Enzymatic Production of Chitooligosaccharide Using a GH Family 46 Chitosanase from Paenibacillus elgii and Its Antioxidant Activity

Abstract

Chitooligosaccharide (COS), a natural antioxidant, is a hydrolysis product of chitosan created using enzymatic or chemical methods. COS has received considerable attention recently, making its efficient bioproduction of great value. This study investigated the optimal conditions for the enzymatic method using a GH family 46 chitosanase from Paenibacillus elgii TKU051 to prepare COS based on the response surface methodology (RSM). The results showed optimal values for chitosan hydrolysis, such as a pH of 5.5, an incubation temperature of 58.3 °C, an [E]/[S] ratio of 118.494 (U/g), and an incubation time of 6.821 h. Under the optimal conditions, the highest reducing sugar level (per substrate, w/w) of the chitosan hydrolysis process that could be reached was 690.587 mg/g. The composition of the obtained COS was analyzed using the thin-layer chromatography (TLC) method, yielding (GlcN)₂ and (GlcN)₃ as the products. The ascorbic acid equivalent antioxidant capacity (AEAC) of the obtained COS was found to be 1246 mg/100 g (via a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay) and 3673 mg/100 g (via an ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical-scavenging assay). This green and efficient bioproduction method may possess excellent potential for application in bioactive COS preparation.

1. Introduction

Chitin and chitosan are the second most prevalent polysaccharides in nature, and they are mainly obtained from the exoskeletons of crustaceans. Chitosan has garnered much interest as a functional biopolymer that can be used for diverse purposes. However, in several applications, the use of chitosan is constrained by its water-insoluble characteristics. Chitooligosaccharide (COS) is typically composed of glucosamines linked by β-1,4 glycosidic bonds, with degrees of polymerization (DP) ranging from 2 to 20 [1]. They are the degraded products resulting from the chemical or enzymatic hydrolysis of chitosan [2]. Due to their distinctive physicochemical characteristics, which include low viscosity, high water solubility, biocompatibility, and biodegradability [3], COSs have garnered significant research interest, setting them apart from chitosan. In terms of biological activity, COS has demonstrated several activities, such as antioxidant, antibacterial, antifungal, anti-inflammatory, anti-cancer, and so on [4,5,6]. As expected, in recent times, research on the applications of COS has focused on various fields, including healthcare [7,8], food [9,10], agriculture [11,12], and other life-sciences-related domains [13].

Chemical hydrolysis, primarily through acid hydrolysis, is widely used in COS production due to its cost and convenience. However, it yields complex components, poses purification challenges, and causes environmental issues. In contrast, enzymatic hydrolysis is non-toxic, controllable, and does not require salt removal. Furthermore, the enzymatic method can enhance chitosan’s functional properties and yield significant quantities of chitooligosaccharides without altering chitosan’s glucose ring or biological activity [14]. Several types of hydrolytic enzymes can break down chitosan to produce COS; among them, chitosanase (EC 3.2.1.132) is known for its high specificity to chitosan, making it particularly well-suited for precise chitosan degradation to yield COS [15,16].

Optimizing enzymatic reactions, which are highly sensitive to temperature, pH, substrate properties, and enzyme type, can be complex and time-consuming. Employing the one-factor-at-a-time (OFAT) approach provides information about individual factors during the experiment but does not consider the potential interactions between various factors that can influence enzyme activity. In contrast, the statistical design of experiments (DoE) is a statistical tool that allows for a faster evaluation of both the effects on and significance of experimental variables with respect to the response [17].

While response surface methodology (RSM) holds significance in optimizing enzyme reaction conditions, there have been limited publications on optimizing reaction conditions for chitosanase in the context of COS preparation. Liu et al. (2014) used the central composite design of RSM to optimize the production of hexaoligochitin using ASCHI61 (a chitinase derived from Aeromonas schubertii) [18]. In a previous study, the fermentation conditions for synthesizing P. elgii TKU051 chitosanase were optimized using RSM’s Box–Behnken design (BBD). P. elgii TKU051 chitosanase is a GH46 chitosanase, and its main reaction products are (GlcN)₂ and (GlcN)₃ [15]. However, the conditions for preparing COS using P. elgii TKU051 chitosanase and assessing the biological activity of the obtained COS have not been investigated. In this study, the optimization of COS preparation utilizing P. elgii TKU051 chitosanase was carried out via RSM-BBD. The key parameters examined encompassed pH, temperature, [E]/[S] ratio, and incubation time. The optimal conditions were forecasted through empirical modeling and statistical analysis and verified through experimental validation. Additionally, the antioxidant potential of the obtained COS was assessed and compared to that of other antioxidants.

2. Results and Discussion

2.1. Optimization via One-Factor-at-a-Time Method

The reducing sugar changes at different pH points are shown in Figure 1a. Initially, the reducing sugar levels increased with an increase in pH (from 3 to 5.5) and reached a maximum value of 30.404 mg/g (reducing sugar/substrate, w/w) when the pH value was 5.5. Then, the reducing sugar levels decreased significantly with a further increase in the pH value (from 5.5 to 8). Thus, the optimal pH was set to pH 5.5 to obtain a high reducing sugar level. Figure 1b shows the effect of temperature on reducing sugar. The highest reducing sugar level (114.193 mg/g) was achieved at 60 °C. To check the optimum [E]/[S] ratio, an experiment was carried out in the range of 5–250 U/g. It was observed that the maximum reducing sugar level (118.713 mg/g) could be reached at an [E]/[S] ratio of 250 U/g (Figure 1c). However, there were no significant differences in the reducing sugar values at 250 U/g compared with those at 100 U/g, 150 U/g, and 200 U/g (112.930 mg/g, 113.867 mg/g, and 114.071 mg/g, respectively). Since a lower [E]/[S] ratio requires less of the enzyme than a higher one, an [E]/[S] ratio of 100 U/g was selected for further experiments. Figure 1d reveals the effect of incubation time on the reducing sugar levels. From 0 h to 4 h, it can be seen that the reducing sugar level increases rapidly. However, over time, the decrease in substrate concentration prevented a higher yield from being obtained [19]. The highest reducing sugar value (660.629 mg/g) was observed at an incubation time of 6 h. After conducting OFAT experiments, the optimal conditions for chitosan hydrolysis were determined: a pH of 5.5, a temperature of 60 °C, an [E]/[S] ratio of 100 U/g, and an incubation time of 6 h. These conditions resulted in the highest reducing sugar level, 660.629 mg/g.

2.2. Optimization via Response Surface Methodology

The preliminary experimental results revealed the efficacy of chitosanase for chitosan hydrolysis under the reaction conditions (concerning pH, temperature, [E]/[S] ratio, and incubation time). However, the effect of the interaction between the factors remains unclear. Therefore, the reaction conditions were optimized based on RSM’s Box–Behnken design (BBD). The BBD was established using SigmaXL software (version 10.02), with 27 runs, including three center points and 24 base runs, as shown in

Table 1. Box–Behnken design and experiment result.

Run	pH	Temperature (°C)	[E]/[S] Ratio (U/g)	Incubation Time (h)	Reducing Sugar (mg/g)
1	6	60	50	6	452.076
2	5.5	60	100	6	648.131
3	5.5	50	100	8	475.012
4	5	60	100	4	431.106
5	6	60	150	6	473.462
6	6	70	100	6	128.964
7	6	60	100	4	453.864
8	5.5	70	150	6	226.115
9	5.5	70	50	6	158.413
10	5	60	100	8	462.393
11	5.5	60	50	4	470.580
12	5.5	70	100	4	217.208
13	5	60	50	6	311.006
14	6	50	100	6	318.300
15	6	60	100	8	544.153
16	5	50	100	6	277.955
17	5.5	60	150	4	601.567
18	5.5	50	150	6	487.753
19	5.5	60	100	6	669.128
20	5.5	50	100	4	366.947
21	5.5	50	50	6	375.350
22	5	70	100	6	98.181
23	5.5	70	100	8	260.548
24	5.5	60	150	8	624.451
25	5.5	60	50	8	609.232
26	5.5	60	100	6	660.545
27	5	60	150	6	561.195

. The influence of the input factors (in regard to pH, temperature, [E]/[S] ratio, and incubation time) on the dependent variable (reducing sugar) was analyzed. The following quadratic equation generated using BBD represents the experimental data:

Reducing sugar (mg/g) = 659.268 + 19.082 × A − 100.991 × B + 49.824 × C + 36.210 × D − 2.391 × A × B −
57.201 × A × C + 14.751 × A × D − 11.175 × B × C − 16.181 × B × D − 28.942 × C × D − 156.628 × A² − 296.867 ×
B² − 51.810 × C² − 31.078 × D²

(1)

where A denotes pH; B denotes temperature (°C); C denotes the [E]/[S] ratio (U/g); and D denotes incubation time (h).

According to

Table 2. Regression and ANOVA analysis.

Parameter Estimates
Term	Coefficient	SE Coefficient	T	p	VIF	Tolerance
Constant	659.268	11.784	55.946	<0.0001
A	19.082	5.892	3.239	0.0071	1.00	1.00
B	−100.991	5.892	−17.140	<0.0001	1.00	1.00
C	49.824	5.892	8.456	<0.0001	1.00	1.00
D	36.210	5.892	6.146	<0.0001	1.00	1.00
A × B	−2.391	10.205	−0.234	0.8187	1.00	1.00
A × C	−57.201	10.205	−5.605	0.0001	1.00	1.00
A × D	14.751	10.205	1.445	0.1740	1.00	1.00
B × C	−11.175	10.205	−1.095	0.2950	1.00	1.00
B × D	−16.181	10.205	−1.586	0.1388	1.00	1.00
C × D	−28.942	10.205	−2.836	0.0150	1.00	1.00
A2	−156.628	8.838	−17.722	<0.0001	1.25	0.80
B2	−296.867	8.838	−33.590	<0.0001	1.25	0.80
C2	−51.810	8.838	−5.862	0.0001	1.25	0.80
D2	−31.078	8.838	−3.516	0.0043	1.25	0.80
Analysis of Variance
Source	Degrees of freedom (DF)	Sum of squares (SS)	Mean square (MS)	F		p
Model	14	738,051.414	52,717.958	126.546		<0.0001
Error	12	4999.084	416.590
Lack of Fit	10	4776.217	477.622	4.286		0.2039
Pure Error	2	222.867	111.434
Total (Model + Error)	26	743,050.498	28,578.865
Model Summary
R2			99.33%
Adjusted R2			98.54%
Predicted R2			96.23%
S (Root Mean Square Error)			20.411

A, pH; B, temperature (°C); C, [E]/[S] ratio (U/g); D, incubation time (h).

, the R², adjusted R², and predicted R² indices (99.33%, 98.54%, and 96.23%, respectively) all show that the regression model is suitable and highly accurate. The low S value (20.411) also strengthens this conclusion. This model can be considered reliable and effective for predicting a response based on the independent variables. Furthermore, the quadratic model equation is significant, with an F-value of 126.546. The very-low p-value (<0.0001) confirms that the model terms were statistically significant. The p-value of the lack-of-fit test result is also insignificant (0.2039), further supporting the proposed model’s suitability.

Each factor in the regression model was tested to evaluate its influence and interaction. Factors with a p-value less than 0.05, such as A, B, C, D, A × C, C × D, A², B², C², and D², were considered statistically significant and affected the quadratic equation. Conversely, factors A × B, A × D, B × C, and B × D were insignificant. Thus, the quadratic equation could be refined as

Reducing sugar (mg/g) = 659.268 + 19.082 × A − 100.991 × B + 49.824 × C + 36.210 × D − 57.201 × A × C −
28.942 × C × D − 156.628 × A² − 296.867 × B² − 51.810 × C² − 31.078 × D²

(2)

where A is pH; B denotes temperature (°C); C stands for the [E]/[S] ratio (U/g); and D is incubation time (h), with an R² = 99.00%, an adjusted R² = 98.37%, a predicted R² = 96.70%, an F-value = 158.215, and a p-value < 0.00001.

The normal probability plot in the BBD matrix reveals a robust linear pattern with minimal deviations (Figure 2a). Furthermore, the data points of the standardized residual plot shown in Figure 2b do not follow any pattern and are symmetrically distributed along the center line. Thus, it could be confirmed that the chosen model is appropriate for the data.

From the quadratic model, three-dimensional (3D) response surface plots could be built to analyze the relationship between reaction parameters and responses [20]. Figure 3a shows that pH and temperature were interpreted in the range of 5–6 and 50–70 °C, respectively, with the [E]/[S] ratio and incubation time fixed at 100 U/g and 6 h. Significantly, raising the pH and temperature led to an increase in the levels of reducing sugar until the maximum value was reached (667.934 mg/g) when the pH and temperature were 5.55 and 58 °C. A continued increase in pH and temperature resulted in a corresponding decrease in the levels of reducing sugar. Figure 3b shows the effect of pH (5–6) and [E]/[S] ratio (50–150 U/g) on reducing sugar when the temperature and incubation time were fixed at 60 °C and 6 h, respectively. It can be seen that the interaction effect between pH and the [E]/[S] ratio significantly affects the response; the highest reducing sugar level (668.709 mg/g) was achieved at pH 5.5 and an [E]/[S] ratio of 125 U/g. Figure 3c presents the effect of pH (5–6) and incubation time (4–8 h) on the production of reducing sugar. The temperature and [E]/[S] ratio were fixed at 60 °C and 100 U/g, respectively. The maximum reducing sugar level (669.806 mg/g) was obtained when the pH and incubation time were 5.5 and 7.2 h, respectively. Figure 3d shows the effect of different values of temperature and [E]/[S] ratio on the production of reducing sugar, revealing a maximum value of 679.551 mg/g at a temperature of 58 °C and a [E]/[S] ratio of 125 U/g. Figure 3e presents the effect of temperature and incubation time on reducing sugar production. The RSM plot was generated with fixed pH (5.5) and [E]/[S] ratio (100 U/g) values and various values of temperature (50–70 °C) and incubation time (4–8 h) to investigate reducing sugar production. The highest reducing sugar level (678.129 mg/g) was achieved at a temperature of 58 °C and an incubation time of 7.2 h. Finally, the interaction effect between the [E]/[S] ratio and incubation time on reducing sugar production is illustrated in Figure 3f. The maximum reducing sugar level (675.455 mg/g) was obtained when an [E]/[S] ratio of 120 U/g and an incubation time of 7 h were used. In conclusion, the optimal values of the factors are as follows: a pH range of 5.50–5.55, a temperature of 58 °C, an [E]/[S] ratio of 120–125 U/g, and an incubation time of 7–7.2 h.

2.3. Model Validation and Product Analysis

From the quadratic equation, the optimal values of pH, temperature, [E]/[S] ratio, and incubation time were estimated to be 5.50, 58.3 °C, 118.494 U/g, and 6.821 h, respectively, with a maximum predicted reducing sugar level of 684.437 mg/g. To validate the predicted model, an experiment was conducted in triplicate under the optimal conditions suggested by the model. The results showed that the reducing sugar level was 690.587 ± 10.937 mg/g, which closely matched the predicted values, demonstrating the accuracy and reliability of the model. There are few reports on the statistical optimization of chitosan hydrolysis using the enzymatic method. By using the central composite design of RSM, the optimal conditions for chitosan hydrolysis using cellulase were determined to be a pH of 5.9, a temperature of 49 °C, an [S] of 0.76%, an [E] of 8.97 U/g, and an incubation time of 180 min [21]. The ideal conditions for the hydrolysis of chitosan via papain determined using the BBD were a temperature of 45 °C, pH 4.5, an [S] of 8.0 g/L, an [E]/[S] ratio of 0.10, and a reaction time of 45 min [22]. In a previous study, P. elgii chitosanase was identified as an endo-type enzyme that catalyzes the hydrolysis of chitosan into low-molecular-weight COS [15]. The yield of COS production varied from 24% to 100% [15]. This indicates that the COS production yield in this study (around 69%) was acceptable. Figure 4 reveals similarities to that study, with the COS composition consisting of (GlcN)₂ and (GlcN)₃. Thus, determining the optimal conditions for chitosan hydrolysis using this enzyme is essential for further application in COS production.

Due to its considerable effectiveness and uncomplicated procedure, chemical hydrolysis is commonly chosen for the degradation of chitin and chitosan. Nonetheless, it poses several obstacles, including difficulty in controlling the reactions, which may yield undesirable by-products that impede the purification of COS. Furthermore, chemical hydrolysis results in a heterogeneous array of molecules exhibiting diverse levels of polymerization and deacetylation, significantly impairing biological function caused by chemical alterations. Sánchez et al. (2017) indicated that COS prepared by the enzymatic method revealed superior antibacterial efficacy relative to the chemical-enzymatic approach [23]. Furthermore, COS-HCl has a bitter taste, restricting its applicability [24]. To address these issues, enzyme-based approaches can be used to help produce COS with desired physicochemical and biological characteristics [25]. In this study, the COS produced consisted of only two main components, (GlcN)₂ and (GlcN)₃; the monomer form (GlcN) was not present. Thus, we have provided an effective method of preparing COSs with low molecule weight.

Furthermore, the primary economic challenge in COS production via enzymatic methods is the cost of enzymes. Our previous study indicated that P. elgii TKU051 could be used to generate chitosanase economically by utilizing seafood by-products as a low-cost nutritional source [15]. In conjunction with the optimal results from this study, producing COS using P. elgii TKU051’ chitosanase seems to be an economically viable and attractive approach.

2.4. Antioxidant Activity of the Chitosan Oligosaccharide

In this experiment, the free radical (ABTS and DPPH)-scavenging activities of the obtained COS (COS1) and a commercial food-grade COS (COS2, Charming & Beauty Co. (Taipei, Taiwan)) were explored. As illustrated in Figure 5, both COS1 and COS2 were shown to have dose-dependent radical-scavenging activity in DPPH and ABTS assays. Compared to the commercial COS, the COS prepared from chitosan hydrolysis using P. elgii TKU051 chitosanase exhibited more significant activity. COS1 was shown to have IC₅₀ values of 4.862 mg/mL and 4.166 mg/mL in ABTS and DPPH assays, respectively, which are significantly less than those of COS2 (13.817 mg/mL in the ABTS assay and 13.304 mg/mL in the DPPH assay). The antioxidant activity of COS relates to its degree of polymerization (DP) and degree of deacetylation (DD). COS with a lower DP and a higher DD could have superior antioxidant capacity [26,27]. In this study, COS1 was produced from 98% DD chitosan and composed of (GlcN)₂ and (GlcN)₃, indicating its potential antioxidant activity. Indeed, its antioxidant capacity, as determined via ABTS and DPPH assays, is better than that of the COS from a commercial source (COS2). It should be noted that COS2 has a DP range of 2–6 and a DD of 75% [28].

It is rather surprising that, despite numerous reports on the antioxidant activity of COS, there appear to be no reports addressing its ascorbic acid equivalent antioxidant capacity (AEAC). This could pose challenges when comparing the antioxidant capacity of COS to that of other antioxidants. Therefore, in this study, the AEAC of the obtained COS was also determined by using the corresponding IC₅₀ values and correlating them with the IC₅₀ of ascorbic acid (AA). The AEAC value was expressed as the equivalent number of milligrams of AA present in 100 g of COS. According to the results of the DPPH assay, the AEAC of COS1 reached a value of 1246.926 ± 26.456 mg AA/100 g, whereas its value according to ABTS was 3673.483 ± 268.560 mg AA/100 g (

Table 3. Ascorbic acid equivalent antioxidant capacity (AEAC) of COS1, COS2, and other antioxidants.

	AEAC (mg AA/100 g)		Ref.
	DPPH Assay	ABTS Assay
COS1	1246.926 ± 26.456	3673.483 ± 268.560
COS2	391.311 ± 19.457	1286.665 ± 32.430
Golden grass (Syngonanthus nitens)	1485 ± 198		[31]
Tangerine peel	913 ± 84		[31]
Banana pulp	165 ± 111		[31]
Banana peel	883 ± 150		[31]
Fresh Gala apples	136.0 ± 6.6	205.4 ± 5.6	[29]
Gooseberry	111.25		[32]
Sideritis hyssopifolia		1.808–2.419	[33]
Star fruit residue	3412 ± 290	3490 ± 310	[34]
Grapefruit peel	9.17 ± 0.19	10.79 ± 0.56	[35]

). A similar phenomenon was also found in fresh Gala apples, with AEAC values of 136.0 ± 6.6 mg/100 g in a DPPH assay and 205.4 ± 5.6 mg/100 g in an ABTS assay [29]. Interestingly, according to the ABTS assay, the AEAC of COS1 (3673.483 ± 268.560) is significantly higher than that of COS2 (1286.665 ± 32.430 mg AA/100 g), fresh Gala apples (205.4 ± 5.6 mg AA/100 g), Sideritis hyssopifolia (1.808–2.419 mg AA/100 g), and grapefruit peel (10.79 ± 0.56 mg AA/100 g) and similar to that of star fruit residue (3490 ± 310 mg AA/100 g). According to the results of the ABTS assay, the AEAC of COS1 (1246.926 ± 26.456 mg AA/100 g) was only lower than that of star fruit residue (3412 ± 290 mg AA/100 g). Therefore, the obtained COS can be deemed an excellent natural antioxidant. It is noteworthy that COS is derived from chitosan, a polymer produced from chitinous materials such as shrimp and crab shells. With the global shrimp harvest projected to reach 7.28 million tons by 2025 [30], the resulting shrimp waste could provide a vast chitosan supply. As a result, using COS as an antioxidant offers certain advantages over plant-based antioxidants, for which supply limitations may be encountered.

3. Materials and Methods

3.1. Chemicals and Reagents

All chemicals used were of the highest quality available. Food-grade chitosan oligosaccharide (COS2) was purchased from Charming & Beauty Co. (Taipei, Taiwan). Ninety-eight percent DD chitosan and Peanibacillus elgii TKU051 chitosanase were obtained from the Microorganisms and Biochemistry Laboratory, Department of Chemistry, Tamkang University, New Taipei, Taiwan. The chitosan solution was prepared by dissolving chitosan powder in 1% (v/v) acetic acid solution.

3.2. Reducing Sugar Assay

The 3,5-dinitrosalicylic acid (DNS) method [36] was used to measure the amount of reducing sugar in the reaction solution.

3.3. Optimization via One-Factor-at-a-Time Method

Initial reaction conditions were pH 7, temperature of 40 °C, [E]/[S] ratio of 100 U/g, and incubation time of 30 min. To investigate the conditions for the hydrolysis of chitosan by P. elgii TKU051 chitosanase, we tested various conditions, such as pH (3–8), temperature (30–80 °C), [E]/[S] ratio (10–250 U/g), and incubation time (0–12 h). Each factor was tested independently, and the condition that corresponded to the highest reducing level sugar was chosen for further experiments. When testing one factor, the other factors were kept constant. All tests were conducted in triplicate.

3.4. Optimization via Response Surface Methodology

Box–Behnken design (BBD) of the response surface methodology (RSM) was employed to optimize the critical parameters in the process of hydrolyzing chitosan using P. elgii TKU051 chitosanase. The parameters, including pH, temperature, [E]/[S] ratio, and incubation time, were varied to assess their impact on hydrolysis efficiency. The ranges of variables are as follows: pH (5, 5.5, and 6), temperature (50 °C, 60 °C, and 70 °C), [E]/[S] ratio (50 U/g, 100 U/g, and 150 U/g), and incubation time (4 h, 6 h, and 8 h). SigmaXL (version 10.02) was employed to analyze data and establish optimal conditions for reducing-sugar formulation. The following quadratic formula represents the impact of the four tested variables:

Reducing sugar (mg/g) = β₀ + β₁ × A + β₂ × B + β₃ × C + β₄ × D + β₁₂ × A × B + β₁₃ × A × C+ β₁₄ × A × D + β₂₃ × B
× C + β₂₄ × B × D + β₃₄ × C × D + β₁₁ × A² − β₂₂ × B² − β₃₃ × C² − β₄₄ × D²

(3)

where A denotes pH; B denotes temperature (°C); C denotes [E]/[S] ratio (U/g); D denotes incubation time (h); β₀ denotes the intercept; β₁, β₂, β₃, and β₄ are linear coefficients; β₁₂, β₁₃, β₁₄, β₂₃, β₂₄, and β₃₄ are interactive coefficients; and β₁₁, β₂₂, β₃₃, and β₄₄ are quadratic coefficients.

3.5. Thin Layer Chromatography Analysis

The TLC method was described in our previous report [28].

3.6. Antioxidant Assay

The DPPH and ABTS radical-scavenging-activity assays were employed according to a previous study [37]. The ascorbic acid equivalent antioxidant capacity (AEAC) was defined as the amount of ascorbic acid (in mg) that produces the same effect as 100 g of a sample [31].

4. Conclusions

In this study, we optimized the reaction conditions of chitosan hydrolysis using P. elgii TKU051 chitosanase. By utilizing RSM-BBD modeling in combination with experimental validation, the ideal reaction conditions for chitosan hydrolysis were found to be pH 5.50, a temperature of 58.3 °C, an [E]/[S] ratio of 118.494 U/g, and an incubation time of 6.821 h. The obtained COS also showed vigorous antioxidant activity; thus, it can be expected to be developed into a natural antioxidant.