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Characterization of Chitinase from White-Rot Fungus Irpex lacteus NK-1
2025 Volume 72 Issue 2 Article ID: 7202107
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2025 Volume 72 Issue 2 Article ID: 7202107
Chitin is a cell wall structural component of many fungi and is important for mycelium growth. Therefore, enzymes like chitinase which break down chitin, are likely important in fungi during morphological changes. Irpex lacteus, a white-rot fungus isolated from wood-rotting fungi, produces several chitinases. Although it produces a range of chitinases, there are currently no characterization reports exploring them, despite the interest and body of published works evaluating carbohydrate degrading enzymes. In this study, IlChi18C was cloned and recombinantly produced using Pichia pastoris as a host. Properties of purified IlChi18C were determined, revealing an optimal pH of 5.0 and temperature of 50 °C when using pNP-N,N'-diacetyl-β-D-chitobioside (pNP-(GlcNAc)2) as a substrate. It is activated in the presence of metal ions such as Mg2+, Ca2+, and Mn2+, but inhibited by DMSO, EtOH, and SDS. The Km and Vmax of IlChi18C for this substrate are 3.48 mM and 5.46 µM min−1, respectively. Using pNP-(GlcNAc)2 and chitin powder as substrates, IlChi18C predominantly exhibited exo-type chitinase activity, releasing chitobiose from the non-reducing ends of chitin chains. It was also observed that this enzyme acts on the fruiting body of Flammulina velutipes, releasing chitobiose as the main product.
GlcNAc, N-acetyl-β-D-glucosamine; GH18, glycoside hydrolase family 18; RNA-Seq, RNA sequencing, SAB, sodium acetate buffer; BRB, Britton-Robinson buffer.
Chitin is a linear polysaccharide chain consisting of N-acetyl-β-D-glucosamine (GlcNAc) units. It is found in the shells of insects and crustaceans, such as crabs and shrimp. Chitin is also a component of fungi cell walls and supports hyphal structure and growth in filamentous fungi [1]. Chitin is the second most abundant biomass after cellulose on earth and is an increasingly important resource, given growth in new applications, such as nanofiber production [2]. The main components of filamentous fungi cell walls vary among taxa: in Ascomycotina and Basidiomycete, they primarily consist of chitin and α- and/or β-glucan, while in Zygomycete they contain chitin and chitosan. Cell walls of basidiomycetes, such as mushrooms, are reported to contain about 10-30 % chitin [3]. Chitin oligosaccharides, including cell wall-derived chitin, are potential food sources, with chitinolytic enzymes implicated in oligosaccharide production. Fungal cell walls contain branched β-1,3-glucan and chitin within a scaffold structure, covalently cross-linked to the non-reducing ends of the branched β-1,3-glucan. The chitin-β-1,3-glucan core complex is speculated to be covalently cross-linked with other polysaccharides and glycoproteins [4, 5]. Additionally, mushroom mycelia cell walls contain many associated proteins, contributing to the complex and strong cellular structure. Investigations of Pleurotus tuber-regium indicate a cell wall structure comprised of the following four layers [6]. The first outermost layer is composed of glycoproteins, the second layer consists of soluble glucans with a small amount of embedded protein. The third layer incorporates highly branched glucan, and the fourth layer is a highly branched glucan-chitin complex. As the innermost layer contains chitin, it is expected to play a fundamental role in the formation of the cell wall.
The mushroom fruiting body developmental process is divided into the following events: fruiting body primordia formation, cap and stalk differentiation, stalk elongation, cap expansion, and aging of the fruiting body. As hyphae morphology changes significantly at each stage, processes involving cell wall decomposition, synthesis, and reconstruction are expected. In Coprinopsis cinerea, there are many carbohydrate-related enzymes involved in stalk elongation [7]. One is chitinase, a glycoside hydrolase family 18 (GH18) enzyme. Chitinases are classified within the CAZy database (http://www.cazy.org) according to amino acid sequence similarities, grouping them into the glycoside hydrolase families GH18 and GH19. However, all chitinases, other than those found in plants and some bacteria, are classified into the GH18 group [8]. Coprinopsis cinerea is thought to have two types of chitinase involved in stalk elongation: an endo- and an exo-type [9]. Chitinase activity is also known to increase during fruiting body cap autolysis [10]. However, there are few reports of gene-cloned mushroom-derived chitinases and clarification of their properties.
Irpex lacteus is a white-rot fungus noted for its wood-decaying properties, the carbohydrate related enzymes of which are long studied by our group [11, 12, 13, 14]. The RNA sequence of I. lacteus NK-1 revealed expression of eight chitinase genes, including four extracellular chitinase genes (IlChi18A, IlChi18B, IlChi18C, and IlChi18D) and four intracellular chitinase genes (unpublished data). Based on primary structure homology, IlChi18A, IlChi18B, and IlChi18C are presumed exo-type enzymes while IlChi18D is presumed as an endo-type enzyme. Irpex lacteus NK-1 produces four types of extracellular chitinases, notably including two types of chitinases with different modes of action. To elucidate the role of each enzyme, it is essential to evaluate their decomposition mechanisms.
In this study, exo-type enzyme chitinase C was selected as a representative of the three, a heterologous expression system was constructed, enzyme purified, properties elucidated, and degradation mechanism investigated.
Materials. All reagents were purchased from NACALAI TESQUE INC. (Kyoto, Japan), Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) or FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) unless otherwise noted. Chito-oligosaccharide was purchased from Megazyme Ltd. (Wicklow, Ireland).
Strains, plasmid, and culture conditions. Irpelx lacteus NK-1 is a strain screened by Nishizawa et al. [15]. Escherichia coli DH5α, purchased from Takara Bio Inc. (Shiga, Japan) was used as a host strain for plasmid extraction. Plasmid pMD19 (simple) (Takara Bio Inc.) was used as a subcloning vector. Pichia pastoris GS115, used for enzyme production, and chitinase expression vector pPICZα were purchased from Invitrogen (USA).
Cloning of IlChi18C. Irpex lacteus NK-1 cells were grown in modified Mandel's medium [16] containing wheat bran as the carbon source at 25 °C with orbital shaking (150 rpm) for 6 days. Total RNA was isolated using TRIZOL® reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's protocol for filamentous fungi. Isolated total RNA was sent to Takara-Bio for RNA sequencing (RNA-Seq) and used for cDNA synthesis. The cDNA sequence encoding the full-length IlChi18C is stored in the DDBJ/EMBL/GenBank database under accession number BFT17578.1.
Primary structure and phylogenetic analysis. The signal peptide of the amino acid sequence was determined using SignalP 6.0 server (https://services.healthtech.dtu.dk/services/SignalP-6.0/). A phylogenetic tree was generated based on chitinase protein amino acid sequencing with the neighbor-joining method using MEGA version 11. Additionally, these sequences were compared by ClustalW (https://www.genome.jp/tools/clustalw/).
Heterologous expression and purification of IlChi18C. The mature protein region of IlChi18C was amplified using polymerase chain reaction (PCR) with primers 5′-AAGAATTCGCGCCTGTCTGCCAGTCC-3′ and 5′-CCTCTAGATTAACAACTTTCGCCAAC-3′ designed using EcoRⅠ and XbalⅠ and a cDNA template for expression in P. pastoris strains. The cDNA was synthesized from total RNA extracted from I. lacteus NK-1 cells cultured in liquid medium (6 wt% wheat bran, 0.7 wt% (NH4)2SO4, 0.3 wt% KH2PO4) using PrimeScript™ II Reverse Transcriptase (Takara Bio Inc.). The amplified DNA fragment was inserted into the expression vector pPICZα (pPICZα_IlChi18C) and introduced into P. pastoris GS115. P. pastoris cells containing pPICZα_IlChi18C were cultured in YPG (1 % yeast extract, 2 % peptone, and 1 % glycerol) medium containing 100 mM potassium phosphate buffer (pH 6.0) at 30 °C in a shaker at 200 rpm for 48 h. After incubation, P. pastoris cells were collected by centrifugation (4,000 × G, 5 min) and resuspended in BMGY (1 % yeast extract, 2 % bacto peptone, and 0.5 wt% methanol) containing 100 mM potassium phosphate buffer (pH 6.0) and cultured at 30 °C for 120 h with rotary shaking at 200 rpm. Methanol was added at 0.5 wt% every 24 h. After incubation, the supernatant was collected by centrifugation (10,000 × G, 10 min, 4 °C) to obtain the crude enzyme solution. Powdered ammonium sulfate was gradually added to the crude enzyme solution to reach 80 % saturation, and after complete dissolution, the mixture stirred at 4 °C overnight. The precipitate obtained by centrifugation (10,000 × G, 20 min, 4 °C) was collected and dissolved in 50 mM sodium acetate buffer (SAB) (pH 5) containing 1.6 M ammonium sulfate, and centrifuged (10,000 × G, 20 min, 4 °C). The enzyme was purified using a hydrophobic column (20 mm ID × 250 mm), Toyopearl® Butyl-650M (TOSOH CORPORATION, Tokyo, Japan), and a gel filtration column (16 mm ID × 600 mm), Sephacryl™ S-200 HR (GE HealthCare Technologies Inc., Chicago, USA). The mobile phase for the hydrophobic column was 50 mM SAB (pH 5) containing 1.6 M ammonium sulfate, and the elution phase was 50 mM SAB (pH 5). Proteins were eluted with a linear gradient of ammonium sulfate from 1.6 M to 0 M over 90 minutes at a flow rate of 3 mL/min. The mobile phase for the gel filtration column was 50 mM SAB (pH 5) containing 0.25 M NaCl.
Protein analysis of the recombinant enzyme. The molecular size of purified IlChi18C was determined by SDS-PAGE [17]. The concentration of purified IlChi18C was determined using the Bradford method [18].
Effect of pH and temperature for IlChi18C activity assays. The optimal pH of IlChi18C was determined as follows: 40 µL of reaction mixture containing Britton-Robinson buffer (BRB) pH 2.0-10.0, 1.25 mM pNP-N,N'-diacetyl-β-D-chitobioside (pNP-(GlcNAc)2), and 3.0 µg of IlChi18C was incubated at 40 °C for 20 min. Then, 200 µL of 0.4 M sodium carbonate was added to the mixture to stop the reaction. The concentration of released pNP was determined by measuring the absorbance at 405 nm.
The optimal temperature for IlChi18C was determined as follows: 40 µL of reaction mixture as mentioned above was incubated for 20 min at 20 °C−80 °C. The reaction was halted and pNP concentration determined as above.
The temperature stability of IlChi18C was determined as follows: in 50 mM BRB (pH 5.0) 3.0 µg of IlChi18C was incubated at 20 °C−60 °C for 3 h, then a 40 µL reaction mixture was incubated as mentioned above at 40 °C for 20 min. The reaction was halted and the concentration of pNP determined as previous. One unit of chitinase activity on the pNP substrate was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per min.
Effect of metal ion and chemicals for IlChi18C activity assays. The effect of additives on IlChi18C was investigated as follows: 40 µL of reaction mixture containing the additive (5 mM CuSO4, 5 mM MgSO4, 5 mM CaCl2, 5 mM MnCl2, 5 mM FeCl3, 5 mM NaCl, 5 mM KCl, 5 mM EDTA, 20 % DMSO, 20 % EtOH, 20 mM β-ME, 20 mM SDS, 0.5 % Tween 20, 0.5 % Triton X-100), 50 mM BRB (pH 5.0), 1.25 mM pNP-(GlcNAc)2 and 3.0 µg of IlChi18C was incubated at 40 °C for 20 min. Upon termination of the reaction, the pNP concentration and chitinase activity were determined as described above.
Kinetic analysis of IlChi18C. The kinetic parameters of IlChi18C for pNP-(GlcNAc)2 (Michaelis constant (Km) and turnover number (kcat)) were measured at concentrations of 0.25-5 mM pNP-(GlcNAc)2 using 0.6 µg of IlChi18C. A 40 µL reaction mixture containing 50 mM SAB pH 5.0 was incubated at 40 °C for 20 min then the reaction halted. The pNP concentration and chitinase activity were determined as described above. Reaction rate constants (Km and Vmax) were determined by nonlinear least-squares regression of the Michaelis-Menten equation. Subsequently, kcat/Km values were calculated from the slope of the s-v plot.
Activity of IlChi18C on various chitin substrates. Products released from pNP-derivative substrates and chitin oligosaccharides by IlChi18C were determined as follows: Mixtures containing 4 mM pNP-(GlcNAc)1-3 and 2-6 mM (GlcNAc)2-6 chitin oligosaccharides in 50 mM SAB (pH 5.0) and an appropriate amount of IlChi18C were incubated at 40 °C for the indicated times, then reactions halted using a boiling bath at 100 °C. Chitin oligosaccharide products were analyzed by HPLC using a post-column method with phosphoric acid-phenylhydrazine [19]. Separation was performed using an Asahipak NH2P-40 3E (3.0 mm ID × 250 mm, Shodex: Resonac Corporation, Tokyo, Japan) column at 40 °C and a flow rate of 0.35 mL/min. The mobile phase was a mixture of solvent A: 98.5 % acetonitrile/1.5 % phosphoric acid (85 %) and solvent B: 98.5 % H2O/1.5 % phosphoric acid (85 %). For the pNP-(GlcNAc)1-3 substate, the following conditions were used: 0-20 min, 93 % solvent A isocratic; 20-25 min, linear from 93 % to 75 % solvent A; 30-45 min, 75 % solvent A isocratic; 45-60 min, 93 % solvent A isocratic for column equilibration. For the oligosaccharide substrates, the following conditions were used: 0-15 min, 75 % solvent A isocratic; 15-20 min, linear from 75 % to 65 % solvent A; 20-25 min, 65 % solvent A isocratic; 25-40 min, 75 % solvent A isocratic for column equilibration. For post-column derivatization, the reaction solution (acetic acid/phenylhydrazine/phosphoric acid = 90/3/110 mL) was mixed with the mobile phase at a flow rate of 0.14 mL/min and to react sugars with phenylhydrazine (reaction coil 0.5 mm ID × 6 m, 140 °C). Fluorescence detection was performed at an excitation wavelength of 330 nm and an emission wavelength of 470 nm.
The hydrolytic activity of IlChi18C towards insoluble chitin substrates was determined using chitin powder (from Crab Shells, NACALAI TESQUE INC.), and colloidal chitin, which was prepared from chitin powder using a modified method from Hsu et al. [20]. A stirred aqueous suspension containing 1 % substrate in 50 mM SAB (pH 5.0) was maintained at 40 °C, then the reaction halted by immersion in a boiling bath at 100 °C. Products released were then analyzed by HPLC in the manner described above. One unit of chitinase activity on chitin or chitin oligosaccharide substrates was defined as the amount of enzyme releasing reducing sugars equivalent to 1 μmol of N,N'-diacetylchitobiose per minute.
Activity of IlChi18C on mushrooms. The hydrolysis activity of IlChi18C towards mushroom fruit bodies was measured using Enokitake mushroom (Flammulina velutipes) purchased from a supermarket. Undried mushroom fruiting bodies were mixed with 5 times the amount of deionized water and boiled for 10 minutes. Then, a 100-mesh nylon bag was used to remove the soluble fraction from the hot water extraction. This operation was repeated five times, with the resulting residue freeze-dried. The residue was ground in a blender and used as the hot water insoluble fraction that passed through a 32-mesh sieve. A mixed aqueous suspension containing 1 % substrate in 50 mM SAB (pH 5.0) and an appropriate amount of IlChi18C was incubated at 40 °C, with the reaction then stopped in a boiling bath at 100 °C. Products released from the hot water-insoluble fraction of Enokitake mushroom were analyzed by HPLC as described above.
Cloning, heterologous expression of IlChi18C. The signal peptide, MWTSNIPLSLLSOVTLTMTTGPSAARA, in the amino acid sequence of IlChi18C was encoded by the N-terminal 1-81 nucleotides. Phylogenetic analysis of the amino acid sequences of eukaryotic chitinase belonging to the family GH18, characterized in the CAZy database (https://www.cazy.org), using NCBI (https://www.ncbi.nlm.nih.gov/) showed that IlChi18C is phylogenetically close to the chitinase from Rhizomucor miehei, with 33 % sequence identity (Fig. 1). IlChi18A and IlChi18B were also phylogenetically close to chitinase from R. miehei, with sequence identities of 36 % and 31 %, respectively. IlChi18D was located away from other chitinases, closer to the groups of Aspergillus niger, Coccidioides posadasii, Clonostachys rosea, and Trichoderma harzianum, with sequence identities of 47 %, 47 %, 45 %, and 45 %, respectively. That RmChiA is an exo-chitinase suggests that the three chitinases from I. lacteus NK-1 (IlChi18A, IlChi18B, and IlChi18C) are also exo-type chitinases [21]. Additionally, Chit42 from T. harzianum is an endo-chitinase, suggesting that IlChi18D may be an endo-type chitinase [22].
Chitinase amino acid sequences with known three-dimensional structures were obtained from the protein data bank with a phylogenetic tree constructed by the neighbor-joining method using the CLUSTAL software in MEGA 11.0.13. To estimate the confidence of the tree topologies, bootstrap resampling analysis for 1,000 replicates was performed. The number after the name of the organism is the PDB ID.
Purification of IlChi18C. After 5 days of induction, the culture solution effected enzymatic action, yielding a hydrolytic activity of 0.22 ± 0.01 U/mL for pNP-(GlcNAc)2. Recombinant IlChi18C was purified from 200 mL of culture medium by hydrophobic chromatography and gel filtration chromatography, yielding 4.91 mg of material and a specific activity of 2.92 ± 0.01 U/mg for pNP-(GlcNAc)2.
Based on the mature IlChi18C protein amino acid sequence, the estimated molecular weight is 45.3 kDa. SDS-PAGE of the expressed protein is shown in Fig. 2. From the amino acid sequence of IlChi18C, three N-glycosidic modifications sites are predicted, with the smear band of approximately 50-90 kDa in the culture supernatant assigned to variously glycosylated IlChi18C (Fig. 2B). By column chromatography, IlChi18C of approximately 50-60 kDa was recovered (Fig. 2C). This purified IlChi18C was treated with endoHf (New England Biolabs Inc., Ipswich, MA, USA) and deglycosylated. Subsequent SDS-PAGE yielded a molecular weight almost identical to the estimated molecular weight (Fig. 2D).
Lanes: A, standard protein molecular weight markers; B, culture broth of the recombinant expression strain; C, purified recombinant IlChi18C; D, deglycosylated recombinant IlChi18C.
Properties of IlChi18C. The optimum pH for IlChi18C hydrolytic activity against pNP-(GlcNAc)2 was 5.0, but stability was observed between pH 4.0-6.0. However, both below pH 4.0 and above 6.0, the hydrolytic activity of IlChi18C rapidly decreased (Fig. 3A). At pH 5.0, maximum IlChi18C hydrolytic activity against pNP-(GlcNAc)2 was observed at 50 °C, with hydrolytic activity rapidly decreasing above 50 °C (Fig. 3B). The thermal stability of IlChi18C was evaluated based on residual activity after 3 hours of treatment at each temperature, with hydrolytic activity against pNP-(GlcNAc)2 decreasing past 40 °C (Fig. 3B).
A) Effect of pH on the activity (●). B) Optimum temperature (▲) and temperature stability (■) after 3 h.
The effects of metal ions, organic solvents, and anionic detergents on IlChi18C hydrolytic activity towards pNP-(GlcNAc)2 were investigated. Compared to the control without additives, IlChi18C activity increased to 139.2 %, 138 %, and 128.2 % in the presence of the metal ions 5 mM Mg2+, Ca2+, and Mn2+, respectively. However, 5 mM Cu2+ and Fe3+ diminished enzyme activity to 90.1 % and 43.5 %. Also, the presence of 20 % DMSO and 20% EtOH reduced activity to 57.4 % and 76.4 %. The chelating agent EDTA did not decrease activity, indicating that IlChi18C is not a metalloenzyme. Additionally, activity was almost completely inhibited by 5 mM SDS, an anionic detergent (Table 1).
Table 1. Effect of metal ions, organic solvents, and chemicals on IlChi18C activity.
| Additives | Relative activity (%) |
| No additive | 100.0±0.4 |
| 5 mM CuSO4 | 90.1±2.6 |
| 5 mM MgSO4 | 139.2±1.6 |
| 5 mM CaCl2 | 138.0±1.1 |
| 5 mM MnCl2 | 128.2±1.6 |
| 5 mM FeCl3 | 43.5±2.3 |
| 5 mM NaCl | 110.2±1.3 |
| 5 mM KCl | 107.4±0.8 |
| 20 % DMSO | 54.7±2.9 |
| 20 % EtOH | 76.4±0.9 |
| 5 mM EDTA | 105.6±2.4 |
| 20 mM SDS | 2.3±1.0 |
| 20 mM β-ME | 99.6±1.1 |
| 0.5 % Tween 20 | 102.2±1.4 |
| 0.5 % Triton X-100 | 98.7±1.3 |
Kinetic parameters. The Km, Vmax, and kcat of IlChi18C using pNP-(GlcNAc)2 as the substrate derived from Fig. 4 were 3.48 mM, 5.46 µM･min−1, and 9.11 s−1, respectively. In comparison, Km (1.25 mM), Vmax (2.74 µM･min−1), and kcat (2.88 s−1) were reported for C. cinerea (ChiEn1) [23]; Km (1.76 mM), Vmax (2.62 µM･min−1), and kcat (11.59 s−1) for C. cinerea (ChiⅢ) [24]; Km (0.03 mM), Vmax (0.28 mM･min−1), and kcat (0.31 s−1) for Bacillus licheniformis (ChiA) [25]; Km (0.14 mM), Vmax (6.60 µM･min−1), and kcat (No description) for Streptomyces violaceusniger (Chitinase) [26]. Chitinases from fungi, such as basidiomycetes, have higher affinity and lower catalytic efficiency than chitinases from bacteria, as indicated by their Km values. This characteristic supports the suggestion that fungal chitinases are involved in cell wall structural modification. The high substrate affinity ensures specificity of activity, while the lower efficiency reduces possible growth inhibition during modification.
Relative activities and hydrolysis products of IlChi18 for various chitin substrates. Hydrolytic activities of IlChi18C towards chitin-derived substrates are shown in Table 2. Hydrolytic activities towards pNP-(GlcNAc)1-3 were evaluated, yielding the highest activity against pNP-(GlcNAc)2. This achieved a 1000-fold higher activity than that for pNP-GlcNAc and 2.5-fold higher than pNP-(GlcNAc)3. In the case of chitin oligosaccharide (GlcNAc)2-6 degradation, IlChi18C is weakly active towards (GlcNAc)2. Activities towards (GlcNAc)4 and (GlcNAc)5 were almost equally high, while the activity towards (GlcNAc)6 was lower. Activity towards the insoluble chitin substrate was considerably lower than that towards oligosaccharide substrates.
Table 2. IlChi18C activities toward chitin substrates.
| Substrates | Specific activity (U/mg) |
| pNP- (GlcNAc)1−3 | |
| pNP- GlcNAc | 1.95±0.41 × 10−3 |
| pNP- (GlcNAc)2 | 2.92±0.01 |
| pNP- (GlcNAc)3 | 1.16±0.03 |
| Chitin-oligosaccharides | |
| N, N '-diacetylchitobiose | 0.15±0.02 × 10−3 |
| N, N ', N ''-triacetylchitotriose | 0.76±0.11 |
| N, N ', N '', N '''-tetraacetylchitotetraose | 8.21±0.09 |
| N, N ', N '', N ''', N ''''-pentaacetylchitopentaose | 8.31±0.12 |
| N, N ', N '', N ''', N '''', N '''''-hexaacetylchitohexaose | 6.41±0.10 |
| insoluble chitin-polysaccharides | |
| chitin powder | 22.14±0.32 × 10−3 |
| colloidal chitin | 45.58±0.55 × 10−3 |
To confirm the cleavage site, reaction products were analyzed using pNP-derivative substrates (Fig. 5). Although activity towards pNP-GlcNAc was extremely low, there was some GlcNAc production (Fig. 5A). During pNP-(GlcNAc)2 degradation, (GlcNAc)2 was initially released, with subsequent pNP-GlcNAc and GlcNAc production (Fig. 5B). Additionally, (GlcNAc)2 and pNP-GlcNAc were primarily released from pNP-(GlcNAc)3 initially, accompanied by a small amount of pNP-(GlcNAc)2 and (GlcNAc)3 (Fig. 5C). From this, the enzyme exhibits specificity towards the non-reducing end chitobiose unit, with some recognition of trisaccharide units for soluble substrates.
A); pNP- (GlcNAc) B); pNP- (GlcNAc)2 C); pNP- (GlcNAc)3
Furthermore, products from the reaction with chitin oligosaccharide (GlcNAc)2-6 were investigated. For (GlcNAc)2 degradation, only a small amount of GlcNAc was detected, despite extended reaction times (Fig. 6A). Also, despite degradation of (GlcNAc)3 to (GlcNAc)2 and GlcNAc, the reactivity was very low (Fig. 6B). However, (GlcNAc)4 underwent rapid degradation to (GlcNAc)2, with small initial quantities of (GlcNAc)3 detected (Fig. 6C). Similarly, (GlcNAc)5 was degraded to (GlcNAc)2 and (GlcNAc)3, while (GlcNAc)6 was degraded to (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4 (Figs. 6D, E). Given this, a model diagram for IlChi18C subsite recognition towards a chitin substrate is shown in Fig. 7. During substrate binding, the recognition of N-acetyl glucosamine at the subsite −2 position is particularly strong, indicating this substrate binding mode is favored. Consequently, if the −2 subsite is involved in binding, products longer than chitobiose are expected. However, the +2 subsite also exhibits substantial binding affinity, leading to the detection of both chitobiose and chitotriose during chitohexose degradation. This suggests that chitobiose may be continuously produced through a progressive mechanism following substrate binding.
A); (GlcNAc)2 B); (GlcNAc)3 C); (GlcNAc)4 D); (GlcNAc)5 E); (GlcNAc)6
A); dimer B); trimer C); tetramer D); pentamer ●); reducing end
Additionally, products from insoluble polymer substrates were identified. When chitin powder was used as a substrate, initial hydrolysis by IlChi18C yields (GlcNAc)2 and GlcNAc (Fig. 8A). From the IlChi18C mechanism described above, (GlcNAc)2 and (GlcNAc)3 are initially produced, with GlcNAc detected from subsequent (GlcNAc)3 degradation. In the case of amorphous colloidal chitin, initial hydrolysis to (GlcNAc)2 and GlcNAc yields a higher rate for GlcNAc production than chitin powder. This is attributed to an IlChi18C affinity increase due to the increased amorphous portion of the polymer substrate and decrease in molecular weight due to the concentrated hydrochloric acid decomposition treatment. In the case of colloidal chitin, the substrate may directly yield GlcNAc, but (GlcNAc)2 decreased after prolonged incubation. These results are reproducible, but the reason behind the increased production of GlcNAc from the colloidal chitin substrate remains unclear, requiring further investigation (Fig. 8B).
A); Chitin powder B); Colloidal chitin C); Mushroom cell wall insoluble fraction
Also of interest is the increased activity of IlChi18C towards mushroom-derived (Enokitake mushroom) cell wall chitin, which achieved a higher chitobiose production ratio than that of other chitin substrates (Fig. 8C). Chitinase IlChi18C is thought to operate via an exo-type decomposition mechanism, with strong recognition at subsites −2 and +2, primarily recognizing the chitobiose unit from the non-reducing end (Fig. 7).
Fungi cell wall structures, such as those of basidiomycetes, are mostly composed of chitin and β-1,3/1,6 glucan [27], with chitinase activity expected for hyphae elongation during various growth stages, alongside carbohydrate decomposition enzymes such as β-1,3-glucanase and β-1,6-glucanase. Chitinase (ChiEn1) from the basidiomycete C. cinerea is considered an endo-type enzyme, but only hydrolyzes soluble chitin or chitin oligomer substrates, not insoluble chitin polymer substrates. Furthermore, the mode of action reflects oligosaccharide length and is not yet determined [23]. Chitinase (ChiIII) from the basidiomycete, C. cinerea, is an endo-type enzyme and known to degrade chitin substrates into (GlcNAc)2 and GlcNAc. Additionally, this chitinase is predominantly expressed in fruiting bodies, with expression levels proportionate to fruiting body maturation [24]. Furthermore, the synergistic action of ChiEn1 and ChiIII in C. cinerea is thought involved in the efficient decomposition of cell wall chitin during autolysis, suggesting that chitinases in fungi, such as basidiomycetes, may play different roles during different growth stages [28]. Thus, IlChi18C may be involved in cell wall structural changes in mushroom fruit-bodies.
Compared to basidiomycetes chitinase activity, many Streptomyces species with strong chitinase activity are thought to use chitin as the sole carbon source, producing large quantities of highly active chitinases. However, the activity of this basidiomycetes chitinase, implicated in mycelial growth or fruit-body formation, may be suppressed.
As the I. lacteus, NK-1 strain has four types of exocytic chitinases, evaluating the other three chitinase (IlChi18A, IlChi18B, and IlChi18D), their synergistic effects on mycelium cell walls, and variances in expression during the growth stage will yield valuable insights into their interactions, influence on organism development, and facilitate new, substrate-specific enzymatic applications.
Takemi Kamijo is an employee of Kyowa Chemical Products Co., Ltd.
