Tjaard Pijning

Verified

Tjaard verified their affiliation via an institutional email.

Learn more

Verified

Tjaard verified their affiliation via an institutional email.

Learn more

• Dr. Ing.
• Research Associate at University of Groningen

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization...

Glucansucrases are extracellular enzymes that synthesize a wide variety of α-glucan polymers and oligosaccharides, such as dextran. These carbohydrates have found numerous applications in food and health industries, and can be used as pure compounds or even be produced in situ by generally regarded as safe (GRAS) lactic acid bacteria in food applic...

The glycoside hydrolase family 70 (GH70) contains bacterial extracellular multidomain enzymes, synthesizing α-glucans from sucrose or starch-like substrates. A few dozen have been biochemically characterized, while crystal structures cover only the core domains and lack significant parts of auxiliary domains. Here we present a systematic overview o...

Proteins featuring the Domain of Unknown Function 1735 are frequently found in Polysaccharide Utilization Loci, yet their role remains unknown. The domain and vicinity analyzer programs we developed mine the Kyoto Encyclopedia of Genes and Genomes and UniProt to enhance the functional prediction of DUF1735. Our datasets confirmed the exclusive pres...

Here we present a systematic overview of the GH70 enzyme space. An initial set of 2673 aligned GH70 sequences (obtained by BLASTp) was reduced to 259 by applying a 95% redundancy filter and addition of biochemically characterized enzymes listed by CAZy. This representative set was(1) classified by predicted enzyme specificity, (2) phylogenetically...

Proline‐specific endoproteases have been successfully used in, for example, the in‐situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline‐specific...

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes from Glycoside Hydrolase Family 70 (GH70) are of interest for the modification of starch into low-glycemic index food ingredients. Compared to the related GH70 GtfB-type α-GTs, found exclusively in lactic acid bacteria (LAB), GtfCs occur in non-LAB, share low sequence identity, lack circular permuta...

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes are of interest for modification of starch into low-glycemic index food ingredients [1]. GtfCs have been classified in Glyco-side Hydrolase family 70 (GH70), together with glucansucrases and GtfB-type α-GTs from lactic acid bacteria (LAB). However, GtfCs have been identified only in non-lactic acid...

Branching sucrases, a subfamily of Glycoside Hydrolase family (GH70), display transglycosidase activity using sucrose as donor substrate to catalyze glucosylation reaction in the presence of suitable acceptor substrates. In this study, the (α1→3) branching sucrase GtfZ-CD2 from Apilactobacillus kunkeei DSM 12361 was demonstrated to glucosylate benz...

Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical...

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information...

Several archaea harbor genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure–function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition, and pharmaceutical applications. In this study, 3D structure of an inulin‐type...

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability‐landscape approach tha...

AmyC, a glycoside hydrolase family 57 (GH57) enzyme of Thermotoga maritima MSB8, has previously been identified as an intracellular α-amylase playing a role in either maltodextrin utilization or storage polysaccharide metabolism. However, the α-amylase specificity of AmyC is questionable as extensive phylogenetic analysis of GH57 and tertiary struc...

GtfB-type glucanotransferases (GT) from GH70 act on starch and starch-derived oligosaccharides, synthesizing α-glucans. The Lactobacillus reuteri 121 4,6-α-GT synthesizes linear isomalto-maltooligosaccharides (IMMP) with up to 95% α-1,6 linked glucosyl units at the non-reducing end; its structure features a tunnel in agreement with its preference f...

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, antidepressants and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from sim...

Glucansucrase Gtf180-ΔN from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize 5 glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with(α1→2)/(α1→3)/(α1→4)-glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased...

An intriguing structural feature of echinocandins is the incorporation of hydroxylated amino acids. Elucidation of the machinery and the mechanism responsible for this modification is critical to generate new echinocandin derivatives with enhanced antifungal activity. In our present study, we biochemically characterized the α-ketoglutarate/Fe2+-dep...

The fructophilic bacterium Lactobacillus kunkeei has promising applications as probiotics promoting the health of both honey bees and humans. Here, we report the synthesis of a highly branched dextran by L. kunkeei DSM 12361 and biochemical characterization of a GH70 enzyme (GtfZ). Sequence analysis revealed that GtfZ harbors two separate catalytic...

Nine GtfB-like 4,6-α-glucanotransferases (4,6-α-GTs) (represented by GtfX of L. aviarius subsp. aviarius DSM 20655) were identified to show distinct characteristics in conserved motifs I-IV. In particular, the “fingerprint” Tyr in motif III of these nine GtfB-type 4,6-α-GTs was found to be replaced by a Trp. In L. aviarius subsp. aviarius DSM20655,...

Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical properties are produced, which have found diverse (...

The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving (α1 → 4)-linkages in maltodextrins/starch and synthesiz...

Crystal structure of the first representative of the GH70 4,6-α-glucanotransferase subfamily, showing overall structure and the main features that distinguishes it from α-amylases of GH13 and glucansucrases of GH70. Reference PDBs : 5JBD (native), 5JBF (complex with maltopentaose). See also Crystal Structure of 4,6-α-Glucanotransferase Supports Die...

The β-galactosidase enzyme from Bacillus circulans ATCC 31382 BgaD is widely used in the food industry to produce prebiotic galactooligosaccharides (GOS). Recently, the crystal structure of a C-terminally truncated version of the enzyme (BgaD-D) has been elucidated. The roles of active site amino acid residues in β-galactosidase enzyme reaction and...

Lactic acid bacteria (LAB) produce α-glucans, using glucansucrases (GSs) from glycoside hydrolase family 70 (GH70), from sucrose. It was proposed that GSs are evolutionarily linked to GH13 enzymes acting on starch-like substrates. Recently, a GH70 subfamily was discovered containing 4,6-α-glucanotransferases (4,6-α-GTs) from Lactobacilli, synthesiz...

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential...

Food processing and refining has dramatically changed the human diet, but little is known about whether this affected the evolution of enzymes in human microbiota. We present evidence that glycoside hydrolase family 70 (GH70) glucansucrases from lactobacilli, synthesizing α-glucan-type extracellular polysaccharides from sucrose, likely evolved from...

Microbial β-galactosidase enzymes are widely used as biocatalysts in industry to produce prebiotic galactooligosaccharides (GOS) from lactose. GOS mixtures are used as beneficial additives in infant formula to mimic the prebiotic effects of human milk oligosaccharides (hMOS). The structural variety in GOS mixtures is significantly lower than in hMO...

Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as...

The glucansucrase GTFA of Lactobacillus reuteri 121 produces an α-glucan (reuteran) with a large amount of alternating (α1 → 4) and (α1 → 6) linkages. The mechanism of alternating linkage formation by this reuteransucrase has remained unclear. GTFO of the probiotic bacterium Lactobacillus reuteri ATCC 55730 shows a high sequence similarity (80%) wi...

The human diet has been subject to dramatic changes due to food processing and refining. however, whether this affected the evolution of enzymes in human microbiota is largely unknown. It was proposed that glycoside hdyrolase family 70 (GH70) glucansucrases (GS) from Lactobacilli, which synthesize α-glucan type extracellular polysaccharides (EPS) f...

Lactic acid bacteria (LAB) are known to produce large amounts of α-glucan exopolysaccharides. Family GH70 glucansucrase (GS) enzymes catalyze the synthesis of these α-glucans from sucrose. The elucidation of the crystal structures of representative GS enzymes has advanced our understanding of their reaction mechanism, especially structural features...

Exopolysaccharides (EPS) of lactic acid bacteria (LAB) are of interest for food applications. LAB are well-known to produce α-glucan from sucrose by extracellular glucansucrases. Various Lactobacillus reuteri strains also possess 4,6-α-glucanotransferase (4,6-α-GTase) enzymes. Purified 4,6-α-GTases (e.g. GtfB) were shown to act on starches (hydroly...

The Glycoside Hydrolase (GH) family 70 originally was established for glucansucrase enzymes, solely found in lactic acid bacteria, synthesizing α-glucan polysaccharides from sucrose (e.g. GtfA). In recent years we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB type of enzymes constitute a f...

α-Glucans produced by glucansucrase enzymes of lactic acid bacteria attract strong attention as novel ingredients and functional biopolymers in the food industry. In the present study, α-helix 4 amino acid residues D1085, R1088 and N1089 of glucansucrase GTF180 of Lactobacillus reuteri 180 were targeted for mutagenesis both jointly and separately....

α-Glucans produced by glucansucrase enymes hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown, even with the recent elucidation of glucansucrase crystal structures. Guided by the crystal structure from glucansucrase GTF180-ΔN of Lactobacillus re...

4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in Glycoside Hydrolase Family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/malto-polysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkag...

Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal structure of the N-terminal truncated GTF180 of Lactobacillus reuteri 180 (GTF180-ΔN) revealed that the polypeptide cha...

Highly conserved GH70 family glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue L940 in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different...

Unlabelled: Glucansucrase enzymes synthesize high-molecular-mass extracellular α-glucan polysaccharides from sucrose. Previously, the crystal structure of truncated glucansucrase glucosyltransferase (GTF)180-ΔN from Lactobacillus reuteri 180 (lacking the N-terminal domain) revealed an elongated overall structure with two remote domains (IV and V)...

Extracellular polysaccharides produced by lactic acid bacteria have interesting properties for food-, non-food and medical applications. Polysaccharides of the α-glucan or β-fructan type are synthesized by glucansucrase and fructansucrase enzymes, respectively. This thesis describes 3D-structural studies on glucan- and fructansucrases, using X-ray...

Penicillin G acylase is the key enzyme used in the industrial production of β-lactam antibiotics. This enzyme hydrolyzes penicillin G and related β-lactam antibiotics releasing 6-aminopenicillanic acid, which is an intermediate in the production of semisynthetic penicillins. To improve the enzymatic activity of Escherichia coli penicillin acylase,...

3D modeling of the B. pumilus lipase mutant L3-3 obtained by DNA shuffling using two B. pumilus parents. Structural observations suggest how the mutations lead to an increase in specific activity and thermostability.

Glucansucrases (EC 2.1.4.5; glucosyltransferases, GTFs) synthesize a variety of high molecular mass glucose polymers with α-glycosidic linkages (α-glucans) from sucrose1. The α-glucans hold great potential in biotechnology, food and health related applications, also due to their potential prebiotic properties. In light of the above, it is crucial...

The reuteransucrase GTFA from Lactobacillus reuteri 121, which belongs to glycosyl hydrolase family GH70, synthesizes branched α-glucans with both α-1,6- and α-1,4-glycosidic linkages (reuteran) from sucrose. The crystal structure of GTFA-ΔN, a 118 kDa fragment of GTFA comprising residues 745-1763 and including the catalytic domain, was determined...

The probiotic bacterium Lactobacillus reuteri 121 produces two fructosyltransferase enzymes, a levansucrase and an inulosucrase. Although these two fructosyltransferase enzymes share high sequence similarity, they differ significantly in the type and size distribution of fructooligosaccharide products synthesized from sucrose, and in their activity...

Many lactic acid bacteria produce extracellular a -glucan polysaccharides using a glucansucrase and sucrose as glucose donor. The structure and the physicochemical properties of the a -glucans produced are determined by the nature of the glucansu-crase. Typically, the a -glucans contain two types of a -glycosidic linkages, for example, (a 1 -2), (a...

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activit...

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltoo...

ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incub...

Fructansucrases (FSs) catalyze a transfructosylation reaction with sucrose as substrate to produce fructo-oligosaccharides and fructan polymers that contain either β-2,1 glycosidic linkages (inulin) or β-2,6 linkages (levan). Levan-synthesizing FSs (levansucrases) have been most extensively investigated, while detailed information on inulosucrases...

Bacterial fructansucrases (FSs) catalyze a transfructosylation reaction with sucrose as substrate to produce fructooligosaccharides (FOS) and fructan polymers. These contain either β-2,1 glycosidic linkages (inulin) or β-2,6 linkages (levan). Bacterial FSs have been classified in glycoside hydrolase family GH68 and share high sequence homologies. N...

Bacterial fructansucrases (FSs) catalyze a transfructosylation reaction with sucrose as substrate to produce fructooligosaccharides (FOS) and fructan polymers. These contain either β-2,1 glycosidic linkages (inulin) or β-2,6 linkages (levan). Bacterial FSs have been classified in glycoside hydrolase family GH68 and share high sequence homologies. N...

Bacterial fructansucrases (FSs) catalyze a transfructosylation reaction with sucrose as substrate to produce fructooligosaccharides (FOS) and fructan polymers. These contain either β-2,1 glycosidic linkages (inulin) or β-2,6 linkages (levan). Bacterial FSs have been classified in glycoside hydrolase family GH68 and share high sequence homologies. N...

Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties a...

Branching enzyme (EC 2.4.1.18; glycogen branching en-zyme; GBE) catalyzes the formation of 1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties a...

Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the...

The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure-function relationships, the enzyme was studied by X-ray crystallography. Crystal structures of native ppLAP at pH 9.5 and pH 5.2, and...

Movie of the crystal structure of the pectin-degrading enzyme Exo-Polygalacturonase from Thermotoga maritima (PDB: 3JUR). Going from the N- to the C-terminus the enzyme, belonging to family GH28 family, folds as a unusually long right-handed parallel beta-helix decorated with several loops.

The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05A resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated s...

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no th...

The structure of truncated inulosucrase from Lactobacillus johnsonii NC533 (InuJ) shows structural conservation of the active site when compared with levansucrases, especially at the fructosyl (-1) site. At the + sites, where acceptor molecules (sucrose or fructo-oligosaccharides) bind, differences occur that likely determine product specificity of...

The structure of truncated inulosucrase from Lactobacillus johnsonii NC533 (InuJ) shows structural conservation of the active site when compared with levansucrases, especially at the fructosyl (-1) site. At the + sites, where acceptor molecules (sucrose or fructo-oligosaccharides) bind, differences occur that likely determine product specificity of...

Lipases are successfully applied in enantioselective biocatalysis. Most lipases contain a lid domain controlling access to the active site, but Bacillus subtilis Lipase A (LipA) is a notable exception: its active site is solvent exposed. To improve the enantioselectivity of LipA in the kinetic resolution of 1,2-O-isopropylidene-sn-glycerol (IPG) es...

In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for....

Bacterial glucansucrases (glucosyltransferases, GTFs) are responsible for the production of extracellular polysaccharides such as α-glucan. These glucose polymers are formed when the glycosidic bond of the substrate (sucrose) is cleaved, and its glucose unit is linked to a growing glucan chain (trans-glycosylation). The type of linkage, which deter...

α-Glucan EPS formation by a glucansucrase crystal incubated with sucrose

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structu...