Adiphol Dilokpimol

VTT Technical Research Centre of Finland | vtt · Protein Production team

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been...

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate‐Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity...

α-L-Arabinofuranosidases (ABFs) are important enzymes in plant biomass degradation with a wide range of applications. The ascomycete fungus Penicillium subrubescens has more α-L-arabinofuranosidase-encoding genes in its genome compared to other Penicillia. We characterized nine ABFs from Glycoside Hydrolase (GH) families GH51, GH54 and GH62 from th...

Xyloglucan is a prominent matrix heteropolysaccharide binding to cellulose microfibrils in primary plant cell walls. Hence, the hydrolysis of xyloglucan facilitates the overall lignocellulosic biomass degradation. Xyloglucanases (XEGs) are key enzymes classified in several Glycoside Hydrolase (GH) families. So far, family GH44 has been shown to con...

Efficient bioconversion of agro-industrial side streams requires a wide range of enzyme activities. Glycoside Hydrolase family 30 (GH30) is a diverse family that contains various catalytic functions and has so far been divided into ten subfamilies (GH30_1-10). In this study, a GH30 phylogenetic tree using over 150 amino acid sequences was contructe...

As important polysaccharide degraders in nature, fungi can diversify their extensive set of carbohydrate-active enzymes to survive in ecological habitats of various composition. Among these enzymes, xylanolytic ones can efficiently and sustainably degrade xylans into (fermentable) monosaccharides to produce valuable chemicals or fuels from, for exa...

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 be...

Aromatic compounds are important molecules which are widely applied in many industries and are mainly produced from nonrenewable sources. Renewable sources such as plant biomass are interesting alternatives for the production of aromatic compounds. Ferulic acid and p-coumaric acid, a precursor for vanillin and p-vinyl phenol, respectively, can be r...

d-xylose reductase is a member of the aldo-keto reductase family, and is involved in d-xylose and l-arabinose conversion through the Pentose Catabolic Pathway (PCP) in fungi. In this study, we biochemically characterized a newly identified second d-xylose reductase (XyrB) from Aspergillus niger. This NADPH-dependent reductase is able to efficiently...

Salicylic acid plays an important role in the plant immune response and its degradation is therefore important for plant pathogenic fungi. However, many non-pathogenic microorganisms can also degrade salicylic acid. In the filamentous fungi Aspergillus niger, two salicylic acid metabolic pathways have been suggested. The first pathway converts sali...

The fungal members of Carbohydrate Esterase family 1 (CE1) from the CAZy database include both acetyl xylan esterases (AXEs) and feruloyl esterases (FAEs). AXEs and FAEs are essential auxiliary enzymes to unlock the full potential of feedstock. They are being used in many biotechnology applications including food and feed, pulp and paper, and bioma...

Penicillium subrubescens is able to degrade a broad range of plant biomass and it has an expanded set of Carbohydrate Active enzyme (CAZyme)-encoding genes in comparison to other Penicillium species. Here we used exoproteome and transcriptome analysis to demonstrate the versatile plant biomass degradation mechanism by P. subrubescens during growth...

Feruloyl esterases (FAEs) have an important role in the enzymatic conversion of lignocellulosic biomass by decoupling plant cell wall polysaccharides and lignin. Moreover, FAEs release anti-oxidative hydroxycinnamic acids (HCAs) from biomass. As a plethora of FAE candidates were found in fungal genomes, FAE classification related to substrate speci...

Ash dieback disease (caused by Hymenoscyphus fraxineus) has affected European ash species (Fraxinus spp.) in recent decades. However, some Asian and American species of Fraxinus and certain genotypes of Fraxinus excelsior are less affected by the disease. We used ITS1-metabacoding to explore the drivers influencing diversity and composition of the...

α-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular α-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All α-galactosidases exhibited high affinity to pNPαGal, and only AglE was not active towards galacto-olig...

Aims: To create an Aspergillus niger mutant with increased tolerance against ferulic acid using evolutionary adaptation. Methods and results: Evolutionary adaptation of Aspergillus niger N402 was performed by consecutive growth on increasing concentrations of ferulic acid in the presence of 25 mmol l-1 D-fructose, starting from 0·5 mmol l-1 and...

The aromatic compound benzoic acid and its derivatives are frequently used preservatives in food and beverages due to their strong antimicrobial properties. Benzoic acid naturally occurs in plants and serves as a building block for primary and secondary metabolites. Several Aspergilli are able to degrade benzoic acid to p-hydroxybenzoic acid by p-h...

Distillers’ grain (DG) is one of the lignocellulosic agricultural waste streams having a high potential for biofuel production, however it is causing health and environmental problems in Southwest region of China because of improper treatment before being used for landfill. Here, we aimed to identify fungal strains that efficiently hydrolyze DG and...

α-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular α-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All α-galactosidases exhibited high affinity to pNPαGal, and only AglE was not active towards galacto-olig...

Cinnamic acid is an aromatic compound commonly found in plants and functions as a central intermediate in lignin synthesis. Filamentous fungi are able to degrade cinnamic acid through multiple metabolic pathways. One of the best studied pathways is the non-oxidative decarboxylation of cinnamic acid to styrene. In Aspergillus niger, the enzymes cinn...

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic...

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, i...

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell‐wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and...

Fig. S1. Phylogenetic tree of fungal CE1 genes based on amino acid sequences.

Table S1. Amino acid sequences used for the CE1 phylogeny.

Fig. S2. (A) Total protein concentration (‐■‐) and activity towards methyl ferulate (‐●‐) followed from the extracellular culture liquid of P. pastoris during fermentation of recombinant AtFaeD. Vertical bars represent standard deviations of three technical replicates in BCA assay. (B) SDS‐PAGE of AtFaeD with and without treatment with Endo Hf, PNG...

Production of value-added compounds from a renewable aromatic polymer, lignin, has proven to be challenging. Chemical procedures, involving harsh reaction conditions, are costly and often result in non-selective degradation of lignin linkages. Therefore, enzymatic catalysis with selective cleavage of lignin bonds provides a sustainable option for l...

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharifica...

4-O-methyl-D-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary...

A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10 mg/L. FaeC was most active at pH 7.0 and 50 °C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, m...

Here we report the genome sequence of the ascomycete saprobic fungus Penicillium subrubescens FBCC1632/CBS132785 isolated from a Jerusalem artichoke field in Finland. The 39.75 Mb genome containing 14,188 gene models is highly similar for that reported for other Penicillium species, but contains a significantly higher number of putative carbohydrat...

Feruloyl esterases (FAEs) represent a diverse group of carboxyl esterases that specifically catalyze the hydrolysis of ester bonds between ferulic (hydroxycinnamic) acid and plant cell wall polysaccharides. Therefore, FAEs act as accessory enzymes to assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomas...

Primers used in this study. (DOCX)

Supplementary Methods. (DOCX)

Supporting Data. All affinity electrophoresis data. (XLSX)

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are no...

A plant glycosyltransferase was utilized to modify the properties of gum arabic as an oil-in-water emulsifier. We previously reported that recombinant beta-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana produced in Pichia pastoris possesses glucuronosyltransferase activity to transfer glucuronic acid (GlcA) from UDP-GlcA to beta-1,3...

Arabinogalactan proteins are abundant cell surface proteoglycans in plants and are implicated to act as developmental markers during plant growth. We previously reported that AtGALT31A, AtGALT29A, and AtGLCAT14A-C, which are involved in the biosynthesis of arabinogalactan proteins, localize not only to the Golgi cisternae but also to smaller compar...

Root hairs are single cells that develop by tip growth, a process shared with pollen tubes, axons, and fungal hyphae. However, structural plant cell walls impose constraints to accomplish tip growth. In addition to polysaccharides, plant cell walls are composed of hydroxyproline-rich glycoproteins (HRGPs), which include several groups of O-glycopro...

The subcellular localization of AtGALT31A-GFP in the Arabidopsis mutant background. The Arabidopsis T-DNA insertional mutant complemented with a C-terminally GFP tagged construct (AtGALT31A-GFP) was transiently transformed with GMII-mRFP. AtGALT31A-GFP (A, green) partially colocalized with GMII-mRFP (B, magenta, overlapping signals; C), but was als...

Subcellular localization and FRET analysis of Y144E AtGALT29A transiently coexpressed with AtGALT31A in N. benthamiana leaf. A) The overlaid images of AtGALT31A-mCer3 (green) together with Y144E AtGALT29A-YFP (magenta) are presented. Some of the colocalized images are indicated (arrows). Scale bar = 10 µm. B) Distribution histograms for pixel by pi...

Localization of N-glycan GTs in small vesicles of 0.4–0.6 µm diameter. N-glycan GTs, GnTI-mRFP (A), GMII-mRFP (B), ST-YFP (C) were occasionally found in the smaller vesicles than the Golgi apparatus (indicated by arrows) similarly to AG GTs (Figures 3). Scale bars = 10 µm.

We report that fluorescently tagged arabinogalactan glycosyltransferases target not only the Golgi apparatus but also uncharacterized smaller compartments when transiently expressed in Nicotiana benthamiana. Approximately 80% of AtGALT31A was found in the small compartments, of which, 45% and 40% of AtGALT29A and AtGlcAT14A co-localized with AtGALT...

Arabinogalactan proteins (AGPs) are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known abou...

Arabinogalactan proteins are abundant cell-surface proteoglycans in plants and are involved in many cellular processes including somatic embryogenesis, cell-cell interactions, and cell elongation. We reported a glucuronosyltransferase encoded by Arabidopsis AtGlcAT14A, which catalyzes an addition of glucuronic acid residues to β-1,3- and β-1,6-link...

Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post- translational modification of proteins in the secretory pathway...

Clonostachys rosea strain IK726 is a mycoparasitic fungus capable of controlling mycotoxin-producing Fusarium species, including F. graminearum and F. culmorum, known to produce Zearalenone (ZEA) and Deoxynivalenol (DON). DON is a type B trichothecene known to interfere with protein synthesis in eukaryotes. ZEA is a estrogenic-mimicing mycotoxin th...

We have characterized a β-glucuronosyltransferase (AtGlcAT14A) from Arabidopsis thaliana, which is involved in the biosynthesis of type II arabinogalactan (AG). This enzyme belongs to Carbohydrate Active Enzyme Glycosyltransferase (GT) family GT14. The protein was localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana....

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a beta-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrop...

ArabinoGalactan Proteins (AGPs) are a complex family of cell wall proteoglycans, which are thought to have major roles in plant growth and development. Genetic approaches studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for...

The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K(M)) towards β-1,4 manno-oligosaccharides with the...

Two β-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k(cat)/K(m)) than BxlB towards para-nitrophenyl β-D-xylop...

Two b-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (kcat/Km) than BxlB towards para-nitrophenyl b-D-xylopyran...

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α...

Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (alpha/alpha)(6)-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with beta-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence ident...

The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g.L(-1) culture) as His-tag fusion in Escherichia coli, catalysed efficient tran...

Lactobacillus acidophilus NCFM maltose phosphorylase (LaMalP) of glycoside hydrolase family 65 catalysed enzymatic synthesis of alpha-(1-->4)-glucosidic disaccharides from maltose and five monosaccharides in a coupled phosphorolysis/reverse phosphorolysis one-pot reaction. Thus phosphorolysis of maltose to beta-glucose 1-phosphate circumvented addi...

A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties...

Germinating barley seeds contain multiple forms of α-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The α-amylases are endo-acting and possess a long substrate binding cleft with a characteristic subsite binding energy profile around the catalytic s...

Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)(8)-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(2...

α Amylases occur widely in plants, animals, and microorganisms. They often act in synergy with other related and degradative enzymes and may also be regulated by proteinaceous inhibitors. Open questions exist on how αamylases interact with polysaccharides. Several enzymes possess secondary carbohydrate binding sites situated on the surface at a cer...

Thai curry is one of the most famous Thai foods. The main ingredient to prepare this kind of food is the curry paste, the mixture of ground spices, which is used for making different kind of curries and food products. Therefore, the quality of these products depends on the curry pastes and their spices. The objective of this research was to determi...