Jorn Dalgaard D Mikkelsen

Jorn Dalgaard D Mikkelsen
Technical University of Denmark | DTU · Department of Chemical and Biochemical Engineering

Professor

About

177
Publications
35,046
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9,215
Citations
Additional affiliations
January 2017 - present
Technical University of Denmark
Position
  • Professor (Full)
Description
  • Designing of novel enzymes for production of healthy food ingredients, including galactan from potato starch side-streams and Human Milk Olgosaccharides from side-stream in the dairy industry

Publications

Publications (177)
Article
Full-text available
The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most la...
Article
Certain enzymes of the glycoside hydrolase family 20 (GH20) exert trans‐glycosylation activity catalyzing transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work aimed to increase trans‐glycosylation activity of two selected...
Article
Full-text available
Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-d-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N′-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-d-glucosaminide (1 → 4)-β-linkages and are thus “exo-chitobiose hydrolases.” In this study, the chiti...
Article
Full-text available
A sialidase (EC 3.2.1.18) from the non-pathogenic Trypanosoma rangeli, TrSA, has been shown to exert trans-sialidase activity after mutation of five specific amino acids in the active site (M96V, A98P, S120Y, G249Y, Q284P) to form the so-called TrSA5mut enzyme. By computational and hypothesis driven approaches additional mutations enhancing the tra...
Data
Inspection of 3D structures of TrSA and TcTS around TrSA G342. Mutation of G342 in TrSA led to low/no expression. The figure shows the conformation of the carboxylic oxygens of TcTS S340 and TrSA S341 adjacent to G341/342 in each of the two enzymes TcTS and TrSA. (Left) In TcTS the residue assumes a conformation with a disallowed phi/psi angle set...
Data
HPAEC-PAD chromatograms of trans-sialylation products. Chromatograms from the analysis of samples at the time of transient maximal 3’SL yield for the enzyme variants TrSA5mut (25 min), TrSA6mut (40 min), TrSA8mut (120 min), TrSA9mut (40 min), TrSA12mut (60 min), TrSA13mut (60 min), TrSA15mut (120 min), and TrSA16mut (60 min). The hydrolysis product...
Article
Full-text available
Pectin production is complex, and final product quality assessment is generally accomplished at the end of the process using time-consuming off-line laboratory analysis. In this study, pectin was extracted from lime peel either by acid or by enzymes. Fourier transform infrared spectroscopy and carbohydrate microarray analysis were performed directl...
Article
Full-text available
Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of...
Data
Homology modeling of active sites of trans-sialidase candidates falling for criterion 1. Active site and aromatic sandwich of TcTS (gray) aligned to homology models of (putative) sialidases from (a) Actinomyces sp. oral taxon 849 str. F0330, (b) Actinomyces odontolyticus, (c) Congregibacter litoralis, (d) Diplosphaera colitermitum, (e) Oceanobacter...
Data
1H NMR spectrum (D2O, 600 MHz) of SialH trans-sialylation products. To confirm the obtained reaction products, mixed samples were analysed by NMR. As expected, the 1H NMR spectrum of the reaction products reveals the presence of 3’-sialyllactose (3’SL) and 6’-sialyllactose (6’SL). Of special interest was the signal at δ 4.537 (Gal H-1) ppm, rendere...
Data
Sequence IDs of the 3D alignments of the 15 sequences selected for homology modeling (HM). Homology models were aligned to the TcTS crystal structure (PDB 1S0I) for evaluation against selection criteria 1 and 2 (Fig 2; S1 Fig). The reason for rejecting a sequence after homology modeling is indicated. Falling for criterion 1: Does not contain all ni...
Data
HPAEC-PAD chromatogram of SialH trans-sialylation products (zoom). The reaction mixture obtained after 90 min of reaction by SialH at 30°C using casein glycomacropeptide (CGMP) as sialyl donor and lactose as acceptor was analysed by HPAEC-PAD and showed and unidentified peak at 9.2 min (blue). External standards of sialic acid (SA; 5.1 min), 6’-sia...
Data
Sequence IDs of the sequences identified in the initial database screening. 46 sequences were identified using the ΩxRDR search motif (Box 1). Of these, 30 were rejected before homology modeling due to redundancy or the fact that they were already known Trypanosoma trans-sialidases (TS), including TcTS. Redundancy indicates that there was more than...
Article
Full-text available
In this study, we introduce enzymatic perturbation combined with Fourier transform infrared (FTIR) spectroscopy as a concept for quantifying casein in subcritical heated skim milk using chemometric multiway analysis. Chymosin is a protease that cleaves specifically caseins. As a result of hydrolysis, all casein proteins clot to form a creamy precip...
Article
Full-text available
This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as...
Data
pH vs Hydrolase activity using pNP-Fuc as substrate. (PDF)
Data
Multiple sequence alignment of α-L-fucosidases reported in this study. (PDF)
Data
SDS-PAGE of recombinant Mfuc1-7 and Thma. (PDF)
Data
Time study of trans-fucosylation catalysed by metagenome-derived α-L-fucosidases. (PDF)
Data
List of primers used for amplification of α-L-fucosidase-encoding genes. (PDF)
Data
Substrate concentration vs. hydrolase activity plots using pNP-Fuc as substrate. (PDF)
Data
Heat inactivation of α-L-fucosidases. (PDF)
Data
Sequence of codon-optimised Thma-encoding gene. (PDF)
Data
Immuno-glycan microarray analysis of fucosylated xyloglucan cleavage. (PDF)
Data
Selected NMR spectra, NMR chemical shifts and comparison of 1H NMR integrals. (PDF)
Data
Substrate specificity of characterized GH29 fucosidases. (PDF)
Article
Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk. Using lactose as a cheap donor, some β-galactosidases (EC 3.2.1.23) can catalyze transgalactosylation to form the human milk oligosaccharide lacto-N-neotetraose (LNnT; Gal-β(1,4)-GlcNAc-β(1,3)-Gal-β(1,4)-Glc). In order to...
Article
Full-text available
A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all...
Article
Rhamnogalacturonan I (RGI) modifying enzymes catalyse the degradation of the RGI backbone and encompass enzymes specific for either the α1,2-bond linking galacturonic acid to rhamnose or the α1,4-bond linking rhamnose to galacturonic acid in the RGI backbone. The first microbial enzyme found to be able to catalyse the degradation of the RGI backbon...
Article
Sialylated galactooligosaccharides (GOS) represent a potential infant formula ingredient, which is believed to contribute with a combination of the beneficial properties of the prebiotic GOS as well as of sialylated human milk oligosaccharides. Sialylated GOS do not exist in natural milk, but can be produced from κ(kappa)-casein glycomacropeptide (...
Article
Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3'- and 6'-sialyllactos...
Article
This paper describes the discovery and characterization of two novel β-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as β-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening...
Data
##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
Data
##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
Article
This study aimed to improve the hydrolase activity of the well-characterised bacterial sialidase from Micromonospora viridifaciens. The enzyme and its mutated versions were produced in Bacillus subtilis and secreted to the growth medium. Twenty amino acid positions in or near the active site were subjected to site-saturation mutagenesis and evaluat...
Article
A new laccase gene (mrlac) from Meiothermus ruber DSM 1279 was successfully overexpressed to produce a laccase (Mrlac) in soluble form in Escherichia coli during simultaneous overexpression of a chaperone protein (GroEL/ES). Without the GroEL/ES protein, the Mrlac overexpressed in E. coli constituted a huge amount of the total cellular protein, but...
Article
This paper reports the identification, heterologous expression in E. coli and characterization of TtMCO from the thermophilic bacterium Thermobaculum terrenum, the first Laccase-like Multi-Copper Oxidase (LMCO) from the distinct Phylum Chloroflexi. TtMCO has only 39% identity to its closest characterized homolog, CotA from Bacillus subtilis, but se...
Article
Separation of 3′-sialyllactose (SL) and lactose is an essential final step for the production of the next generation of infant formulas containing sialyllated prebiotics. Due to the difference in molecular weight (MW) between SL and lactose and the charge density of SL, nanofiltration could provide a rapid, inexpensive alternative for the separatio...
Article
Full-text available
Enzymatic conversion of pectinaceous biomasses such as potato and sugar beet pulp at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, and increased substrate solubility and lowers the risk of contamination. Such high-temperature processing requires development of thermostable enzymes. Talaromyces stipi...
Patent
Full-text available
The invention encompasses the use of a lipolytic enzyme obtainable from one of the following genera: Streptomyces, Corynebacterium and Thermobifida in various methods and uses, wherein the lipolytic enzyme hydrolyzes a glycolipid or a phospholipid or transfers an acyl group from a glycolipid or phospholipids to an acyl acceptor. The present inventi...
Patent
Full-text available
Plant polysaccharides, relating to the field of natural prebiotic components, can comprise structures similar to human milk oligosaccharides. A method for enzymatic hydrolysis of gum tragacanth from the bush-like legumes of the genus Astragalus, using a combination of pectin hydrolases and a xylogalacturonan hydrolase, is described. Fractions with...
Article
The objective of the present study was to transform "Waste to Food" using enzymes to recover value-added food ingredients from biomass. Six commercial cellulases were screened to generate proof of concept that enzymes are selective and efficient catalysts for opening of lime peel biomass to recover pectin. The most efficient enzyme preparation was...
Article
Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including e.g. human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredi...
Article
Abstract Laccases (EC 1.10.3.2) are copper-containing oxidoreductases that have a relatively high redox potential which enables them to catalyze oxidation of phenolic compounds, including lignin-derived phenolics. The laccase-catalyzed oxidation of phenolics is accompanied by concomitant reduction of dioxygen to water via copper catalysis and invol...
Patent
Full-text available
A method of oxidizing a saccharide, by contacting the saccharide with an alcohol dehydrogenase (ADH) enzyme selected from a quinone redox cofactor-dependent ADH, a nicotinamide adenine dinucleotide (NAD+) redox cofactor-dependent ADH, a nicotinamide adenine dinucleotide phosphate (NADP+) redox cofactor-dependent ADH, and any combination thereof is...
Article
Full-text available
Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage pat...
Article
Full-text available
The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain B...
Article
A xyloglucan-specific endo-1,4β-glucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae has been cloned, expressed in Escherichia coli, purified and characterised. The XcXGHA enzyme belongs to CAZy family GH74 and has catalytic site residues conserved with other xyloglucanases in this family. At its optimal reaction conditions, pH 7.0 and 4...
Article
Full-text available
Pectin is abundant in various agro-industrial bio-resources such as citrus peel, apple pomace, cranberry pulp and sugar beet pulp. These materials can therefore be considered as a source of potential bioactive pectic oligosaccharides. This chapter reviews the various extraction and purification methods for production of the pectic oligosaccharides...
Article
Biocatalytic trans-sialylation is relevant for the design of biomimetic oligosaccharides such as human milk oligosaccharides. t-Butanol and ionic liquids, EAN (ethylammonium nitrate), [MMIm][MeSO4] (1,3-dimethylimidazolium methyl sulfate), and [C2OHMIm][PF6] (1-(2-hydroxyethyl)-3-methylimidazolium hexafluorophosphate), were examined as co-solvents...
Article
Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial...
Article
Full-text available
This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for tran...
Article
Based on a differential pre-screening of 44 white-rot fungi on a lignocellulose-supplemented minimal medium, four basidiomycetes were selected for further study: Ganoderma lucidum, Polyporus brumalis, Polyporus ciliatus and Trametes versicolor. Only G. lucidum was able to grow vividly on malt extract or minimal media supplemented with alkali lignin...
Article
A Trypanosoma cruzi trans-sialidase (E.C. 3.2.1.18) was cloned into Pichia pastoris and expressed. The pH and temperature optimum of the enzyme was determined as pH 5.7 and 30°C. Using casein glycomacropeptide (CGMP) and lactose as sialyl-donor and acceptor respectively, the optimal donor/acceptor ratio for the trans-sialidase catalysed 3'-sialylla...
Article
This study examined a recombinant Pasteurella multocida sialyltransferase exhibiting dual trans-sialidase activities. The enzyme catalyzed trans-sialylation using either 2-O-(p-Nitrophenyl)-α-D-N-acetylneuraminic acid or casein glycomacropeptide (whey protein) as the sialyl donor and lactose as the acceptor, resulting in production of both 3'-sialy...
Patent
Full-text available
The invention encompasses the use of a lipolytic enzyme obtainable from one of the following genera: Streptomyces, Corynebacterium and Thermobifida in various methods and uses, wherein the lipolytic enzyme hydrolyzes a glycolipid or a phospholipid or transfers an acyl group from a glycolipid or phospholipids to an acyl acceptor. The present inventi...
Article
Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine in...
Article
Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which are active on specific substrates generating a mult...
Article
A novel Emericella nidulans endo-β-1,4-galactanase (EnGAL) demonstrates a strong capacity to generate high levels of very potent prebiotic oligosaccharides from potato pulp, a by-product of the agricultural potato-starch industry. EnGAL belongs to glycoside hydrolase family 53 and shows high (72.5%) sequence identity to an endo-β-1,4-galactanase fr...
Article
The recent advances in multi-way analysis provide new solutions to traditional enzyme activity assessment. In the present study enzyme activity has been determined by monitoring spectral changes of substrates and products in real time. The method relies on measurement of distinct spectral fingerprints of the reaction mixture at specific time points...
Patent
Full-text available
The present application provides a method of reducing and/or removing diglyceride from an edible oil, comprising admixing an edible oil with an acyl acceptor substrate and a diglyceride:glycerol acyltransferase, wherein the diglyceride:glycerol acyltransferase is characterized as an enzyme which in an edible oil is capable of transferring an acyl g...
Article
Tragacanth gum is a plant derived hydrocolloid that has a long history of use in food, pharma, and cosmetics. The gum is mainly produced in the Middle East and permitted for food use in the US and Europe. Tragacanth gum consists of complex, heterogeneous polysaccharides, which contain different highly substituted pectin-like structural elements. En...
Article
Gum tragacanth derived from the plant "goat's horn" (Astragalus sp.) has a long history of use as a stabilizing, viscosity-enhancing agent in food emulsions. The gum contains pectinaceous arabinogalactans and fucose-substituted xylogalacturonans. In this work, gum tragacanth from Astragalus gossypinus was enzymatically depolymerized using Aspergill...
Article
Based on a differential pre-screening of 44 white-rot fungi on a lignocellulose-supplemented minimal medium, four basidiomycetes were selected for further study: Ganoderma lucidum, Polyporus brumalis, Polyporus ciliatus and Trametes versicolor. Only G. lucidum was able to grow vividly on malt extract or minimal media supplemented with alkali lignin...