Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Microorganisms
2025 May 28;136:. doi: 10.3390/microorganisms13061235.
Show Gene links
Show Anatomy links
Multi-Method Combined Screening of Agarase-Secreting Fungi from Sea Cucumber and Preliminary Analyses on Their Agarases and Agar-Oligosaccharide Products.
He S
,
Lu T
,
Sun X
,
Ban F
,
Zhou L
,
Liu Y
,
Feng Y
,
Zhang Y
.
???displayArticle.abstract???
Agar can be degraded into agar-oligosaccharides by physical, chemical, and biological methods, but the further industrial application of agar-oligosaccharides has been limited by the environmental pollution of traditional agar-oligosaccharides preparation methods and the lack of novel agarase. In this study, we reported the screening of 12 strains with agar-degrading activity from sea cucumber intestine and mucus using a combination of Gram's iodine staining and 3,5-dinitrosalicylic acid (DNS) method, during which five fungal strains exhibited high agarase activity. Their production of different agarases and agar-oligosaccharides could be visualized by zymogram assay and thin-layer chromatography. A strain ACD-11-B with the highest agarase activity showed 99.79% similarity to Aspergillus sydowii CBS593.65 for ITS rDNA sequence. Strain ACD-11-B produced five possible agarases with predicted molecular weights of 180, 95, 43, 33, and 20 kDa, approximately. The optimal temperature and pH of the crude enzyme production by strain ACD-11-B were 40 °C and 6.0. The crude enzyme was stable at 30 °C, and Ca2+, K+, and Na+ could increase the activity of the crude enzyme. Its agarases demonstrated remarkable salt tolerance and substrate specificity, with neoagarobiose (NA2) identified as the main degradation product. These results indicate that the fungal strain ACD-11-B can secrete agarases with potential in industrial applications, making it a new producer strain for agarase production.
KCXFZ20240903093925033 the Sustainable Development Program of Shenzhen Science and Technology Major Program, 2025A1515010941 Guangdong Provincial 580 Natural Science Foundation, 2021ZDZX2064, 582 2024ZDZX2086 Special Project in Key Fields of 581 Guangdong Provincial Higher Education Institutions, 2022E05010 Zhanjiang Marine Youth Talent Innovation Project, JCYJ20220530162014032 the Basic Research Project of Shenzhen Science and Technology Innovation Commission, 2024B01265 Zhanjiang Science and Technology Plan Projects
Figure 1. Screening and identification of agarase-secreting strains. (A) The strains showed the distinct zones of agar degradation screened by Gram’s iodine staining. Pictures 1–12 are strains NY5-1, NY-13, NY-7, CD-1, ACD-2, ACD-3, ACD-5, ACD-7, ACD-8, ACD-11-Q, ACD-11-B, and ACD-12, respectively. (B) Taxonomical distribution of the agarase-secreting fungal strains.
Figure 2. Neighbor-joining phylogenetic tree of the 12 agarase-secreting fungi based on the ITS rDNA sequences. The neighbor-joining method was used to construct the phylogenetic tree using Mega 7 software. (Bootstrap values were calculated using 1000 replications).
Figure 3. Quantification of agarase activity of the strains fermented in shaking flasks. (A) The clearance zones of twelve strains were determined by Oxford cup diffusion method using Gram’s iodine staining (Oxford cups were removed after staining; unlabeled clearance zones were produced by other sea cucumber symbiotic fungi, which had low agarase activities); (B) quantification of agarase activities evaluated by Oxford cup diffusion method and DNS method. The enzyme activities of the strain ACD-11-B detected by both methods were taken as 100%, respectively.
Figure 4. SDS–PAGE and agarase zymogram analysis of extracellular proteins of the strains ((A1,A2) ACD-11-B; (B1,B2) ACD-11-Q; (C1,C2) ACD-12; (D1,D2) NY-7; (E1,E2) ACD-7). Lane (M), protein marker; for each strain, lane 1: SDS–PAGE of extracellular protein of strain with staining by CBB R-250 (for (D1)) or Fast Silver Stain Kit (for (A1,B1,C1,E1)); lane 2: zymogram analysis of extracellular protein of strain. The places indicated by the blue arrows represent locations where the strain may produce agarase. After the enzymatic reaction, the agar plates were stained using Gram’s iodine staining.
Figure 5. Agar hydrolytic product analysis of agarase-secreting strains. (A) TLC analysis of the hydrolysates by the crude agarases of strains ACD-11-B, ACD-12, NY-7, ACD-7, and ACD-11-Q. Lane M: standard marker; lane A: agar without enzyme treatment; lane 1–8: agar with enzyme incubation for 0 min, 15 min, 30 min, 1 h, 6 h, 12 h, 24 h, 48 h, respectively. The places indicated by black arrows represent the locations of the main enzymatic products. (B) HPLC analysis of the 24 h hydrolysates by crude agarase of ACD-11-B.
Figure 6. Characterization of the crude agarase of strain ACD-11-B. (A) The effect of temperature on the enzymatic activity of crude agarase. The activity was tested from 30 °C to 80 °C. (B) The effect of temperature on the enzymatic stability of crude agarase. The residual activity was detected after incubating at 30 °C, 40 °C, or 50 °C at different times (0–6, 12, 24, 36, and 48 h). (C) The effect of pH on the enzymatic activity of crude agarase. The activity was tested by incubating crude agarase at 40 °C in these buffers: citric acid/Na2HPO4 buffer (pH 3.0–8.0); Tris-HCl buffer (pH 7.0–9.0); NaOH/Gly buffer (pH 9.0–11.0). (D) The storage stability of crude agarase under different pH conditions. The remaining activity was detected after incubating at 4 °C for 12 h in specific buffers (pH 3.0–11.0). (E) The effect of metal ions and chemical agents on the activities of crude agarase. (F) Effects of NaCl on the activity of crude agarase. (G) Substrate specificity of crude agarase towards agar, sodium alginate, carrageenan, and cellulose. For all of the above plots, the maximal activity of crude agarase was taken as 100%, and other values were indicated as the ratio of the maximum. Data are expressed as SD ± mean (n = 3), “*” indicates significant differences between the sample-treated group and the control group (“*” represents p < 0.05, “**” represents p < 0.01, “***” represents p < 0.001).
