
==== Front
J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02123-9
10.1016/j.jbc.2024.107622
107622
Research Article
Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII
Qu Mingbo 12
Guo Xiaoxi 1
Ando Toshio tando@staff.kanazawa-u.ac.jp
2∗
Yang Qing qingyang@caas.cn
34∗
1 MOE Key Laboratory of Bio-Intelligent Manufacturing, School of Bioengineering, Dalian University of Technology, Dalian, China
2 Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan
3 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
4 Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China
∗ For correspondence: Toshio Ando; Qing Yang tando@staff.kanazawa-u.ac.jpqingyang@caas.cn
03 8 2024
9 2024
03 8 2024
300 9 10762212 6 2024
17 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation has been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Keywords

chitin
carbohydrate-binding module
chitinase
HS-AFM
multi-modular
single-molecule
Abbreviations

β-MPs β-chitin microparticles

β-NCs β-chitin nanocrystals

β-NFs β-chitin nanofibers

CD catalytic domains

CBM carbohydrate-binding module

HS-AFM high-speed atomic force microscopy

OfChtII chitinase II from Ostrinia furnacalis

SEM scanning electron microscopy

TEM transmission electron microscopy

Reviewed by members of the JBC Editorial Board. Edited by Robert Haltiwanger
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pmcChitin, a water-insoluble crystalline fibril comprised of β-1,4-linked N-acetylated glucosamine (GlcNAc) units, serves as an indispensable constituent of crustacean shells, insect exoskeletons, and the cell walls of fungi. Chitin is identified as the second most ubiquitous carbohydrate polymer in nature, second only to cellulose, and its annual production is estimated to be around 100 billion tons (1). Chitin is expected to find diverse applications in a wide range of fields, including agriculture, food, paper, textiles, cosmetics, and the biomedical industry (2). However, its physical and chemical stability renders it recalcitrant to degradation, leading to the disposal of most chitin as waste (3). Overcoming its recalcitrance is a bottleneck for efficient utilization of chitin-containing biomass.

The enzymatic degradation of recalcitrant chitin has emerged as an attractive process due to its eco-friendly nature. The chitin degradation system has been identified in organisms that utilize (4) or contain chitin (5). It typically comprises chitinases, which are assisted by lytic polysaccharide monooxygenases (6) and β-N-acetylglucosaminidases (7). The key chitinases involved are endo-acting chitinases and processive exo-acting chitinases (8). Endo-acting chitinases attack chitin chains randomly, while the processive exo-acting chitinases attack either the reducing end or the non-reducing end of chitin chains and consecutively repeat the catalytic cycle for certain times without release of its substrate (9, 10, 11). The chitin degradation systems from the bacterial Serratia marcescens (10, 12, 13, 14) and insect Ostrinia furnacalis (15, 16, 17) are the most well-studied ones. They both possess a chitinase cocktail containing both endo- and exo-acting chitinases. The major difference between the two chitin degradation systems is the presence of a multi-modular endo-acting chitinase (ChtII) in insects, in contrast to a non-reducing end processive exo-acting chitinase (ChiB) in bacteria.

The insect ChtIIs contain 3 to 5 glycoside hydrolase family 18 (GH18) catalytic domains (CD) and 5 to 7 carbohydrate-binding modules of family 14 (CBM14s). The GH18 CD catalyzes the biodegradation of β-1,4-glycosidic bonds in amino polysaccharides via a substrate-assisted retention mechanism (7). The CBM14s are short modules of approximately 70 residues belonging to the type C class of CBMs. They are abundant across all domains of life and have been found to bind explicitly to chitin (18). ChtIIs are highly conserved in insects and have been proven to be essential for insect development (5, 19, 20). The two active CDs (OfChtII-C1 and OfChtII-C2) of OfChtII from O. furnacalis have been demonstrated to follow an endo-acting catalytic mechanism according to their 3D crystal structures, and no synergistic action between these CDs was observed (16). The addition of three CBMs to the N-terminus of CD to form multi-modular OfChtII-B4C1 truncation exhibits higher activity compared to OfChtII-C1 (16, 21). Besides ChtII, multi-modular endo-acting chitinases are also widely distributed in microorganisms, such as ChiW from Paenibacillus sp. strain FPU-7 (22), ChiA74 from Bacillus thuringiensis (23), CsChiG from Chitiniphilus shinanonensis (24), and Chitinase A from Stenotrophomonas maltophilia (25). A few studies into the biochemical properties of these chitinases have proved that they may be particularly powerful in degrading chitin.

One important feature of these chitinases is that they possess multiple CBMs. The roles of CBMs have been investigated by using deletion derivatives lacking one or all of them. The loss of the C-terminal CBM of chitinase A1 from Bacillus circulans WL-12 deprives the enzyme of the ability to bind to insoluble chitin and significantly reduces the ability to hydrolyze colloidal chitin (26). The deletion of the C-terminal CBM of ChiA74 from B. thuringiensis reduces the enzymatic activity and substrate binding for colloidal chitin (16). The removal of two N-terminal CBMs of ChiG from C. shinanonensis reduces the enzymatic activity for α-chitin and colloidal chitin by 4 to 5 folds (24). The deletion of CBMs at the C-terminus of Chi92 from Aeromonas hydrophila JP101 reduces the adhesion and catalytic activity for unprocessed and colloidal chitins (27). The deletion of CBM from human chitotriosidase decreases its activity toward chitin (28). The truncation containing only the CD of chitinase 5 from Manduca sexta exhibits lower activity toward the insoluble polymer compared to the full length (29). Besides endo-acting chitinases, the CBMs are also proven to facilitate the catalysis of exo-acting processive chitinases like SmChiA and SmChiB (30, 31, 32). All these studies demonstrate that the main role of CBMs is to increase the affinity to chitin, thus promoting the hydrolytic activity of chitinases. However, a deeper understanding of how CBMs elevate the binding ability and catalytic activity of chitinases is still very limited.

In the present study, we investigated the functions of CBMs in a multi-modular endo-acting OfChtII from O. furnacalis, and then clarified the mechanism by which OfChtII deconstructs chitin. OfChtII truncations containing different numbers of CBMs were constructed and their activities and interactions with different chitin substrates were compared with each other using biochemical and nanoscale analyses. The results revealed that CBMs significantly enhanced the catalytic and binding activity of OfChtII by increasing substrate association without affecting the dissociation process. Notably, the OfChtII-B4C1 truncation containing three CBMs was proved to be more active in chitin deconstruction. Microscopic analyses further demonstrated that it preferentially cut surface-exposed fibers at their bent portions, resulting in mobile fiber segments released from the composite structure. Consequently, the fibrillar organization in the attacked regions became destabilized and more vulnerable to degradation, leading to the deconstruction of chitin. It is proposed that OfChtII-B4C1 follows a fragment-generating mechanism during chitin degradation, which provides important information for designing highly efficient endo chitinases as well as other glycoside hydrolases.

Results

Impact of CBM on the biochemical properties of OfChtII truncations

Three truncated OfChtIIs from O. furnacalis containing zero (OfChtII-C1), one (OfChtII-B6C1), and three CBMs (OfChtII-B4C1) at the N-terminus of CD were designed (Fig. 1A) and recombinantly expressed using Pichia pastoris. These recombinant proteins were purified to homogeneity (Fig. 1B) and their properties were compared to each other to investigate the impact of CBMs on the activity of chitinases.Figure 1 OfChtII truncations and their propertiestoward β-chitin microparticles.A, modular structural organization of OfChtII and its truncations. B, SDS-PAGE analysis of purified OfChtII truncations. M: standard protein marker; B4C1: OfChtII-B4C1; B6C1: OfChtII-B6C1; C1: OfChtII-C1. C, time course of total reducing sugars generated by OfChtII truncations during hydrolysis of β-chitin microparticles. D, Binding isotherms of OfChtII truncations on β-chitin microparticles.

The β-chitin microparticles (β-MPs) composed of compacted chitin fibers were first used as a substrate. A time-dependent analysis of product accumulation revealed that OfChtII-B4C1, with three CBMs, exhibited the highest hydrolytic activity toward β-MPs among the three OfChtII truncations (Fig. 1C). In contrast, OfChtII-C1, containing only the CD, exhibited the lowest activity. With the extension of incubation time (longer than 12 h), the products generated by OfChtII-C1 tend to plateau, whereas this tendency was reduced with the increasing number of CBMs in the OfChtII truncations (Fig. 1C). Subsequently, the binding amounts of three OfChtII truncations toward β-MPs were assessed by Langmuir isotherm, and they were demonstrated to increase with the augmentation of CBMs (Fig. 1D). These results demonstrated that CBMs enhance both the hydrolytic activity and substrate binding of the OfChtII truncations.

Chitin substrates deconstruction by OfChtII truncations

To further elucidate the significant role of CBMs in modulating the catalytic properties of OfChtII truncations, the β-MPs after hydrolysis were precipitated, washed with distilled water, dehydrated with ethanol, freeze-dried, and then observed using scanning electron microscopy (SEM). The results revealed that, in comparison to the untreated control, OfChtII-C1 could hardly disrupt the compact structure of β-MPs. Only a minuscule amount of fragmented fractions were detected, predominantly located at the edges of the compact particles (Fig. 2, 96 h, arrowhead). In contrast, OfChtII-B6C1 could generate more fragmented fractions than OfChtII-C1. It showed some capacity to break the compact structure of β-MPs, but not as extensively as OfChtII-B4C1 did. Remarkably, OfChtII-B4C1 exhibited the most profound effect. At the same hydrolysis time, it could deconstruct the compact particles more extensively, resulting in a much higher yield of fragmented fractions compared to the other two truncations, OfChtII-C1 and OfChtII-B6C1 (Fig. 2, arrowhead). These fragmented fractions were observed to be dispersed throughout the samples or located at the boundaries of the compacted particles. At an extended incubation time, these phenomena were more pronounced, and more fragmented fractions could be observed (Fig. 2, 96 h).Figure 2 Ultrastructure changes of β-chitin microparticles after hydrolysis by OfChtII truncations.A, β-chitin microparticles after degradation for 24 h. B, β-chitin microparticles after degradation for 96 h. The arrowhead indicates fragmented fractions generated by OfChtII truncations. Scale bars and magnifications are indicated in each column. The “1000”, “3000”, “5000” and “10,000” on the top images indicate amplification times. The area enclosed by the dashed box in the left image was amplified and presented in the images on the right.

To further elucidate the impact of OfChtII-B4C1 toward β-MPs, these β-MPs hydrolyzed by OfChtII-B4C1 were directly deposited onto the copper mesh after dehydration and subsequently observed by transmission electron microscopy (TEM). Compared to the untreated β-MPs that exhibited a compacted structure, the OfChtII-B4C1-treated samples revealed the presence of small and fragmented nanoscale chitin fibers surrounding the compacted particles (Fig. S1, top right) or forming a meshwork structure within the sample (Fig. S1, bottom right). These chitin fibers observed by TEM likely originated from the fragmented fractions identified by SEM during the freeze-drying process for TEM sample preparation. These results further demonstrated the pivotal role of CBMs in facilitating the deconstruction of compacted structures of β-MPs during the catalysis of OfChtII-B4C1, ultimately leading to the formation of fragmented nanoscale chitin fibers.

Effects of OfChtII truncations on β-chitin nanofiber

To further investigate how CBMs facilitate the degradation of nanosized chitin fibers observed in SEM, β-chitin nanofibers (β-NFs) were produced through the homogenization of β-MPs. The activities of OfChtII truncations toward β-NFs were then investigated. The results indicated that OfChtII truncations exhibited higher activity toward β-NFs (Fig. 3A) during the initial stages of hydrolysis, as compared to their respective activities toward β-MPs (Fig. 1C). However, as the incubation period exceeded 12 h, the level of products generated tended to plateau. Among the three OfChtII truncations, OfChtII-B4C1 still exhibited the highest hydrolytic activity toward β-NFs (Fig. 3A), while OfChtII-C1 displayed the lowest. The binding abilities of OfChtII truncations to β-NFs were then assessed by Langmuir isotherm (Fig. 3B). This analysis revealed that the binding amounts of OfChtII truncations also increased with the augmentation of CBMs within their structures. These results indicated that CBMs also enhanced both the hydrolytic activity and substrate binding of chitinases toward nanosized β-NF.Figure 3 Effects of OfChtII-B4C1 on β-chitin nanofibers.A, time course of total reducing sugar generated by OfChtII truncations during hydrolysis of β-chitin nanofibers. B, binding isotherms of OfChtII truncations on β-chitin nanofibers. C, ultrastructure changes of β-chitin nanofibers after hydrolyzed by OfChtII-B4C1. Scale bars and magnifications were indicated in each column. CK stands for the control sample without the addition of an enzyme. The area enclosed by the dashed box in the left image was amplified and presented in the images on the right.

Subsequently, SEM analysis was applied to observe the alterations in β-NFs after hydrolysis by OfChtII-B4C1. In the control group without chitinase treatment, β-NFs assembled during the freeze-drying process, forming incompact aggregations on the surface of the silicon wafer (Fig. 3C, top left). Nanosized fibers were still discernible on the surface of these aggregations (Fig. 3C, top right). However, upon hydrolysis of β-NFs with OfChtII-B4C1, a notable reduction in the size of these aggregations was observed, and small fibers became visible (Fig. 3C, bottom right). TEM analysis was then applied to better observe these samples. For the untreated β-NFs, a vast network structure of chitin fibers was observed (Fig. S2). After treatment with OfChtII-B4C1, a large network structure remained discernible, but the fibers therein appeared to be shorter (Fig. S2, top right). Additionally, smaller networks comprised of shorter nanofibers were visible within the sample (Fig. S2, bottom right). These results demonstrated that CBMs facilitated the degradation of β-NFs and OfChtII-B4C1 was capable of effectively degrading β-NFs into shorter fragments.

The impact of CBMs studied at the single-molecule level

To gain deeper insights into how CBMs promote the catalytic activities of OfChtII truncations, high-speed atomic force microscopy (HS-AFM) was applied to analyze the interactions between these enzymes and β-NFs at the single-molecule level and nanoscale. β-NFs were adsorbed onto the surface of a highly oriented pyrolytic graphite disc (HOPG). The single chitin fiber appeared as bright sticks in the HS-AFM images. Upon the addition of OfChtII truncations, bulges representing the enzyme molecules were observed to attach to and detach from the surface of β-NFs (Fig. S3). Notably, no processive movements were observed, indicating that these enzymes were non-processive chitinases. To further describe their movement, the kon (defined as the number of newly bound enzymes per unit of chitin surface area, duration, and enzyme concentration, expressed in μM−1 μm−2 s−1) and koff (defined as the reciprocal of the residence time on the β-NFs) were analyzed. The kon distribution was fitted to a Gaussian curve to estimate its mean value. Meanwhile, the residence time distribution exhibited a single-exponential decay, from which koff was estimated (Fig. 4). The results revealed that on β-NFs, the addition of a single CBM to CD resulted in about a 34-fold increase in kon compared to CD alone. Furthermore, the addition of three CBMs to CD enhanced the kon by 130-fold (Fig. 4, Table 1). In contrast, the koff of OfChtII truncations was almost unaffected by the addition of CBMs (Fig. 4, Table 1). Therefore, on β-NFs, CBMs significantly strengthen the substrate-enzyme association while having minimal impact on enzyme dissociation from the substrate.Figure 4 Binding parameters of OfChtII truncations on β-chitin nanofibers.A–C, distribution of kon for OfChtII-C1, OfChtII-B6C1 and OfChtII-B4C1 respectively. It was fitted with a single Gaussian distribution curve and the fitting gave mean value and standard deviation for kon respectively. D–F, Distribution of binding time for OfChtII-C1, OfChtII-B6C1, and OfChtII-B4C1 respectively. It was fitted with a single exponential decay function. The fitting gave mean value and fitting error for koff respectively.

Table 1 Parameters of OfChtII truncations on different β-chitin substrates

Substrate	OfChtII truncations	Time resolution (frame/second)	Number of molecules	kon (μM−1 μm−2s−1)	1/koff (s)	koff (s−1)	
β-NC	C1	10	363	5.42 × 101	0.176	5.68	
B6C1	6.7	307	4.36 × 101	0.170	5.88	
B4C1	6.7	481	3.27 × 102	0.173	5.78	
β-NF	C1	10	179	2.36 × 102	0.177	5.65	
B6C1	10	252	8.06 × 103	0.199	5.03	
B4C1	10	598	3.08 × 104	0.182	5.49	

Chitin fiber deconstruction by OfChtII-B4C1

To further characterize the nanoscale effects on chitin deconstruction of multiple CBMs introduced to OfChtII, the process of β-NFs degradation by OfChtII-B4C1 was observed with HS-AFM. After OfChtII-B4C1 was added to β-NFs attached to the HOPG substrate, the temporal progression of β-NFs degradation was observed. The degradation was initiated at locations where the surface-exposed fibers exhibited a bent conformation (Fig. 5A, arrow), followed by cutting at these bent regions (Fig. 5A, 30 s) as well as further fragmentation into multiple sections. The degradation process led to the destabilization of the chitin fiber network organization (Fig. 5A, from 30 s to 40 s), resulting in the releasing of fiber segments from the chitin fiber network or the HOPG surface (Figs. 5A, 50s, Movie S1 and S2). At a higher imaging rate of 10 frames per second, an enzyme molecule was observed to bind to one chitin fiber and subsequently cut it off (Fig. 5B, Movie S3 and S4). These findings further confirmed that OfChtII is an endo-acting chitinase, and provide insights into how the chitin fiber network is degraded. OfChtII cuts chitin fibers, releases small fragments into the solution, and disrupts the compacted structure of chitin. The small chitin fragments observed by SEM and TEM must be generated in this way.Figure 5 HS-AFM images showing hydrolysis of β-chitin nanofibers by OfChtII-B4C1.A, hydrolysis process of β-chitin nanofibers by OfChtII-B4C1. The original images were captured at 1 frame per second for an area of 500 × 500 nm2 with 100 × 100 pixels. The images were extracted from supporting movie S1. Arrows point to cuts generated by OfChtII-B4C1 hydrolysis. The small chitin fibers going to be released were circled. B, hydrolysis process of one β-chitin nanofiber by OfChtII-B4C1. The original images were captured at 10 frames per second for an area of 50 × 50 nm2 with 100 × 100 pixels. The images were extracted from the supporting movie S2. Arrows point to OfChtII-B4C1 molecules and circles indicate the chitin fragment that is releasing. Scale bars were provided in each column.

Impact of CBMs on highly crystalline β-chitin nanocrystal

The CBMs promote the substrate association of OfChtII with β-NFs, thereby increasing the hydrolysis activity of the chitinase. To investigate if these effects of CBMs can be applied equally to chitin with higher crystallinity, we applied the highly crystallized β-chitin nanocrystals (β-NCs) isolated from tubeworm for HS-AFM analysis. In the HS-AFM images, β-NCs appeared as straight fibers. When OfChtII truncations were added, new bulges representing enzyme molecules were observed to attach to and detach from the β-NC surface (Fig. S4). No processive movement of these molecules was observed. The kon value of each OfChtII truncation was lower on β-NCs compared to β-NFs, which must be related to the high crystallinity of β-NCs. Among the different OfChtII truncations, the kon of OfChtII-B4C1 was over five times higher than that of OfChtII-B6C1 and OfChtII-C1 (OfChtII-B4C1 > OfChtII-B6C1 ≈ OfChtII-C1). The koff values were nearly identical for all three species (Fig. 6, Table 1). These findings suggest that for the highly crystallized β-NC substrate, the addition of a single CBM is insufficient to significantly affect enzyme association and dissociation, whereas the addition of three CBMs can enhance the association. It indicates that chitinases with multiple CBMs possess specific advantages in degrading the crystalline part of chitin. Additionally, the similar koff values suggest that the residence time on the surfaces of β-NFs and β-NCs is possibly determined by the duration of the catalytic reaction, which is independent of the substrate crystallinity and the number of CBMs.Figure 6 Binding parameters of OfChtII truncations on β-chitin nanocrystals.A–C, distribution of kon for OfChtII-C1, OfChtII-B6C1, and OfChtII-B4C1 respectively. It was fitted with a single Gaussian distribution curve and the fitting gave mean value and standard deviation for kon respectively. D–F, distribution of binding time for OfChtII-C1, OfChtII-B6C1, and OfChtII-B4C1 respectively. It was fitted with a single exponential decay function. The fitting gave mean value and fitting error for koff respectively.

Discussion

The ChtIIs from insects are highly conserved among insects and feature a unique modular structure organization with multiple CDs and CBMs. They are essential for the biodegradation of chitin in insect cuticles and have been proven to be effective in deconstructing compacted chitin. Previous studies have shown that the CDs of ChtII follow an endo-acting catalytic mechanism and do not synergize with each other (16). It seems that the unique property of ChtII is dependent on the multiple CBMs, but the function of which remains not fully understood. In the combination of biochemical and microscopic analyses, we investigated the function of CBMs in OfChtII truncations with varying numbers of CBMs. Our findings not only clarified the role of CBMs in OfChtII but also provided a mechanistic understanding of how this multi-modular chitinase degrades chitin, as discussed subsequently.

The impact of CBMs on OfChtII

CBMs are widely distributed in both exo-acting and endo-acting chitinases. Upon deletion of the CBM in ChiA (33), ChiCW (34), and ChiA74 (16) from B. circulans, Bacillus cereus, and B.thuringiensis respectively, a reduction in chitinase activity and substrate binding was observed. The function of group 14 CBM (CBM14) in OfChtII had been elucidated. Previously, we delved into the functionalities of chitin-binding proteins that harbor three CBM14s (CPAP3), confirming their ability to interact with chitin (35). Similarly, the effector protein Avr4 from the tomato pathogen Cladosporium fulvum possesses a CBM14 domain. It has been proved to bind chito-oligosaccharide, and its structure in complex with chitohexaose has been resolved (18). The deletion of CBM14 from human chitotriosidase decreases its activity toward chitin (28). The truncation containing only the CD of chitinase 5 from M. sexta and O. furnacalis exhibited lower activity toward the insoluble polymer compared to the full length (29, 36). Our biochemical analysis concurs with these findings, indicating that the deletion of CBMs significantly reduces both the catalytic and binding activities of OfChtII truncations toward chitin substrates, especially toward the more complex substrate β-MPs. To further illustrate how CBMs affect the interactions between OfChtII truncations and chitin substrates, HS-AFM was applied to image the dynamic process of OfChtII truncations at the single-molecule level, which enabled direct estimation of the kon and koff of these enzymes on chitin. On β-NFs, the kon of OfChtII truncations increased significantly with the augmentation of CBMs (OfChtII-B4C1 > OfChtII-B6C1 > OfChtII-C1), while the koff remained unaffected (Table 1). Interestingly, on β-NCs with high crystallinity, the addition of a single CBM to CD was insufficient to enhance the kon. However, the addition of three CBMs to the CD exhibited a 5-fold increase in kon than the single CD addition. These results clearly indicate that CBMs possess a critical role in the initial interaction with chitin substrate, rather than the dissociation of OfChtII truncations from chitin substrate. These features are consistent with findings in the cellulose degradation system. For the endoglucanase TrCel5A from Trichoderma reesei, the absence of CBM diminished its ability to hydrolyze cellulose (37). For the processive cellulase CfCel6B, the addition of CBM resulted in a 25-fold increase in kon compared to the CD alone. Their results indicate that CBMs play a critical role in the initial interaction between CfCel6B and crystalline cellulose (38). For the processive cellulase TrCel6A, the CBM was proved to guide the CD to the hydrophobic surface of crystalline cellulose, increasing the probability of catching a hydrolyzable cellulose chain (39). For the endo-acting chitinase OfChtII in this work, the addition of CBMs appears to provide more opportunities (kon) for the chitinases to identify the hydrolyzable sites on a chitin fiber. However, the catalytic duration time (1/koff) of these chitinases was not affected, which is very likely determined by the CD domain. Furthermore, a single CBM was insufficient to contribute to the binding of CD to the crystalline chitin, suggesting that chitinases with multiple modular organizations may possess advantages in degrading substrates with high crystallinity.

The mechanism of chitin deconstruction by endo-acting OfChtII-B4C1 on the nanoscale

It has been widely accepted that endo-acting chitinases hydrolyze a chitin chain in the middle and generate new chitin chain ends for exo-acting chitinases, resulting in a synergy between endo- and exo-acting chitinases. Our previous study demonstrated this synergy that the endo-acting chitinases OfChtI and OfChtII-B4C1 increase both the chitin-binding activity and the processivity of exo-acting chitinase OfChi-h during the degradation of β-NCs, leading to the formation of an instantaneous cluster of OfChi-h that efficiently degrades chitin fiber (9). OfChtII-B4C1 with multiple modular has been proven to break the compacted structure of chitin (21) but still lacks a mechanistic understanding of how the multi-module chitinase directs the nanoscale deconstruction of the chitin substrate. Here, HS-AFM was applied to track the nanoscale deconstruction of chitin fibers. OfChtII-B4C1 was observed to cut surface-exposed fibers preferentially at their bent portions. The resulting more mobile fiber segments are eventually released from the composite structure. The fibrillar organization in the regions affected is destabilized and becomes more vulnerable. During the degradation of cellulose, several mechanisms of cellulases are postulated according to observations during the cellulose deconstruction process. One is conceptualized as a “thinning” or “surface ablation” mechanism (40, 41), primarily achieved by processive cellulases that lead to the direct ablation of microfibrils from the cellulose surface (40). Similarly, the processive chitinase OfChi-h, involved in chitin degradation, has also been proved to follow similar machinery as previously described (9). Another proposed mechanism is the “cavity-forming”, which is achieved by a cellulase CbCelA from Caldicellulosiruptor bescii with multiple domains (42). Furthermore, there is a specialized fiber-fragmenting machinery that relies on the cellulosome. It causes fiber degradation in the transversal direction (43, 44, 45). When comparing the mode of action for OfChtII-B4C1 to the catalytic mechanisms of cellulases, it exhibits the most resemblance to the fiber-fragmenting machinery achieved by the cellulosome. The full-length OfChtII comprises an extra one active CD (OfChtII-C2), three inactive CDs, and three CBMs. Our previous work has demonstrated that OfChtII-C2 exhibits chitinase activity similar to the first OfChtII-C1 (16). As for the inactive CDs, our preliminary findings suggest their capacity to bind chitin, suggesting a potential role in facilitating chitin degradation. Despite attempts to obtain a larger portion of the full-length enzyme, we have encountered challenges due to insufficient quantities for functional characterization. Based on the current data, we proposed that the additional modules in OfChtII would contribute to enzyme association and even deconstruction of chitin structures as our SEM and TEM observations for OfChtII-B4C1. The mode of action of the full-length OfChtII is anticipated to be similar to that of OfChtII-B4C1. However, the precise catalytic mechanism of the full-length OfChtII remains to be elucidated.

Conclusion

This work demonstrated that the carbohydrate-binding modules enhances the catalytic activity of endo-acting chitinase by increasing substrate association without affecting the dissociation process, and demonstrated that multi-modular endo-acting chitinase deconstructs the compact structure of chitin as a fiber fragmenting machinery. This insight into endo-acting chitinases can be generalized to any enzymatic degradation of solid substrates and facilitates the design of chitinases with high efficiency.

Experimental procedures

Chitin substrates preparation

To prepare β-chitin nanofibers (NFs), 1 g β-chitin microparticles (β-MPs, a kindly gift from Prof. Yuguang Du from the Institute of Process Engineering, Chinese Academy of Science) were suspended in 100 ml distilled water and passed through high-pressure homogenizer with an operating pressure of 1000 bar. The number of passes through the homogenizer at high pressure was about 20. Finally, β-NFs were filtered to get a final suspension of 10 mg/ml in distilled water containing 20 mM sodium azide and stored at 4 °C before use. The β-chitin nanocrystals (β-NCs) were purified from Satsuma tubeworms (Lamellibrachia satsuma), which were collected from Kagoshima Bay and kindly gifted by Prof. Yu Ogawa. It was prepared according to the method described previously (46).

Enzyme preparation

Three truncated OfChtIIs from O. furnacalis containing zero (OfChtII-C1), one (OfChtII-B6C1), and three CBMs (OfChtII-B4C1) respectively at the N-terminus of CD were designed (Fig. 1A) and recombinantly expressed using the yeast P. pastoris (17, 47). These recombinant chitinases were secreted into the culture supernatant and were purified by a combination of ammonium sulfate precipitation and immobilized metal ion affinity chromatography (IMAC), as described previously (47). The concentrations of proteins were determined by the Bradford protein assay. The yields for the truncations OfChtII-C1, OfChtII-B6C1 and OfChtII-B4C1 were 1 mg/L, 0.1 mg/L and 2 mg/L respectively. All purified enzymes were dissolved in the sample buffer (50 mM sodium phosphate, 150 mM NaCl, pH 6.0) before use.

Enzymatic activity assays

The enzyme activities of the OfChtII truncations toward β-NFs or β-MPs were evaluated in a total volume of 1.0 ml reaction system comprising 50 mM sodium phosphate (pH 6.0), 150 mM sodium chloride, 20 mM sodium azide, 1 μM enzyme, and 2 mg/ml substrate in a 2 ml Eppendorf tube. Each tube was incubated horizontally in an incubator at 30 °C and 200 rpm, and samples were collected at different time points after incubation. For detecting the reducing sugar generated, a 0.1 ml sample was taken from the well-mixed digestion mixtures. Subsequently, 0.3 ml potassium ferricyanide (2 mg/ml) was added, and the mixture was boiled for 15 min. The amount of reducing sugar generated was determined by measuring the absorbance at 420 nm, which corresponded to the potassium ferricyanide consumption by (GlcNAc)2, the major product of chitinase (21). Three independent biological replicates were performed for each condition.

Substrate binding assay

The binding abilities of OfChtII truncations on β-NFs or β-MPs were evaluated through measurements of the free enzyme concentration at varying enzyme loading concentrations. Each sample contained a constant substrate concentration of 2 mg/ml β-NFs or β-MPs, along with 1 to 2 μM enzyme in 50 mM sodium phosphate buffer (pH 6.0), and 150 mM sodium chloride. Following incubation at 4 °C for 60 min, the samples were centrifuged at 17,000g for 20 min to remove the substrate. The supernatant was then collected to detect the protein concentration of the free enzyme using the Bradford assay. The concentration of the bound enzyme was calculated by the total enzyme concentration (E0) minus the concentration of free enzyme in equilibrated systems (Efree). Three independent biological replicates were performed for each condition.

High-speed AFM observations

HS-AFM observations were performed using a laboratory-built customized HS-AFM apparatus and operated with tapping mode. A small cantilever (BL-AC10DS-A2; Olympus Corporation, Tokyo, Japan) was prepared and used for AFM imaging as described previously (9). The AFM images were taken at a speed of 1 to 10 frames/s.

When performing HS-AFM observations, 2 μl of 0.1 mg/ml β-NFs or β-NCs suspension in water was deposited onto a freshly cleaved highly oriented pyrolytic graphite (HOPG) substrate. Following incubation for 10 min, the HOPG was rinsed three times with 18 μl sample buffer containing 50 mM sodium phosphate (pH 6.0), and 150 mM sodium chloride. Initially, β-NFs or β-NCs on the HOPG substrate were observed in a 70 μl sample buffer. Upon confirming the chitin was stable on the HOPG substrate, tenfold concentrated enzymes were prepared and added to the liquid chamber in the HS-AFM to achieve the desired protein concentration for analysis. The images were recorded immediately after the addition of enzymes.

Data analyses

The HS-AFM images were processed and analyzed by the Image J program (National Institute of Health) as described previously (9). Initially, the background of the images was subtracted, and the frames were aligned to correct for any drift of the scanner in the x- and y-directions. Subsequently, the individual moving molecules were tracked through the MTrackJ plugin in Image J. The duration time of binding was calculated according to the number of frames in which the molecules were observed on chitin fiber. The kon values of the enzymes were calculated as the number of newly bound molecules normalized by enzyme concentration, the area of microfibril, and the observation time according to Equation 1:(1) kon=nc·t·S

Where n represents the number of newly bound molecules, c is the concentration of the enzyme, t is the observation time during which n molecules newly bind, and S is the top area of chitin microfibril. The mean value of kon was estimated based on the curve–fitting of its distribution to a single Gaussian function according to Equation 2:(2) f(x1)=a1e−(x1−b1)22c12

Where x1 stands for kon, a1 is the height of the curve’s peak, b1 is the mean value and c1 is the standard deviation. The duration time of binding was calculated by curve-fitting of its distribution to the exponential decay function according to Equation 3:(3) f(x2)=a2e(−x2/b2)

Where x2 represents the duration time, a2 is the intercept on the y-axis, and b2 is the estimated duration time (1/koff) of the enzyme. Consequently, 1/b2 corresponds to the estimated koff. All the fittings were performed using the Igor Pro software, and the associated fitting errors were estimated. At least three independent chitin nanofibers were observed to estimate the parameters of OfChtII truncations.

Electron microscope analysis of chitin substrate

After digestion by OfChtII truncations, the residual chitin samples were precipitated by centrifugation. The resulting pellets were washed with 1.0 ml distilled water three times to remove salt, followed by three additional washes with 1.0 ml 100% ethanol to eliminate residual water. To observe the β-MPs by SEM, the samples were first freeze-dried, and then an appropriate amount of powder was applied onto a conductive gel stick on a silicon wafer (Zhongjingkeyi Technology, China). To observe the β-NFs by SEM, the samples were first applied on a silicon wafer, and then freeze-dried. All these samples were sputter-coated with gold-palladium to enhance electrical conductivity before observation using SEM. The images were taken on a SU3800 SEM (Hitachi, Tokyo, Japan) at an electron beam voltage of 15 kV.

To observe the β-MPs and β-NFs by TEM, the chitin samples in ethanol were first sonicated for 1 min. Subsequently, 5 μl samples were directly deposited onto a 200 mesh copper grid (Zhongjing-keyi Technology). The samples were then dried at room temperature, and images were captured using a JEM2000EX TEM (JEOL, Tokyo, Japan) with a 120 kV electron beam.

Data availability

Data supporting the reported results will be available from the corresponding author (Qing Yang).

Supporting information

This article contains supporting Information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supporting information

Movie S1

Movie S2

Movie S3

Movie S4

Supporting Figures

Author contributions

M T. A. and Q. Y. writing–review & editing; T. A. methodology; Q. Y. supervision; M. Q. writing–original draft; M. Q. investigation; M. Q. and X. G. data curation.

Funding and additional information

This work was supported by the 10.13039/100014717 National Natural Science Foundation of China (grant number 32170501 to M. Q.), 10.13039/501100012166 National Key R&D Program of China (grant number 2022YFD1700204 to Q. Y.) and the 10.13039/501100012226 Fundamental Research Funds for the Central Universities of China (grant number DUT22LK13 to M. Q.).
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References

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