Chitin is the second most abundant natural polymer in nature, second only to cellulose (Figure 1). It is widely distributed in the exoskeletons of crustaceans, insects, and fungal cell walls. Its structure, composed of N-acetyl-D-glucosamine units linked by β-1,4-glycosidic bonds, is highly crystalline and water-resistant, making it virtually impossible to directly utilize in the wild[1].

Figure 1. Sources of chitin and structure of chitosan[1].

However, chitin and its derivatives, including chito-oligosaccharides and N-acetyl-D-glucosamine, possess significant biological activities, such as immune regulation, antibacterial and anti-tumor properties, and anti-blood pressure and cholesterol-lowering properties (Figure 2). These bioactive properties have made chitin and its derivatives not only a hot topic in basic research but also a valuable resource for bioindustry development[2].

Figure 2. Potential roles of chitin in biomedical applications. Green arrows indicate potential beneficial applications of chitin with strong evidence. Red arrows demonstrate potential detrimental effect of chitin with strong evidence. Black arrows delineate the potential effects of chitin with slight evidence. Chitin has been generally utilized as biomaterials for neural treatment, wound dressing and nanoparticle component for drug delivery. In addition, chitin has been used for improving intestinal barrier function, increasing beneficial gut microbiota, partially inhibiting cardiovascular diseases, inhibiting cancer growth, and inhibiting oxidative stress-induced aging. However, chitin may have detrimental effects. For instance, chitin exposure can induce asthma and acts as an inflammatory inducer[3].

Chitinase, a key biocatalyst for chitin degradation, hydrolyzes long-chain chitin into usable oligosaccharides and monosaccharides.[4-6] We can see the specific enzymatic degradation pathway of chitin from Figure 3. It can be seen that after chitin degradation, many high value-added products will be produced.

Figure 3. Chitin convergence and degradation pathway. First, chitin can be converted to chitosan through deacetylation and further degraded to N-glucosamines (GlcpN) through glucosaminidase enzymes. Second, chitin can be degraded by chitinases and N-acetylglucosaminidase enzymes to N-acetylglucosamines (GlcpNAc). Enzymes involved in the chitin cycle are classified in glycosyl hydrolase (GH) families and represented in blue. The metagenome reads are mapped towards these enzymes and the RPKG normalized counts representing the number of hits are illustrated by the arrow thickness in the figure. If there was a statistical effect of chitin addition, a second arrow (orange) is drawn representing the normalized count of the genes related to the chitin-added samples[7].

International studies have shown that chitinases are secreted by a variety of microorganisms, including bacteria (such as Bacillus and Vibrio) and fungi (such as Aspergillus and Penicillium)[8-9]. However, these natural microorganisms typically have limited enzyme production under experimental conditions and lack stability under industrial conditions, making them difficult to meet large-scale production requirements. For example, one research team has achieved remarkable results by screening high-producing chitinase strains from marine environments and heterologously expressing them, but optimization of yield and enzyme activity remains a challenge[10].

Figure 4. (a) Chitinolytic activity of some local isolates belonging to actinomycetes on chitin agar composed of colloidal chitin (10.0 g/l), NH₄Cl (5 g/l), MgSO₄·7H₂O (0.5 g/l), KH₂PO₄ (2.4 g/l), K₂HPO₄ (0.6 g/l), and bacteriological agar (15 g/l). The medium was adjusted to pH 7.0 and incubation of cultures was done at 30 °C up to 6 days. Clear zones are indicative of chitinase production and hydrolysis of chitin. (b) Time course of enzyme production[10].

Currently, numerous studies are advancing the screening and application of chitinases[11]. For example, high-producing chitinase strains are being isolated from soil, waste aquatic byproducts, and marine sediments, and enzyme activity is being enhanced through fermentation process optimization[12]. Despite this, the enzyme production capacity of wild-type strains remains limited, failing to meet the requirements of economical industrial production. On the other hand, many teams have begun using genetic engineering to clone high-efficiency chitinase genes into Escherichia coli or other hosts for recombinant expression, achieving higher enzyme yields and controllability[13-14]. This strategy not only significantly improves enzyme activity but also enhances its potential for industrial application by optimizing parameters such as expression vectors, induction conditions, culture temperature, and inducer concentration.
In practical applications, the functions of chitinases extend beyond product production and also have environmental and biocontrol benefits. Its application in marine environments can be as a detergent, treating chitin-containing waste and reducing ecological pollution. In agriculture and biocontrol, chitinases can degrade insect exoskeletons or fungal cell walls, achieving biological control of pests and pathogenic fungi. In industrial production, enzymatic degradation of chitin can efficiently produce physiologically active chito-oligosaccharides and their derivatives, providing high-value-added raw materials for food, pharmaceutical, and biochemical products in Figure 5[15].

Figure 5. Environmental and public health effects of chitinous waste dumping scenarios. The bottom left pane describes the enrichment of water bodies with nutrients (N: nitrogen, P: phosphorus) and the subsequent depletion of biologically available oxygen (O₂: molecular oxygen)[15].

Based on current research both domestically and internationally, our core issues include:
1. High-yield expression: The production of chitinases secreted by natural microorganisms is limited, making it difficult to meet industrial demand. High yields require recombinant expression systems.
2. Optimizing enzyme activity and stability: To be applicable in diverse industrial environments, the enzyme must possess excellent thermal and pH stability, as well as tolerance to metal ions and additives.
3. Synergistic degradation efficiency: A single chitinase is insufficient for efficient degradation of natural chitin. The synergistic use of multiple enzymes may achieve higher yields, requiring exploration of the optimal combination and ratio.
4. Recycling and Immobilization: Industrial applications require cost reduction and increased enzyme reusability, making enzyme immobilization a viable solution.
In summary, building on research both domestically and internationally, this study focuses on the efficient recombinant expression of chitinases from multiple sources, screening for optimal reaction conditions, synergistic effects, and immobilized applications. The goal is to establish an efficient, stable, and industrially scalable chitinase system, providing a solution for the high-value-added utilization of natural chitin resources through degradation.

This project employed a multi-layered design strategy to achieve a comprehensive system from gene screening to industrial application.
1. Screening and Recombinant Expression of Chitinase Genes from Multiple Sources
Given the differences in substrate specificity, catalytic efficiency, and stability among different chitinases, this study screened three chitinase genes from different sources with complementary functions:
AsCDA: Derived from marine microorganisms, it exhibits deacetylation activity, partially deacetylating chitin and increasing the diversity of downstream oligosaccharides.
Cts2p: Primarily exhibits exochitinase activity, effectively cleaving long chitin chains into oligomers.
FjChiB: It possesses endochitinase activity, breaking down the intermediate structure of chitin chains and accelerating overall degradation.
All three genes were cloned into the pET-28a(+) expression vector for recombinant expression. By optimizing expression conditions (inducer concentration, temperature, induction time, etc.), the expression level and solubility of each enzyme in the E. coli host were improved.
2. Enzyme Characterization and Optimal Condition Screening
To ensure efficient enzyme application under industrial conditions, this project systematically characterized the properties of three recombinant chitinases:
1) Optimal Temperature and pH: Enzyme activity was measured at different temperatures (20–70°C) and pH buffers (pH 2–11). The optimal temperature and pH values were determined using relative enzyme activity curves.
2) Effects of Metal Ions and Additives: The effects of common metal ions (Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Mn²⁺, Zn²⁺, etc.) on enzyme activity were tested to provide a reference for industrial application.
3. Synergistic Effects of Free Enzymes and Ratio Optimization
Given the limited efficiency of single enzymes in degrading natural chitin, the project designed pairwise and triple-enzyme degradation experiments:
A. Pairwise combinations: AsCDA + Cts2p, AsCDA + FjChiB, Cts2p + FjChiB.
B. A three-enzyme combination: AsCDA + Cts2p + FjChiB. The synergistic effects of different combinations were evaluated by measuring the reducing sugar content in the degradation products (using the DNS method or a commercial kit).
C. The optimal volume ratios of 2:1, 1:1, 0.5:1, 1:0.5, and 1:2 were further explored for each pairwise combination to determine which ratio yielded the highest enzyme activity, providing a reference for subsequent industrial applications.
4. Enzyme immobilization and reuse studies
To improve the economics and ease of operation for industrial applications, this project immobilized the enzymes in the optimal combination ratio on a carrier:
• Enzymes were immobilized using the epoxy carrier LX-1000EP to enhance their thermal stability and reusability.
• Immobilization efficiency was confirmed by SDS-PAGE, and the reducing sugar assay was used in a continuous cycle experiment to evaluate the number of reuses and residual activity of the immobilized enzymes.

The overall goal of this project is to establish a chitinase system that is efficient in production, stable in application, and suitable for industrial scalability, thereby achieving efficient degradation and value-added utilization of natural chitin resources. Specific objectives include:
1.Screening highly efficient chitinase genes (AsCDA, Cts2p, and FjChiB) from various sources and constructing a pET-28a(+) recombinant expression system for efficient heterologous expression.
2.Optimizing culture and induction conditions to increase enzyme yield and activity; systematically analyzing the enzymes' optimal temperature, pH, and response to metal ions to provide experimental parameters for subsequent applications.
3.Exploring the synergistic degradation effects of the three enzymes and identifying the optimal combination and volume ratio.
4.Immobilizing the enzymes with the optimal synergistic ratio and evaluating their activity retention over multiple reaction cycles will provide a theoretical basis for industrial continuous production.

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