Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.)

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree (Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC₅₀-values as low as 1 μg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.     

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin

Chemically prepared hevein domains (HDs), N-terminal domain of an antifungal protein from Nicotiana tabacum (CBP20-N) and an antimicrobial peptide from Amaranthus caudatus (Ac-AMP2), were examined for their affinity for chitin, a beta-1,4-linked polymer of N-acetylglucosamine. An intact binding domain, CBP20-N, showed a higher affinity than a C-terminal truncated domain, Ac-AMP2. The formation of a pyroglutamate residue from N-terminal Gln of CBP20-N increased the affinity. The single replacement of any aromatic residue of Ac-AMP2 with Ala resulted in a significant reduction in affinity, suggesting the importance of the complete set of three aromatic residues in the ligand binding site. The mutations of Phe18 of Ac-AMP2 to the residues with larger aromatic rings, i.e. Trp, beta-(1-naphthyl)alanine or beta-(2-naphthyl)alanine, enhanced the affinity, whereas the mutation of Tyr20 to Trp reduced the affinity. The affinity of an HD for chitin might be improved by adjusting the size and substituent group of stacking aromatic rings.     

H NMR study of the interaction of N,N′,N″-triacetyl chitotriose with Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus

The interaction between Ac-AMP2, a lectin-like small protein with antimicrobial and antifungal activity isolated from Amaranthus caudatus, and N,N′,N″-triacetyl chitotriose was studied using ¹H NMR spectroscopy. Changes in chemical shift and line width upon increasing concentration of N,N′,N″-triacetyl chitotriose to Ac-AMP2 solutions at pH 6.9 and 2.4 were used to determine the interaction site and the association constant K_a. The most pronounced shifts occur mainly in the C-terminal half of the sequence. They involve the aromatic residues Phe¹⁸, Tyr²⁰ and Tyr²⁷ together with their surrounding residues, as well as the N-terminal Val-Gly-Glu segment. Several NOEs between Ac-AMP2 and the N,N′,N″-triacetyl chitotriose resonances are reported.     

Antimicrobial peptides fromMirabilis jalapa andAmaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco

The cDNAs encoding the seed antimicrobial peptides (AMPs) fromMirabilis jalapa (Mj-AMP2) andAmaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolleet al., Plant Mol Biol 28:713–721 (1995) and 22:1187–1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195–1206 (1991)]; an Ac-AMP2wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing eitherwild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. Thein vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with eitherBotrytis einerea orAlternaria longipes.     

Evolution of Chitin-Binding Proteins in Invertebrates

Analysis of a group of invertebrate proteins, including chitinases and peritrophic matrix proteins, reveals the presence of chitin-binding domains that share significant amino acid sequence similarity. The data suggest that these domains evolved from a common ancestor which may be a protein containing a single chitin-binding domain. The duplication and transposition of this chitin-binding domain may have contributed to the functional diversification of chitin-binding proteins. Sequence comparisons indicated that invertebrate and plant chitin binding domains do not share significant amino acid sequence similarity, suggesting that they are not coancestral. However, both the invertebrate and the plant chitin-binding domains are cysteine-rich and have several highly conserved aromatic residues. In plants, cysteines have been elucidated in maintaining protein folding and aromatic amino acids in interacting with saccharides [Wright HT, Sanddrasegaram G, Wright CS (1991) J Mol Evol 33:283–294]. It is likely that these residues perform similar functions in invertebrates. We propose that the invertebrate and the plant chitin-binding domains share similar mechanisms for folding and saccharide binding and that they evolved by convergent evolution. Furthermore, we propose that the disulfide bonds and aromatic residues are hallmarks for saccharide-binding proteins. Received: 2 March 1998 / Accepted: 17 July 1998     

The importance of CH/pi interactions to the function of carbohydrate binding proteins

It is suggested that the interactions between the hydrophobic C-H groups of carbohydrate residues and the pi-electron systems of aromatic amino-acid residues play an important role in the ligand-recognition function of carbohydrate-binding proteins. This review focuses on our recent structural and functional studies of human lysozyme and hevein-domain type lectins (wheat-germ agglutinin and Ac-AMP2) aimed at understanding how CH/pi interactions are involved in the actual binding events.     

The solution structure of horseshoe crab antimicrobial peptide tachystatin B with an inhibitory cystine-knot motif.

Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 A, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin.     

Detection of the antimicrobial peptide gene in different Amaranthus species

Using primers to amplify the gene AMP2 in Amaranthus caudatus, we found the gene to be present in seven other species of the Amaranthus genus (A. albus, A. cruentus, A. blitum, A. hybridus, A. hypochondriacus, A. retroflexus and A. tricolor), in which it had not been described previously. The PCR products were sequenced and it was established that all the sequences were identical, except for two polymorphisms. These single nucleotide polymorphisms occurred at nucleotide positions 45 and 246. This exchange of one nucleotide for another was manifested in an amino acid change in both cases. Due to the fact that both polymorphisms lay outside the region encoding the chitin-binding peptide domain, which is crucial for antimicrobial peptide function, they will not likely affect the proper functioning of the peptide. With the exception of the above-mentioned polymorphisms, all sequences were identical to the sequence of the AMP2 gene that codes for the A. caudatus Ac-AMP2 (antimicrobial peptide isolated from Amaranthus caudatus seeds). The detection of sequences with high degree of sequence similarity to A. caudatus AMP2 gene leads us to the assumption that an antimicrobial peptide could also be produced by other amaranth species.     

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.     

Structure of the antimicrobial peptide tachystatin A

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded beta-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist omega-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that omega-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and omega-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and omega-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe(9) of tachystatin A might be an essential residue for binding to chitin.     

Primary structure, isoforms, and molecular modeling of a chitin-binding mistletoe lectin

From mistletoe Viscum album L. extracts three chitin-binding lectin isoforms, cbML1, cbML2, and cbML3, were isolated and their primary structure determined. All three cbML isoforms are composed of two protein chains of 48 or 49 amino acid residues, linked by an intermolecular disulfide bond. The sequence of each single cbML chain is characterized by a relatively high number of cysteine and glycine residues, 9 and 6, respectively, and contains four intramolecular disulfide bridges. On the basis of the combined interpretation of sequencing and MALDI MS data, the following results for the three cbML isoforms were obtained: the first one consists of two identical truncated polypeptide chains (1--48), the second is a heterodimer, containing one truncated (1--48) and one full-length chain (1--49), and the third is composed of two full length chains (1--49). The cbML sequence shows 55% identity to hevein, a single-chain chitin-binding protein of 43 amino acids, one of the most predominant proteins in natural rubber latex. On the basis of the NMR data on hevein from Hevea brasiliensis the three-dimensional structure of cbML3 was modelled. The 26 sequence changes between cbML3 and hevein were accommodated with only little perturbation in the main chain folding. A comparison of the primary structures of cbML3 and hevein is shown and the effects of the sequence changes are discussed. Differences have been identified in the loop region of the molecule and the potential interface region of cbML3 supporting the dimer formation. The high-affinity chitin-binding site seems to be highly conserved.     

A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag

Site-directed mutagenesis was carried out to investigate the roles of a number of highly conserved residues of the chitin-binding domain (ChBD) of Bacillus circulans chitinase A1 (ChiA1) in the binding of chitin. Analysis of single alanine replacement mutants showed that mutation of an exposed tryptophan residue (Trp(687)) impaired the binding to chitin, while mutation of other highly conserved residues, most carrying aromatic or hydrophobic side chains, did not significantly affect the binding activity. Interestingly, replacement of Trp(687) with phenylalanine significantly reduced chitin-binding activity at lower salt concentrations (0-1 M NaCl) but allowed strong binding to chitin at 2 M NaCl. Since Trp(687) is conserved among the ChBDs belonging to the bacterial ChiA1 subfamily, the data presented suggest a general mechanism in which this exposed tryptophan, which is located in the cleft formed between two beta-sheets as revealed by the solution structure [J. Biol. Chem. 275 (2000) 13654], makes a major contribution to ligand binding presumably through hydrophobic interactions. Furthermore, modulation of the chitin-binding activity by the conserved amino acid replacement (W687F) and a shift in the ionic strength of buffer has led to the development of an elutable affinity tag for single column purification of recombinant proteins.     

Potato lectin: a modular protein sharing sequence similarities with the extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors)

Potato (Solanum tuberosum) lectin, is a chimeric chitin-binding protein comprised of a lectin domain fused to a hydroxyproline-rich glycoprotein domain. Here peptide sequence information from both domains is presented. A partial sequence of a major tryptic peptide T2: Leu-Pro-Ser-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-(His)-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp- was similar to the ‘P3’ type extensin major repetitive sequence: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-suggesting common evolutionary origins for the extensins and the hydroxyproline-rich glycoprotein (HRGP) domain of potato lectin. Furthermore, alignment of three chymotryptic peptides from potato lectin, C1: Cys-Gly-Thr-Thr-Ser-Asp-Tyr, C2: Cys-Ser-Pro-Gly-Tyr, and C8: Thr-Gly-Glu-Cys-Cys-Ser-Ile with similar sequences from the hevein lectin family indicates that they have homologous chitin-binding domains, and hence have common evolutionary origins. Finally, all plant chitin-binding domains examined bore a remarkable sequence similarity, particularly in the spacing of Cys residues, to the disintegrins (platelet aggregation inhibitors) which occur in crotalid and viperid snake venoms. As such, sequence similarities not only identify potato lectin as a member of both the hevein and extensin families of plant proteins, but also suggest that an archetypal polypeptide module gave rise to both the plant chitin-binding domain and the reptile disintegrins.     

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.     

Cys/Gly-rich proteins with a putative single chitin-binding domain from oat (Avena sativa) seeds

Through a reliable and repeatable procedure based on solid-phase extraction techniques, a protein fraction (P fraction) rich in Cys/Gly residues was extracted and captured from oat (Avena sativa L.) seeds. Quantitative amino acid analysis and MS of the P fraction indicated that it contains a series of heterogeneous Cys/Gly-rich proteins with molecular masses of 3.6-4.0 kDa. Preliminary results from bioassays showed that these proteins possess weak to moderate antifungal properties to some fungal strains. From this fraction, a new polypeptide, designated avesin A, was purified and sequenced by Edman degradation. Avesin A consists of 37 amino-acid residues, with 10 glycine residues and eight cysteine residues forming disulfide bridges, and contains a single chitin-binding domain, which indicates that avesin A is a new member of the putative chitin-binding proteins. Avesin A is the first identified hevein-like small protein from cereal grains.     

Chitin-binding proteins in invertebrates and plants comprise a common chitin-binding structural motif

Tachycitin, a 73-residue polypeptide having antimicrobial activity is present in the hemocyte of horseshoe crab (Tachypleus tridentatus). The first three-dimensional structure of invertebrate chitin-binding protein was determined for tachycitin using two-dimensional nuclear magnetic resonance spectroscopy. The measurements indicate that the structure of tachycitin is largely divided into N- and C-terminal domains; the former comprises a three-stranded beta-sheet and the latter a two-stranded beta-sheet following a short helical turn. The latter structural motif shares a significant tertiary structural similarity with the chitin-binding domain of plant chitin-binding protein. This result is thought to provide faithful experimental evidence to the recent hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.     

Production and characterization of recombinant tachycitin,the Cys-rich chitin-binding protein

Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The (1)H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929-17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, (15)N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone (15)N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.     

Functional analyses of the chitin-binding domains and the catalytic domain of <Emphasis Type="Italic">Brassica juncea</Emphasis> chitinase BjCHI1

We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar Pieris rapae feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2, showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using -mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by -mannosidase. BjCHI1s ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0 of wild-type specific activity. H211N and R361A resulted in considerable (>91) activity loss, implying these charged residues are also important in catalysis. E234A showed 36 retention of activity and substitution Y269D, 50. The least affected mutants were E349A and D360A, with 73 and 68 retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.     

萝卜块根中两个具溶菌酶活性的几丁质结合蛋白的纯化及其特性

通过亲和层析和羧甲基一纤维素离子交换层析从萝卜的块根中分离到两个具溶菌酶活性的酶组份：CBP1和CBP2。两者经SDS-PAGE均显示单一蛋白染色条带,其对应的分子量分别为26．9kD和24．8kD。两种蛋白除有溶菌酶活性外,还有几丁质酶活性,但无壳聚糖酶活性。各种类型的几丁质对CBP1和CBP2都有较强的吸附作用,而在还原／非还原的单向SDS-PAGE中却观察不到两者分子中存在二硫键。     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.