Oppdagelse og karakterisering av kitin-aktive enzymer

In the shift from a fossil-based to a bio-based economy, exploration of renewable recourses is needed. Chitin is considered the second most abundant polysaccharide on Earth, after cellulose, and its water-soluble derivatives chitosan and chitooligosaccharides (CHOS) have several applications, for example in medicine, agriculture, and the food industry. Today, the extraction of chitin from chitin-rich biomasses and the subsequent production of chitosan and CHOS involve harsh chemicals. It is of interest to replace the current chemical processing technology with enzyme-driven processes, since this would be more environmentally friendly. In addition, enzymes can be used to produce well-defined chitosans and CHOS, which is of interest, since the bioactivity of these compounds depends on properties such as the fraction of acetylation (FA), the degree of polymerization (DP) and the pattern of acetylation (PA). Investigation of proteins utilized by microorganisms during growth on chitin might provide insight into natural chitin conversion and may yield enzymes that can aid in industrial valorization of chitin-rich biomasses. Paper I describes the characterization of a carbohydrate esterase family 4 (CE4) deacetylase, which was selected because of its potential application in the production of CHOS with defined FA and PA. To utilize these enzymes in an optimal way, good understanding of their substrate interactions and specificities is needed. Paper I includes the first enzyme-substrate complex of a CE4 deacetylase with an open active site, providing valuable insight into how the enzyme interacts with its substrate. The enzyme is able to deacetylate a variety of substrates at varying positions. This broad specificity and the presence of seemingly few subsites occupied by the substrate indicate that it may be difficult to use or develop this type of CE4 enzymes for enzymatic tailoring of the PA of CHOS. The genome of Cellvibrio japonicus encodes a large array of carbohydrate-active enzymes, including several putative chitinases and other enzymes possibly involved in chitin degradation. Whether these enzymes are actually involved in chitin utilization by this Gram-negative bacterium had not been investigated at the start of the work described in this thesis. Paper II describes a study of proteins that C. japonicus secretes during growth on chitin, using a novel, plate-based proteomics approach which yielded secretome samples with a relatively low fraction of cytoplasmic proteins. This study revealed that the four glycosyl hydrolase family 18 (GH18) chitinases encoded in the C. japonicus genome are produced in high amounts, indicating that these enzymes are all involved in natural chitin turnover. Chitin degradation studies showed that C. japonicus has considerable chitinolytic power. The proteomics study revealed several proteins without an obvious role in chitin degradation that also are produced in high amounts during growth on chitin, thus providing a list of proteins that could be targeted in future searches for proteins that degrade chitin-rich biomass. Paper III describes an in-depth investigation of the GH18 chitinases encoded by C. japonicus. Knockout studies showed that one of the chitinases, CjChi18D, is crucial for the bacterium’s ability to utilize chitin as a carbon source. Biochemical characterization showed that CjChi18D is the most efficient chitin degrader, which could explain its crucial role. Comparative studies of the four enzymes indicated different and putatively complementary functions, as exemplified by CjChi18C having the by far highest activity against chitohexaose. Indeed, when combining enzymes, synergistic effects on chitin degradation efficiency were observed. Transcriptomic analysis showed that the four GH18 chitinases and a chitin-active LPMO, CjLPMO10A, are strongly up-regulated when C. japonicus grows on chitin, along with several other putatively chitin-active enzymes as well as a few proteins of unknown function, which are up-regulated to a lesser extent. Serratia marcescens produces one the best studied chitinolytic machineries, involving three chitinases, a lytic polysaccharide monooxygenase, and a chitobiase. However, the genome sequence of one of the most frequently studied S. marcescens strains was not available at the start of this thesis work, and possible involvement of other proteins in chitin utilization had not been investigated. Paper IV describes the genome sequence of S. marcescens BJL200 and a proteomics investigation of proteins secreted during growth on chitin. The genome sequence showed that S. marcescens encodes a fourth chitinase, SmChiD, but the proteomics data indicated that this chitinase is not important in chitin utilization. Indeed, biochemical characterization of SmChiD supported the notion that this enzyme is not important for chitin conversion and, thus, likely has another, yet unknown, biological role. Taken together, the results presented in this thesis provide novel insight into chitin-active enzymes encoded by bacteria. Paper I provides insights into the substrate binding of CE4 deacetylases with an open active site. Papers II-IV reveal chitin-active enzymes, in particular hydrolases, that play key roles in natural chitin conversion. Additionally, Papers II-IV yield a list with proteins without an obvious role in chitin degradation, which may be targeted in future studies of the degradation of chitin-rich biomasses. Further studies on tailoring CE4 deacetylases for modification of chitosan and CHOS and on more efficient chitin conversion using enzymes derived from S. marcescens and C. japonicus are currently in progress.I overgangen fra en fossil-basert til en bio-basert økonomi må bruken av fornybare ressurser utforskers. Kitin er, etter cellulose, ansett som den biomassen det fins mest av på jorden, og de vannløselige kitin-derivatene kitosan og kitooligosakkarider har mange applikasjoner innen eksempelvis medisin, jordbruk og matindustri. Ekstraksjonsprosessen av kitin fra kitinrik biomasse og videre produksjon av kitosan og kitooligosakkarider involverer i dag farlige kjemikalier. Det er derfor ønskelig å erstatte dagens kjemiske prosess med en enzymdrevet prosess da dette vil være mer miljøvennlig. I tillegg kan enzymer brukes til å produsere godt definerte kitosaner og kitooligosakkarider, noe som er av interesse siden bioaktiviteten til disse forbindelsene er avhengig av egenskaper slik som fraksjon av acetylering, grad av polymerisering og acetyleringsmønster. Å undersøke hvilke proteiner mikroorganismer bruker når de vokser på kitin kan gi innsikt i naturlig kitin-nedbrytning og kan gi relevante enzymer som trengs for industriell valorisering av kitin-rik biomasse

Discovery and characterization of enzymes acting on chitin

In the shift from a fossil-based to a bio-based economy, exploration of renewable recourses is needed. Chitin is considered the second most abundant polysaccharide on Earth, after cellulose, and its water-soluble derivatives chitosan and chitooligosaccharides (CHOS) have several applications, for example in medicine, agriculture, and the food industry. Today, the extraction of chitin from chitin-rich biomasses and the subsequent production of chitosan and CHOS involve harsh chemicals. It is of interest to replace the current chemical processing technology with enzyme-driven processes, since this would be more environmentally friendly. In addition, enzymes can be used to produce well-defined chitosans and CHOS, which is of interest, since the bioactivity of these compounds depends on properties such as the fraction of acetylation (FA), the degree of polymerization (DP) and the pattern of acetylation (PA). Investigation of proteins utilized by microorganisms during growth on chitin might provide insight into natural chitin conversion and may yield enzymes that can aid in industrial valorization of chitin-rich biomasses. Paper I describes the characterization of a carbohydrate esterase family 4 (CE4) deacetylase, which was selected because of its potential application in the production of CHOS with defined FA and PA. To utilize these enzymes in an optimal way, good understanding of their substrate interactions and specificities is needed. Paper I includes the first enzyme-substrate complex of a CE4 deacetylase with an open active site, providing valuable insight into how the enzyme interacts with its substrate. The enzyme is able to deacetylate a variety of substrates at varying positions. This broad specificity and the presence of seemingly few subsites occupied by the substrate indicate that it may be difficult to use or develop this type of CE4 enzymes for enzymatic tailoring of the PA of CHOS. The genome of Cellvibrio japonicus encodes a large array of carbohydrate-active enzymes, including several putative chitinases and other enzymes possibly involved in chitin degradation. Whether these enzymes are actually involved in chitin utilization by this Gram-negative bacterium had not been investigated at the start of the work described in this thesis. Paper II describes a study of proteins that C. japonicus secretes during growth on chitin, using a novel, plate-based proteomics approach which yielded secretome samples with a relatively low fraction of cytoplasmic proteins. This study revealed that the four glycosyl hydrolase family 18 (GH18) chitinases encoded in the C. japonicus genome are produced in high amounts, indicating that these enzymes are all involved in natural chitin turnover. Chitin degradation studies showed that C. japonicus has considerable chitinolytic power. The proteomics study revealed several proteins without an obvious role in chitin degradation that also are produced in high amounts during growth on chitin, thus providing a list of proteins that could be targeted in future searches for proteins that degrade chitin-rich biomass. Paper III describes an in-depth investigation of the GH18 chitinases encoded by C. japonicus. Knockout studies showed that one of the chitinases, CjChi18D, is crucial for the bacterium’s ability to utilize chitin as a carbon source. Biochemical characterization showed that CjChi18D is the most efficient chitin degrader, which could explain its crucial role. Comparative studies of the four enzymes indicated different and putatively complementary functions, as exemplified by CjChi18C having the by far highest activity against chitohexaose. Indeed, when combining enzymes, synergistic effects on chitin degradation efficiency were observed. Transcriptomic analysis showed that the four GH18 chitinases and a chitin-active LPMO, CjLPMO10A, are strongly up-regulated when C. japonicus grows on chitin, along with several other putatively chitin-active enzymes as well as a few proteins of unknown function, which are up-regulated to a lesser extent. Serratia marcescens produces one the best studied chitinolytic machineries, involving three chitinases, a lytic polysaccharide monooxygenase, and a chitobiase. However, the genome sequence of one of the most frequently studied S. marcescens strains was not available at the start of this thesis work, and possible involvement of other proteins in chitin utilization had not been investigated. Paper IV describes the genome sequence of S. marcescens BJL200 and a proteomics investigation of proteins secreted during growth on chitin. The genome sequence showed that S. marcescens encodes a fourth chitinase, SmChiD, but the proteomics data indicated that this chitinase is not important in chitin utilization. Indeed, biochemical characterization of SmChiD supported the notion that this enzyme is not important for chitin conversion and, thus, likely has another, yet unknown, biological role. Taken together, the results presented in this thesis provide novel insight into chitin-active enzymes encoded by bacteria. Paper I provides insights into the substrate binding of CE4 deacetylases with an open active site. Papers II-IV reveal chitin-active enzymes, in particular hydrolases, that play key roles in natural chitin conversion. Additionally, Papers II-IV yield a list with proteins without an obvious role in chitin degradation, which may be targeted in future studies of the degradation of chitin-rich biomasses. Further studies on tailoring CE4 deacetylases for modification of chitosan and CHOS and on more efficient chitin conversion using enzymes derived from S. marcescens and C. japonicus are currently in progress

The gastric mucosa of Atlantic salmon (Salmo salar) is abundant in highly active chitinases

Atlantic salmon (Salmo salar) possesses a genome containing 10 genes encoding chitinases, yet their functional roles remain poorly understood. In other fish species, chitinases have been primarily linked to digestion, but also to other functions, as chitinase‐encoding genes are transcribed in a variety of non‐digestive organs. In this study, we investigated the properties of two chitinases belonging to the family 18 glycoside hydrolase group, namely Chia.3 and Chia.4, both isolated from the stomach mucosa. Chia.3 and Chia.4, exhibiting 95% sequence identity, proved inseparable using conventional chromatographic methods, necessitating their purification as a chitinase pair. Biochemical analysis revealed sustained chitinolytic activity against β‐chitin for up to 24 h, spanning a pH range of 2 to 6. Moreover, subsequent in vitro investigations established that this chitinase pair efficiently degrades diverse chitin‐containing substrates into chitobiose, highlighting the potential of Atlantic salmon to utilize novel chitin‐containing feed sources. Analysis of the gastric matrix proteome demonstrates that the chitinases are secreted and rank among the most abundant proteins in the gastric matrix. This finding correlates well with the previously observed high transcription of the corresponding chitinase genes in Atlantic salmon stomach tissue. By shedding light on the secreted chitinases in the Atlantic salmon's stomach mucosa and elucidating their functional characteristics, this study enhances our understanding of chitinase biology in this species. Moreover, the observed capacity to effectively degrade chitin‐containing materials implies the potential utilization of alternative feed sources rich in chitin, offering promising prospects for sustainable aquaculture practices

A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin

publishedVersio

Structure and function of a CE4 deacetylase isolated from a marine environment

Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family

A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin

publishedVersio

Structure-based sequence alignment of CE4 deacetylases.

<p>The structure-based sequence alignment was obtained using PyMod 1.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref043" target="_blank">43</a>]. Fully conserved residues are shown on a green background. The asterisks indicate residues involved in metal binding (blue) and in catalysis (pink). MT1-5 indicate the five conserved motifs in CE4 deacetylases. Colored horizontal bars indicate the different loops described by Andrés et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref016" target="_blank">16</a>]. The deacetylases included in the alignment are: <i>Sp</i>PgdA, PDB id 2C1G [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref024" target="_blank">24</a>]; <i>Cl</i>CDA, PDB id 2IW0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref034" target="_blank">34</a>]; <i>An</i>CDA, PDB id 2Y8U [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref015" target="_blank">15</a>]; <i>Sl</i>CE4, PDB id 2CC0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref033" target="_blank">33</a>]; <i>Bs</i>PdaA, PDB id 1W17 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref044" target="_blank">44</a>]; <i>Vc</i>CDA, PDB id 4NY2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref016" target="_blank">16</a>]. For clarity, the alignment only shows the sequence area of the five motifs and the loops. Sequence numbering is based on the primary gene product, including the signal peptide for the proteins harboring a signal peptide.</p

Activity of <i>Ar</i>CE4A against different substrates.

<p>Activity of <i>Ar</i>CE4A against different substrates.</p

Cartoon representation of the ArCE4A showing the disrupted (β/α)8 barrel topology.

<p>The N- and C-terminus of the protein are marked and the metal ion in the active site is shown as a brown sphere, with the metal coordinating triad in sticks.</p

Structure of <i>Ar</i>CE4A determined by X-ray crystallography.

<p>(a) The His-His-Asp metal binding triad and the catalytic base (in sticks, PDB id: 5LFZ) with the Ni²⁺ ion as brown sphere. The Ni²⁺ ion shows octahedral coordination involving three amino acids and three water molecules (red spheres); interactions are shown as black dashed lines with distances in Å. The water molecule interacting with Asp55 is proposed to act as a nucleophile attacking the carbonyl carbon in the acetyl group. (b) Electron density map of the (GlcNAc)₂ ligand. This illustrates the lack of electron density for the remainder of the tetramer used in the co-crystallization. (c) <i>Ar</i>CE4A in complex with (GlcNAc)₂ (PDB id: 5LCG) showing active site with the ligand bound in subsites 0 and +1 (grey carbons). Residues involved in substrate binding and catalysis are shown as sticks (purple carbons). Interactions between the protein and the substrate are shown as dashed lines in pink with distances in Å. (d) Superposition of <i>Ar</i>CE4A (purple carbons) and <i>Cl</i>CDA (green carbons; PDB id: 2IW0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187544#pone.0187544.ref034" target="_blank">34</a>]), showing the extra loop containing Trp79 and nearby Phe53 (in sticks) in <i>Cl</i>CDA in what could be subsite -2. Subsites occupied by the ligand are labeled 0 and +1. (e) Cross-eyed stereo view of a superposition of the two structures (5LFZ in teal, 5LCG in purple) showing the active site cleft, and how the Ni²⁺ ion (brown sphere) and the three water molecules (red spheres) in 5LFZ are located relative to (GlcNAc)₂ in 5LCG. Interactions involving the Ni²⁺ ion are shown as dashed black lines. Interactions between the proposed nucleophilic water and Asp55 and the carbonyl carbon in the acetyl group are shown as pink dashed lines.</p