Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Chitosanase Assay
2.3. Production Condition Screening
2.4. Production Optimization
2.5. Chitosanase Purification
2.6. Mass Spectrometry and Protein Identification
2.7. Characterization of Paenibacillus elgii TKU051 Chitosanase
2.8. Thin-Layer Chromatography (TLC) Analysis of Hydrolysis Products
3. Results and Discussion
3.1. Screening of Carbon/Nitrogen Source and Culture Factors for the Chitosanase Productivity of Paenibacillus elgii TKU051
3.2. Production Optimization of Paenibacillus elgii TKU051 Chitosanase
3.3. Enzyme Purification and Identification
3.4. Biochemical Characteristic of Paenibacillus elgii TKU051 Chitosanase
3.5. Hydrolysis Products
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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Run | Coded Levels | Uncoded Levels | Chitosanase Activity (U/mL) | ||||||
---|---|---|---|---|---|---|---|---|---|
X1 | X2 | X3 | X4 | X1 | X2 | X3 | X4 | ||
1 | −1 | 0 | 1 | 0 | 4 | 8.6 | 40 | 6 | 1.140 |
2 | 0 | 0 | −1 | −1 | 5 | 8.6 | 34 | 5 | 0.712 |
3 | 0 | 0 | 0 | 0 | 5 | 8.6 | 37 | 6 | 1.966 |
4 | 0 | 0 | 0 | 0 | 5 | 8.6 | 37 | 6 | 1.740 |
5 | 0 | 1 | −1 | 0 | 5 | 9.6 | 34 | 6 | 0.826 |
6 | 0 | 1 | 0 | −1 | 5 | 9.6 | 37 | 5 | 1.162 |
7 | 0 | 1 | 0 | 1 | 5 | 9.6 | 37 | 7 | 0.426 |
8 | −1 | 0 | 0 | −1 | 4 | 8.6 | 37 | 5 | 1.300 |
9 | 0 | 0 | −1 | 1 | 5 | 8.6 | 34 | 7 | 0.230 |
10 | 1 | 0 | 0 | 1 | 6 | 8.6 | 37 | 7 | 0.656 |
11 | 1 | 0 | 0 | −1 | 6 | 8.6 | 37 | 5 | 1.106 |
12 | 0 | −1 | −1 | 0 | 5 | 7.6 | 34 | 6 | 0.910 |
13 | 0 | −1 | 0 | 1 | 5 | 7.6 | 37 | 7 | 0.376 |
14 | 0 | 0 | 1 | −1 | 5 | 8.6 | 40 | 5 | 1.426 |
15 | −1 | 0 | 0 | 1 | 4 | 8.6 | 37 | 7 | 0.053 |
16 | 1 | 1 | 0 | 0 | 6 | 9.6 | 37 | 6 | 1.576 |
17 | −1 | 1 | 0 | 0 | 4 | 9.6 | 37 | 6 | 0.680 |
18 | 1 | −1 | 0 | 0 | 6 | 7.6 | 37 | 6 | 1.063 |
19 | −1 | 0 | −1 | 0 | 4 | 8.6 | 34 | 6 | 0.736 |
20 | −1 | −1 | 0 | 0 | 4 | 7.6 | 37 | 6 | 1.413 |
21 | 0 | −1 | 1 | 0 | 5 | 7.6 | 40 | 6 | 0.613 |
22 | 0 | 1 | 1 | 0 | 5 | 9.6 | 40 | 6 | 1.486 |
23 | 0 | 0 | 1 | 1 | 5 | 8.6 | 40 | 7 | 0.283 |
24 | 1 | 0 | 1 | 0 | 6 | 8.6 | 40 | 6 | 1.480 |
25 | 0 | 0 | 0 | 0 | 5 | 8.6 | 37 | 6 | 1.880 |
26 | 1 | 0 | −1 | 0 | 6 | 8.6 | 34 | 6 | 0.683 |
27 | 0 | −1 | 0 | −1 | 5 | 7.6 | 37 | 5 | 0.944 |
Term | Coefficient Estimate | Standard Error Coefficient | t Value | Pr (>|t|) | |
---|---|---|---|---|---|
Constant | −102.340 | 19.185 | −5.335 | 0.0002 | *** |
X1 | −1.664 | 1.421 | −1.171 | 0.2644 | |
X2 | 2.406 | 1.614 | 1.491 | 0.1618 | |
X3 | 3.674 | 0.627 | 5.858 | 0.0001 | *** |
X4 | 9.819 | 1.466 | 6.696 | <0.0001 | *** |
X1X2 | 0.312 | 0.077 | 4.027 | 0.0017 | ** |
X1X3 | 0.033 | 0.026 | 1.270 | 0.2281 | |
X1X4 | 0.199 | 0.077 | 2.576 | 0.0243 | * |
X2X3 | 0.080 | 0.026 | 3.093 | 0.0093 | ** |
X2X4 | −0.042 | 0.077 | −0.543 | 0.5971 | |
X3X4 | −0.055 | 0.026 | −2.137 | 0.0539 | |
X12 | −0.332 | 0.067 | −4.955 | 0.0003 | *** |
X22 | −0.383 | 0.067 | −5.722 | 0.0001 | *** |
X32 | −0.056 | 0.007 | −7.495 | <0.0001 | *** |
X42 | −0.733 | 0.067 | −10.949 | <0.0001 | *** |
Source | Degrees of Freedom | Sum of Squares | Mean Square | F Value | Pr (>F) |
---|---|---|---|---|---|
Fo | 4 | 2.423 | 0.606 | 25.314 | <0.0001 |
TWI | 6 | 0.931 | 0.155 | 6.483 | 0.003 |
PQ | 4 | 3.303 | 0.826 | 34.504 | <0.0001 |
Residuals | 12 | 0.287 | 0.024 | ||
Lack of fit | 10 | 0.261 | 0.026 | 2.007 | 0.378 |
Pure error | 2 | 0.026 | 0.013 |
Factor | Before Optimization | After Optimization | |
---|---|---|---|
By OFAT | By RSM | ||
Amount of SPP (%) | 1 | 5 | 5.278 |
Initial pH | 7.6 | 8.6 | 8.93 |
Incubation temperature (°C) | 37 | 37 | 38 |
Incubation time (day) | 3 | 6 | 5.73 |
Chitosanase activity (U/mL) | 0.777 ± 0.076 | 1.899 ± 0.031 | 2.023 ± 0.072 |
Step | Total Protein (mg) | Total Activity (U) | Specific Activity (U/mg) | Recovery (%) | Purification (fold) |
---|---|---|---|---|---|
Cultural supernatant | 3541.060 | 772.933 | 0.218 | 100 | 1.000 |
(NH4)2SO4 precipitation | 642.982 | 314.496 | 0.489 | 41 | 2.241 |
High S column | 17.938 | 96.945 | 5.404 | 13 | 24.759 |
CM column | 1.782 | 44.588 | 25.019 | 6 | 114.619 |
Sephacryl S-200 column | 0.990 | 30.490 | 30.795 | 4 | 141.081 |
Matched Peptides Sequence | Identified Protein and Coverage Rate | Strain |
---|---|---|
74LINKPEQDDLNWIKYYGYCEDIEDERGYTIGLFGATTGGSR114 132GASNPSADGALKRLGINGKMKGSILEIKDSEK163 172LQNDAAWR179 193YSVEQAR199 252RTLVVDTNKYNKPPNGK268 273QWDTLVDMGK282 287NVDSEIAQVTDWEMK301 | Chitosanase CHIS_BACCI (GH46) 43% | Bacillus circulans |
Enzyme Source | Chitosan | DP of Major Product | Detection Method | References |
---|---|---|---|---|
P. elgii TKU051 | 98% DDA | 2–3 | TLC | This study |
S. hygroscopicus R1 | 95% DDA | 2–6 | TLC | [59] |
Renibacterium sp. Y82 | 95% DDA | 2–3 | TLC and ESI-MS | [15] |
Paenibacillus sp. TKU047 | 98% DDA | 2–4 | TLC | [8] |
P. dendritiformis | - | 2 | TLC and ESI-MS | [49] |
Bacillus sp. Q1098 | ≥95% DDA | 2–3 | TLC and ESI-MS | [52] |
P. barengoltzii | 85% DDA | 2–3 | TLC | [30] |
Bacillus cereus TKU034 | 90% DDA | 3–9 | MALDI-TOF-MS | [62] |
Pseudoalteromonas sp. SY39 | - | 2–3 | TLC and HPLC | [63] |
Bacillus sp. DAU101 | - | 2–6 | TLC | [53] |
Bacillus mycoides TKU038 | 85% DDA | 1–8 | HPLC and MALDI-TOF-MS | [64] |
Bacillus thuringiensis | ≥90% DDA | 2–5 | ESI-Q-TOF and HPAEC-PAD | [65] |
Streptomyces niveus | 94% DDA | 2–4 | TLC and HPLC | [13] |
Serratia sp. QD07 | ≥95% DDA | 2–3 | TLC | [14] |
Bacillus amyloliquefaciens | 2–3 | TLC | [66] |
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Doan, C.T.; Tran, T.N.; Tran, T.P.H.; Nguyen, T.T.; Nguyen, H.K.; Tran, T.K.T.; Vu, B.T.; Trinh, T.H.T.; Nguyen, A.D.; Wang, S.-L. Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii. Polymers 2023, 15, 3724. https://doi.org/10.3390/polym15183724
Doan CT, Tran TN, Tran TPH, Nguyen TT, Nguyen HK, Tran TKT, Vu BT, Trinh THT, Nguyen AD, Wang S-L. Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii. Polymers. 2023; 15(18):3724. https://doi.org/10.3390/polym15183724
Chicago/Turabian StyleDoan, Chien Thang, Thi Ngoc Tran, Thi Phuong Hanh Tran, Thi Thanh Nguyen, Huu Kien Nguyen, Thi Kim Thi Tran, Bich Thuy Vu, Thi Huyen Trang Trinh, Anh Dzung Nguyen, and San-Lang Wang. 2023. "Chitosanase Production from the Liquid Fermentation of Squid Pens Waste by Paenibacillus elgii" Polymers 15, no. 18: 3724. https://doi.org/10.3390/polym15183724