosylations have different behaviors both in terms of enzyme activity and of immobilization performance.
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| 3. |
- Bonzom, Cyrielle, 1987, et al.
(author)
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Glycosylation influences activity, stability and immobilization of the feruloyl esterase 1a from Myceliophthora thermophila
- 2019
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In: AMB Express. - : Springer Science and Business Media LLC. - 2191-0855. ; 9:1
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Journal article (peer-reviewed)abstract
- Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host.
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| 4. |
- Cerullo, Gabriella, et al.
(author)
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The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties
- 2018
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In: Catalysts. - : MDPI AG. - 2073-4344. ; 8:242
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Journal article (peer-reviewed)abstract
- Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.
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| 5. |
- Granchi, Zoraide, et al.
(author)
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Genome Sequence of the Thermophilic Biomass-Degrading Fungus Malbranchea cinnamomea FCH 10.5
- 2017
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In: Genome Announcements. - 2169-8287. ; 5:33
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Journal article (peer-reviewed)abstract
- We report here the annotated draft genome sequence of the thermophilic biomass-degrading fungus Malbranchea cinnamomea strain FCH 10.5, isolated from compost at a waste treatment plant in Vietnam. The genome sequence contains 24.96 Mb with an overall GC content of 49.79% and comprises 9,437 protein-coding genes.
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| 6. |
- Hüttner, Silvia, 1984, et al.
(author)
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Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds
- 2017
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 101:13, s. 5301-5311
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Journal article (peer-reviewed)abstract
- The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)d-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 degrees C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.
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| 7. |
- Hüttner, Silvia, 1984, et al.
(author)
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Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea
- 2017
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834 .- 1754-6834. ; 10:1
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Journal article (peer-reviewed)abstract
- Background: Genome and transcriptome sequencing has greatly facilitated the understanding of biomass-degrading mechanisms in a number of fungal species. The information obtained enables the investigation and discovery of genes encoding proteins involved in plant cell wall degradation, which are crucial for saccharification of lignocellulosic biomass in second-generation biorefinery applications. The thermophilic fungus Malbranchea cinnamomea is an efficient producer of many industrially relevant enzymes and a detailed analysis of its genomic content will considerably enhance our understanding of its lignocellulolytic system and promote the discovery of novel proteins. Results: The 25-million-base-pair genome of M. cinnamomea FCH 10.5 was sequenced with 225x coverage. A total of 9437 protein-coding genes were predicted and annotated, among which 301 carbohydrate-active enzyme (CAZyme) domains were found. The putative CAZymes of M. cinnamomea cover cellulases, hemicellulases, chitinases and pectinases, equipping the fungus with the ability to grow on a wide variety of biomass types. Upregulation of 438 and 150 genes during growth on wheat bran and xylan, respectively, in comparison to growth on glucose was revealed. Among the most highly upregulated CAZymes on xylan were glycoside hydrolase family GH10 and GH11 xylanases, as well as a putative glucuronoyl esterase and a putative lytic polysaccharide monooxygenase (LPMO). AA9-domain-containing proteins were also found to be upregulated on wheat bran, as well as a putative cutinase and a protein harbouring a CBM9 domain. Several genes encoding secreted proteins of unknown function were also more abundant on wheat bran and xylan than on glucose. Conclusions: The comprehensive combined genome and transcriptome analysis of M. cinnamomea provides a detailed insight into its response to growth on different types of biomass. In addition, the study facilitates the further exploration and exploitation of the repertoire of industrially relevant lignocellulolytic enzymes of this fungus.
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| 8. |
- Hüttner, Silvia, 1984, et al.
(author)
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Fungal Glucuronoyl and Feruloyl Esterases for Wood Processing and Phenolic Acid Ester/Sugar Ester Synthesis
- 2015
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In: Biotrans 2015, Vienna, Austria, 26-30 July 2015.
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Conference paper (other academic/artistic)abstract
- Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) and glucuronoyl esterases(GEs, E.C. 3.1.1.-, CAZy family CE15) are involved in the degradation of plantbiomass by hydrolysing ester linkages in plant cell walls, and thus have potential use inbiofuel production from lignocellulosic materials and in biorefinery applications withthe aim of developing new wood-based compounds [1, 2]. GEs and FAEs are present inthe genomes of a wide range of fungi and bacteria.Under conditions of low water content, these enzymes can also carry out(trans)esterification reactions, making them promising biocatalysts for the modificationof compounds with applications in the food, cosmetic and pharmaceutical industry.Compared to the chemical process, enzymatic synthesis can be carried out under lowerprocess temperatures (50-60°C) and results in fewer side products, thus reducing theenvironmental impact.We characterised new FAE and GE enzymes from mesophilic, thermophilic and coldtolerantfilamentous fungi produced in Pichia pastoris. The enzymes were characterisedfor both their hydrolytic abilities on various model substrates (methyl ferulate, pNPferulate)- for potential applications in deconstruction of lignocellulosic materials andextraction of valuable compounds - as well as for their biosynthetic capacities. Wetested and optimised the FAEs’ transesterification capabilities on ferulate esters in a 1-butanol-buffer system, with the aim of using the most promising candidates for theproduction of antioxidant compounds with improved hydrophobic or hydrophilicproperties, such as prenyl ferulate, prenyl caffeate, glyceryl ferulate and 5-O-(transferuloyl)-arabinofuranose.
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| 9. |
- Hüttner, Silvia, 1984, et al.
(author)
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Genome sequence of Rhizomucor pusillus FCH 5.7, a thermophilic zygomycete involved in plant biomass degradation harbouring putative GH9 endoglucanases
- 2018
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In: Biotechnology Reports. - : Elsevier BV. - 2215-017X. ; 20
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Journal article (peer-reviewed)abstract
- We report here the annotated draft genome sequence of the thermophilic zygomycete Rhizomucor pusillus strain FCH 5.7, isolated from compost soil in Vietnam. The genome assembly contains 25.59 Mb with an overall GC content of 44.95%, and comprises 10,898 protein coding genes. Genes encoding putative cellulose-, xylan- and chitin-degrading proteins were identified, including two putative endoglucanases (EC 3.2.1.4) from glycoside hydrolase family 9, which have so far been mostly assigned to bacteria and plants.
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| 10. |
- Hüttner, Silvia, 1984, et al.
(author)
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Immobilisation on mesoporous silica and solvent rinsing improve the transesterification abilities of feruloyl esterases from Myceliophthora thermophila
- 2017
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In: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; 239, s. 57-65
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Journal article (peer-reviewed)abstract
- The immobilisation of four feruloyl esterases (FAEs) (FaeA1, FaeA2, FaeB1, FaeB2) from the thermophilic fungus Myceliophthora thermophila C1 was studied and optimised via physical adsorption onto various mesoporous silica particles with pore diameters varying from 6.6 nm to 10.9 nm. Using crude enzyme preparations, enrichment of immobilised FAEs was observed, depending on pore diameter and protein size. The immobilised enzymes were successfully used for the synthesis of butyl ferulate through transesterification of methyl ferulate with 1-butanol. Although the highest butyl ferulate yields were obtained with free enzyme, the synthesis-to-hydrolysis ratio was higher when using immobilised enzymes. Over 90% of the initial activity was observed in a reusability experiment after nine reaction cycles, each lasting 24 h. Rinsing with solvent to remove water from the immobilised enzymes further improved their activity. This study demonstrates the suitability of immobilised crude enzyme preparations in the development of biocatalysts for esterification reactions.
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