Chitin Microfibrils Research Articles

Production and characterization of Trichoderma asperellum chitinases and their use in synergy with Bacillus thuringiensis for lepidopteran control.

Despite their known negative effects on ecosystems and human health, synthetic pesticides are still largely used to control crop insect pests. Currently, the biopesticide market for insect biocontrol mainly relies on the entomopathogenic bacterium Bacillus thuringiensis (Bt). New biocontrol tools for crop protection might derive from fungi, in particular from Trichoderma spp., which are known producers of chitinases and other bioactive compounds able to negatively affect insect survival. In this study, we first developed an environmentally sustainable production process for obtaining chitinases from Trichoderma asperellum ICC012. Then, we investigated the biological effects of this chitinase preparation - alone or in combination with a Bt-based product - when orally administered to two lepidopteran species. Our results demonstrate that T. asperellum efficiently produces a multi-enzymatic cocktail able to alter the chitin microfibril network of the insect peritrophic matrix, resulting in delayed development and larval death. The co-administration of T. asperellum chitinases and sublethal concentrations of Bt toxins increased larval mortality. This synergistic effect was likely due to the higher amount of Bt toxins that passed the damaged peritrophic matrix and reached the target receptors on the midgut cells of chitinase-treated insects. Our findings may contribute to the development of an integrated pest management technology based on fungal chitinases that increase the efficacy of Bt-based products, mitigating the risk of Bt-resistance development. © 2024 Society of Chemical Industry.

Advances in understanding insect chitin biosynthesis

Chitin, a natural polymer of N-acetylglucosamine chains, is a principal component of the apical extracellular matrix in arthropods. Chitin microfibrils serve as structural components of natural biocomposites present in the extracellular matrix of a variety of invertebrates including sponges, molluscs, nematodes, fungi and arthropods. In this review, we summarize the frontier advances of insect chitin synthesis. More specifically, we focus on the chitin synthase (CHS), which catalyzes the key biosynthesis step. CHS is also known as an attractive insecticidal target in that this enzyme is absent in mammals, birds or plants. As no insect chitin synthase structure have been reported so far, we review recent studies on glycosyltransferase domain structures derived from fungi and oomycetes, which are conserved in CHS from all species containing chitin. Auxiliary proteins, which coordinate with CHS in chitin biosynthesis and assembly, are also discussed.

Preparation of Flexible Calcium Carbonate by In Situ Carbonation of the Chitin Fibrils and Its Use for Producing High Loaded Paper.

Flexible calcium carbonate (FCC) was developed as a functional papermaking filler for high loaded paper, which was a fiber-like shaped calcium carbonate produced from the in situ carbonation process on the cellulose micro-or nanofibril surface. Chitin is the second most abundant renewable material after cellulose. In this study, a chitin microfibril was utilized as the fibril core for making the FCC. Cellulose fibrils for the preparation of FCC were obtained by fibrillation of the TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) treated wood fibers. The chitin fibril was obtained from the β-chitin from the born of squid fibrillated in water by grinding. Both fibrils were mixed with calcium oxide and underwent a carbonation process by the addition of carbon dioxide, thus the calcium carbonate attached on the fibrils to make FCC. When used in papermaking, both the FCC from chitin and cellulose gave a much higher bulk and tensile strength simultaneously than the conventional papermaking filler of ground calcium carbonate, while maintaining the other essential properties of paper. The FCC from chitin caused an even higher bulk and higher tensile strength than those of the FCC from cellulose in paper materials. Furthermore, the simple preparation method of the chitin FCC in comparison with the cellulose FCC may enable a reduction in the use of wood fibers, process energy, and the production cost of paper materials.

PREPARATION OF NANOCRYSTALS AND CHITIN MICROFIBRILS

Chitin nanocrystals and microfibrils were prepared based on the strong acid hydrolysis method. The change in the molecular structure was studied by Fourier-transform infrared and Raman spectroscopy. The surface morphology of chitin materials was observed using scanning electron and optical microscopy. The separation of nano-crystals and microfibril crystalline regions from native chitin may increase the scope of its application for the creation of bioactive composites and scaffolds for tissue culture. As a result of the conducted experimental work, it was possible to loosen the amorphous areas and partially separate nano-crystals and chitin microfibrils. Stable aqueous suspensions of chitin nano-crystals and microfibrils were obtained, which can be used for further experimental work.

Biomimetic Chiral Photonic Materials with Tunable Metallic Colorations Prepared from Chiral Melanin-like Nanorods for UV Shielding, Humidity Sensing, and Cosmetics.

Many biological species combine the helical organization of cellulose or chitin microfibrils with broadband light absorption of black melanin to produce brilliant structural colors with metallic and glossy effects and other diverse functions. In this work, based on core-shell CNC@PDA chiral nanorods consisting of cellulose nanocrystals (CNCs) as the core and melanin-like polydopamine (PDA) as the shell that can form well-defined chiral liquid crystal phases, we report chiral photonic materials that closely mimic the unique coloration mechanisms and functionalities mastered by several biological species. The photonic films formed by such single CNC@PDA nanorods have brilliant iridescent structural colors originating from selective reflection of circularly polarized lights by the helical organization of CNC@PDAs across the films. Furthermore, the colors of such films have background-independent brightness, high visibility, and metallic effects that arise from the light absorption of the PDA component. Especially, the color ranges and metallic effects of the films can be conveniently tuned by varying the thickness of the PDA shell. In addition, the UV absorption and hygroscopic properties of PDA endow these CNC@PDA films with efficient broadband UV shielding and sensitive humidity-induced dynamic color changes. Due to the mussel-like superior adhesion of PDA, CNC@PDA-based photonic coatings can be formed conformably onto diverse kinds of substrates. A shiny eye shadow with viewing angle-dependent colorful patterns was used to demonstrate the potential applications. With combinations of multiple unique properties in one photonic material fabricated from a single building block, these CNC@PDA-based films are expected to have potential applications in cosmetics, UV protection, anticounterfeiting, chiral reflectors, etc.

Preparation of Composite Materials from Self-Assembled Chitin Nanofibers.

Although chitin is a representative abundant polysaccharide, it is mostly unutilized as a material source because of its poor solubility and processability. Certain specific properties, such as biodegradability, biocompatibility, and renewability, make nanofibrillation an efficient approach for providing chitin-based functional nanomaterials. The composition of nanochitins with other polymeric components has been efficiently conducted at the nanoscale to fabricate nanostructured composite materials. Disentanglement of chitin microfibrils in natural sources upon the top-down approach and regeneration from the chitin solutions/gels with appropriate media, such as hexafluoro-2-propanol, LiCl/N, N-dimethylacetamide, and ionic liquids, have, according to the self-assembling bottom-up process, been representatively conducted to fabricate nanochitins. Compared with the former approach, the latter one has emerged only in the last one-and-a-half decade. This short review article presents the preparation of composite materials from the self-assembled chitin nanofibers combined with other polymeric substrates through regenerative processes based on the bottom-up approach.

Behavioral and ultrastructural effects of novaluron on Aedes aegypti larvae.

Chitin synthesis inhibitors (CSI) are supposed to inhibit formation of chitin microfibrils in newly synthesized cuticle during molting process. Conversely, there has been comparatively few data on morphological effects of CSI on non-target insect organs. In this work, the effects of the CSI novaluron on behavior and midgut of A. aegypti were evaluated. Toxicity bioassays revealed that novaluron is toxic to A. aegypti larva with LC50=18.57mgL-1 when exposed in aqueous solution for 24h. Novaluron treated larvae were less active and spent more time resting compared to the control group. Histopathology showed that midguts of novaluron-treated larvae had cytoplasm vacuolization and damaged brush border. Cytotoxic effects in midguts of treated larvae induced necrosis, autophagy and damage to mitochondria. Despite being chitin synthesis inhibitor, novaluron did not induce alterations in the integument of A. aegypti larvae. Fluorescence microscopy revealed that the number of digestive cells were higher in novaluron-treated larvae than in control, in response to digestive cell apoptosis. The present study highlights the importance of novaluron against A. aegypti larvae by causing injuries to non-target organs, altering behaviors, inducing cell death and inhibiting cell proliferation.

AOP Report: Inhibition of Chitin Synthase 1 Leading to Increased Mortality in Arthropods.

AOP Report: Inhibition of Chitin Synthase 1 Leading to Increased Mortality in Arthropods.

Formulation of an exoskeleton degrading bacterial consortium from seafood processing effluent for the biocontrol of crustacean parasite Alitropus typus

Parasitic infestations on cultured fish due to the crustacean isopod Alitropus typus has been on the rise in recent years, causing large scale mortality, leading to significant economic loss to the farmer. Crustaceans are encased by an exoskeleton composed of chitin, protein and lipid microfibril frameworks, in which calcium carbonate is deposited. A strategy focused on the degradation of the exoskeletal framework utilizing nonpathogenic microorganisms that produce a wide variety of hydrolytic enzymes may be an environment-friendly and safe alternative to control these pests. The present study was aimed to formulate a microbial consortium having chitinase, protease, lipase and urease producing bacteria from seafood processing effluents that can potentially degrade the exoskeleton of A. typus. Based on the qualitative and quantitative assessment of the extracellular enzymes produced by the isolates, a novel consortium was prepared with three strains that were not antagonistic to each other and were nonpathogenic. The chitinase producing - Stenotrophomonas maltophilia and Bacillus altitudinis that produced protease and lipase as well; and non-chitinase producing Klebsiella pneumoniae were taken in the ratio of 1:1:2 respectively (109 CFU/mL). The result showed 100 % mortality of the isopods within five days when applied at a concentration of 2% (v/v) of 107 CFU/mL without any adverse effect on the fish host Oreochromis niloticus. Analysis of the ultrastructural alterations of the parasites by Environmental Scanning Electron microscopy (ESEM) showed noticeable exoskeletal damages. The microbial members of the consortium displayed remarkable chemotactic properties towards A. typus. The results suggest that the microbial consortium acts as a potential parasiticide that can be used for the control of A. typus infestation in aquaculture ponds., thus benefitting the aquaculture industry especially the small-scale farmers.

Scalar nanostructure of the Candida albicans cell wall; a molecular, cellular and ultrastructural analysis and interpretation

Despite the importance of fungal cell walls as the principle determinant of fungal morphology and the defining element determining fungal interactions with other cells, few scalar models have been developed that reconcile chemical and microscopic attributes of its structure. The cell wall of the fungal pathogen Candida albicans is comprised of an amorphous inner skeletal layer of β(1,3)- and β(1,6)-glucan and chitin and an outer fibrillar layer thought to be dominated by highly mannosylated cell wall proteins. The architecture of these two layers can be resolved at the electron microscopy level, but the visualised structure of the wall has not yet been defined precisely in chemical terms. We have therefore examined the precise structure, location and molecular sizes of the cell wall components using transmission electron microscopy and tomography and tested predictions of the cell wall models using mutants and agents that perturb the normal cell wall structure. We demonstrate that the fibrils are comprised of a frond of N-linked outer chain mannans linked to a basal layer of GPI-proteins concentrated in the mid-wall region and that the non-elastic chitin microfibrils are cantilevered with sufficient lengths of non-fibrillar chitin and/or β-glucan to enable the chitin-glucan cage to flex, e.g. during morphogenesis and osmotic swelling. We present the first three-dimensional nano-scalar model of the C. albicans cell wall which can be used to test hypotheses relating to the structure–function relationships that underpin the pathobiology of this fungal pathogen.

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea.

The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by β-1,6-branched β-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and β-1,3-glucans under turgor pressure in vivo.

Preparation and properties of microfibrillated chitin/gelatin composites.

Microfibrillated chitin/gelatin composite films were prepared by solvent casting method, and the nano-sized microfibrillated chitin as reinforce phase to improve oxygen resistance, water-resistant and mechanical performance in this system. The morphologies were analyzed by scanning electron microscope (SEM), and the mechanical properties were investigated by texture analyzer. Oxygen permeability property, optical property and swelling property were investigated. The results indicated that the elastic modulus and tensile strength of microfibrillated chitin/gelatin composite film reached 2.2 GPa and 74.5 MPa respectively when the content of microfibrillated chitin is 8 wt%. The swelling ratio decreased to 11.63 with the 6 wt% content of microfibrillated chitin. In addition, chitin microfibrils effectively enhanced the oxygen resistance of composite film without obvious loss of transmittance. This work expects to provide a pathway to improve gelatin performance.

Microscopy of crustacean cuticle: formation of a flexible extracellular matrix in moulting sea slaters Ligia pallasii

AbstractStructural and functional properties of exoskeleton in moulting sea slaters Ligia pallasii from the Eastern Pacific coast were investigated with CT scanning and electron microscopy. Ultrastructure of preecdysial and postecdysial cuticular layers was described in premoult, intramoult and postmoult animals. Cuticle is a flexible extracellular matrix connected to the epidermal cells through pore channels. During premoult epicuticle and exocuticle are formed and during intramoult and postmoult endocuticular lamellae are deposited and the cuticle is progressively constructed by thickening and mineralization. Cuticle permeability, flexibility and waterproofing capacity change accordingly. Elaboration of epicuticular scales connected to an extensive network of nanotubules, establish its anti-adhesive and hydrophobic properties. Labelling with gold conjugated WGA lectins on Tokuyashu thawed cryosections exposes differences in chitin content between exocuticle and endocuticle. Histochemical staining of cuticle shows presence of acidic carbohydrates/glycoconjugates and lipoproteins in epicuticular layer. Chitin microfibrils are formed at the microvillar border of epidermal cells with abundant Golgi apparatus and secretory vesicles. Numerous spherules associated with nanotubules were observed in the ecdysial space in intramoult animals. The mineral component of the cuticle as visualized with CT scanning indicates progressive mineral resorption from the posterior to the anterior half of the body in premoult animals, its translocation from the anterior to posterior part during intramoult and its progressive deposition in the posterior and anterior exoskeleton during postmoult. Cuticle of sea slaters is a unique biocomposite and biodynamic material constantly reconstructed during frequent moults, and adapted to specific physical and biotic conditions of the high intertidal rocky zone.

Chitin distribution in the Oithona digestive and reproductive systems revealed by fluorescence microscopy.

Among copepods, which are the most abundant animals on Earth, the genus Oithona is described as one of the most numerous and plays a major role in the marine food chain and biogeochemical cycles, particularly through the excretion of chitin-coated fecal pellets. Despite the morphology of several Oithona species is well known, knowledge of its internal anatomy and chitin distribution is still limited. To answer this problem, Oithona nana and O. similis individuals were stained by Wheat Germ Agglutinin-Fluorescein IsoThioCyanate (WGA-FITC) and DiAmidino-2-PhenylIndole (DAPI) for fluorescence microscopy observations. The image analyses allowed a new description of the organization and chitin content of the digestive and reproductive systems of Oithona male and female. Chitin microfibrils were found all along the digestive system from the stomach to the hindgut with a higher concentration at the peritrophic membrane of the anterior midgut. Several midgut shrinkages were observed and proposed to be involved in faecal pellet shaping and motion. Amorphous chitin structures were also found to be a major component of the ducts and seminal vesicles and receptacles. The rapid staining protocol we proposed allowed a new insight into the Oithona internal anatomy and highlighted the role of chitin in the digestion and reproduction. This method could be applied to a wide range of copepods in order to perform comparative anatomy analyses.

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 μm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M μm and 8.1 × 10-10 M μm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

Involvement of cecropin B in the formation of the Aedes aegypti mosquito cuticle

In this study, we found a mosquito antimicrobial peptide (AMP), Aedes aegypti cecropin B (Aacec B), was expressed constitutively in pupae. Knockdown in the pupae of Aacec B using double-stranded RNA (dsRNA) resulted in high mortality, the emergence of deformed adults and an impairment of pharate adult cuticle formation with fewer lamellae being deposited and the helicoidal pattern of the chitin microfibrils being disorganized. Simultaneous injection of Aacec B dsRNA and Aacec B peptide into pupae significantly reduced this mortality and no deformed adults then emerged. The expression levels of Ae. aegypti prophenoloxidase (AaPPO) 3 and AaPPO 4 were significantly reduced in the Aacec B knockdown pupae. Exogenous Aacec B peptide significantly enhanced the transcription of AaPPO 3 in pupae. Knockdown of AaPPO 3 in pupae caused effects similar to Aacec B-knockdown. The Aacec B peptide could be detected in both the cytoplasm and nuclei of pupal cells and was able to bind to the TTGG(A/C)A motif in AaPPO 3 DNA both in vitro and in vivo. These findings suggest that Aacec B plays a crucial role in pharate adult cuticle formation via the regulation of AaPPO 3 gene expression in pupae.

Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation

The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects.

Fabrication and mechanical properties of composite based on β-chitin and polyacrylic acid

Squid β-chitin has been exfoliated in aqueous acrylic acid (AA), after which a composite film of chitin microfibrils in polyacrylic acid (PAA) has been prepared by in situ polymerization of the AA. The segregated chitin fibrils in the composite are 4–6nm in diameter, with an aspect ratio >250. After drying cast films of the composites containing 1, 2 and 3% (w/w) chitin at 140°C for four hours, there was a dramatic resistance to swelling in water, in that the dried films showed only small changes in shape and properties after four hours immersed in water.The most profound impact of the reinforcement on the mechanical properties is observed at high relative humidity (RH), when the PAA is in the rubbery state. At 97.5% RH and room temperature, the elastic moduli of the composites with 1, 2 and 3% (w/w) chitin were 150, 230 and 2100MPa respectively, compared to 65MPa for pure PAA. The main contribution to the filler-reinforcing effect is the high aspect ratio of fibrils and non-covalent interactions, but the stability in water suggests the presence of chemical bonding between the PAA and chitin.

Helicoidal Organization of Chitin in the Cuticle of the Migratory Locust Requires the Function of the Chitin Deacetylase2 Enzyme (LmCDA2)

In the three-dimensional extracellular matrix of the insect cuticle, horizontally aligned microfibrils composed of the polysaccharide chitin and associated proteins are stacked either parallel to each other or helicoidally. The underlying molecular mechanisms that implement differential chitin organization are largely unknown. To learn more about cuticle organization, we sought to study the role of chitin deacetylases (CDA) in this process. In the body cuticle of nymphs of the migratory locust Locusta migratoria, helicoidal chitin organization is changed to an organization with unidirectional microfibril orientation when LmCDA2 expression is knocked down by RNA interference. In addition, the LmCDA2-deficient cuticle is less compact suggesting that LmCDA2 is needed for chitin packaging. Animals with reduced LmCDA2 activity die at molting, underlining that correct chitin organization is essential for survival. Interestingly, we find that LmCDA2 localizes only to the initially produced chitin microfibrils that constitute the apical site of the chitin stack. Based on our data, we hypothesize that LmCDA2-mediated chitin deacetylation at the beginning of chitin production is a decisive reaction that triggers helicoidal arrangement of subsequently assembled chitin-protein microfibrils.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.