Abstract
Alginate lyases are enzymes that hydrolyze alginate polysaccharides. Alginate lyase finds application as a biocatalyst in the production of biochemicals and biofuels and, as an agent in the control of alginate fouling. Furthermore, alginate lyases could be used as potential therapeutic agents for treating P. aeruginosa infections, notably in the treatment of cystic fibrosis by disruption of alginate biofilm and therefore enhancing the efficacy of anti-pseudomonal antibiotics. However, the commercial application of the enzyme is constrained due to high production costs and poor productivity. This issue could be addressed by employing high-yielding strains, optimizing the process parameters and media, and controlling systems. Marine microbes are known to produce alginate lyase for the utilization of seaweed alginate. In this perspective, different sponges and seaweeds were collected from Tuticorin, Gulf of Mannar, Tamilnadu, India, and primarily screened for alginate-degrading bacteria using Gram’s Iodine method. Further screening of potential isolates was done by evaluation of alginate lyase activity using DNS assay. The potential marine bacterium was identified as Enterobacter tabaci RAU2C using biochemical tests followed by 16S rRNA sequencing. The initial pH for the production of alginate lyase was optimized. The Plackett–Burman design was used to screen nitrogen and mineral sources for the production of alginate lyase. Central Composite Design was formulated using Design Expert software for five variables to optimise the concentration of media components. Response Surface Methodology was used to estimate optimal concentrations, which were then confirmed by experiments. ANFIS modelling was established using MATLAB to further analyze and predict the composition of variables for enhancing enzyme activity. The optimized media constituents were found to be sodium alginate-5 g/l, peptone-15 g/l, ammonium sulfate-8 g/l, sodium chloride-30 g/l, and dipotassium hydrogen phosphate 4 g/l with an enzyme activity of 12.5 U/ml. Furthermore, the process parameters such as production time, inoculum volume, and age of the inoculum were also optimized for maximizing the alginate lyase production.
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig1_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig2_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig3_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig4_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig5_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig6_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig7_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig8_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig9_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig10_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig11_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig12_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig13_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig14_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig15_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig16_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig17_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig18_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig19_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig20_HTML.png)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs10499-023-01302-5/MediaObjects/10499_2023_1302_Fig21_HTML.png)
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this manuscript.
References
Aarstad OA, Tondervik A, Sletta H, Skjak-Braek G (2012) Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes. Biomacromol 13:106–116. https://doi.org/10.1021/bm2013026
Alkawash MA, Soothill JS, Schiller NL (2006) Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS 114:131–138. https://doi.org/10.1111/j.1600-0463.2006.apm_356.x
Ashour SM, Kheiralla ZMH, Eldiwany AI, Maany Dina A (2014) Production, purification and characterization of polysaccharide lytic enzymes of a marine isolate, Bacillus cereus NRC-20 and their application in biofilm removal. African Journal of Microbiology Research 8(26):2492–2504. https://doi.org/10.5897/ajmr12.953
Beltagy EA, El-Borai A, Lewiz M et al (2016) Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04. Biologia Futura 67:305–317. https://doi.org/10.1556/018.67.2016.3.8
Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA (2008) Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta 76(5):965–977. https://doi.org/10.1016/j.talanta.2008.05.019
Buchanan RE, Gibbons NE (1994) Bergey’s manual of determinative bacteriology. 9:1268
Courtois J (2009) Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology. Curr Opin Microbiol 12:261–273. https://doi.org/10.1016/j.mib.2009.04.007
Draget KI, Skjåk-Bræk G, Stokke BT (2006) Similarities and differences between alginic acid gels and ionically crosslinked alginate gels. Food Hydrocoll 20:170–175. https://doi.org/10.1016/j.foodhyd.2004.03.009
Ertesvåg H, Høidal HK, Skjåk-Braek G, Valla S (1998) The Azotobacter vinelandii mannuronan C-5-epimerase AlgE1 consists of two separate catalytic domains. J Biol Chem 273(47):30927–30932. https://doi.org/10.1074/jbc.273.47.30927
Farrell EK, Tipton PA (2012) Functional characterization of AlgL, an alginate lyase from Pseudomonas aeruginosa. Biochemistry 51:10259–10266. https://doi.org/10.1021/bi301425r
Gan CY, Latiff AA (2011) Optimisation of the solvent extraction of bioactive compounds from Parkiaspeciosa pod using Response surface methodology. Food Chem 124(3):1277–1283. https://doi.org/10.1016/j.foodchem.2010.07.074
Ganesan V, Gurumani V, Kunjiappan S, Panneerselvam T, Somasundaram B, Kannan S, Chowdhury A, Saravanan G, Bhattacharjee C (2018) Optimization and analysis of microwave-assisted extraction of bioactive compounds from Mimosa pudica L. using RSM & ANFIS modeling. J Food Meas Charact 12(1):228–242. https://doi.org/10.1007/s11694-017-9634-y
Huang L, Zhou J, Li X, Peng Q, Lu H, Du Y (2013) Characterization of a new alginate lyase from newly isolated Flavobacterium sp S20. J Ind Microbiol Biotechnol 40(1):113–122. https://doi.org/10.1007/s10295-012-1210-1
Huang G, Wen S, Liao S, Wang Q, Huang S (2019) Characterization of a bifunctional Alginate lyase as a new member of the polysaccharide lyase family 17 from a marine strain BP-2. Biotechnol Lett 41:1187–1200. https://doi.org/10.1007/s10529-019-02722-1
Hutcheson SW, Zhang H, Suvorov M (2011) Carbohydrase systems of Saccharophagus degradans degrading marine complex polysaccharides. Mar Drugs 9:645–665. https://doi.org/10.3390/md9040645
Inoue A, Mashino C, Kodama T, Ojima T (2011) Protoplast preparation from Laminaria japonica with recombinant alginate lyase and cellulase. Marine Biotechnol 13:256–263
Islan GA, Bosio VE, Castro GR (2013) Alginate lyase and ciprofloxacin co-immobilization on biopolymeric microspheres for cystic fibrosis treatment. Macromol Biosci 13:1238–1248. https://doi.org/10.1002/mabi.201300134
Iwamoto M, Kurachi M, Nakashima T, Kim D, Yamaguchi K, Oda T, Iwamoto Y, Muramatsu T (2005) Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells. FEBS Lett 579(20):4423–4429. https://doi.org/10.1016/j.febslet.2005.07.007
Kawada A, Hiura N, Tajima S, Takahara H (1999) Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells. Arch Dermatol Res 291:542–547. https://doi.org/10.1007/s004030050451
Khoramnia A, Lai OM, Ebrahimpour A, Tanduba CJ, Voon TS, Mukhlis S (2010) Thermostable lipase from a newly isolated Staphylococcus xylosus strain; process optimization and characterization using RSM and ANN. Electron J Biotechnol 13:1–16. https://doi.org/10.2225/vol13-issue5-fulltext-22
Lange B, Wingender J, Winkler UK (1989) Isolation and characterization of an alginate lyase from Klebsiellaaerogenes. Arch Microbiol 152:302–308
Li JW, Dong S, Song J, Li CB, Chen XL et al (2011a) Purification and characterization of a bifunctional alginate lyase from Pseudoalteromonas sp. SM0524. Mar Drugs 9:109–123. https://doi.org/10.3390/md9010109
Li L, Jiang X, Guan H, Wang P (2011b) Preparation, purification and characterization of alginate oligosaccharides degraded by alginate lyase from Pseudomonas sp. HZJ 216. Carbohydr Res 346:794–800. https://doi.org/10.1016/j.carres.2011.01.023
Li H, Zhu S, Liu X, Gong J, Jiang M, Xu Z, Shi J (2014) Identification of an alginate lyase producing strain Halomonas sp. WF6 and fermentation optimization. China Biotechnology 34(9):94–101. https://doi.org/10.13523/j.cb.20140914
Loganathan C, Girija K (2013) Hybrid learning for adaptive neuro-fuzzy interference system. Int J Eng Sci 2(11):6–13
Ma Y, Ji T, Li H, Chen W, Gao S, Liu S (2009) Culture optimization and characterization of an alginate-lyase from Pseudoalteromonas sp. LJ1. Acta Microbiol Sin 49:1086–1094
Maddaox IS, Richert SH (1977) Use of Response surface methodology for the rapid optimization of microbiological media. J Appl Bacteriol 43:197–204. https://doi.org/10.1111/j.1365-2672.1977.tb00743.x
Nguyen TNT, Chataway T, Araujo R, Puri M, Franco CMM (2021) Purification and characterization of a novel alginate lyase from a marine Streptomyces species isolated from seaweed. Mar Drugs 19:590. https://doi.org/10.3390/md19110590
Nibu Y, Satoh T, Nishi Y, Takeuchi T, Murata K, Kusakabe I (1995) Purification and characterization of extracellular alginate lyase from Enterobacter cloacae M-1. Biosci Biotechnol Biochem 59:632–637. https://doi.org/10.1271/bbb.59.632
Ochiai A, Hashimoto W, Murata K (2006) A biosystem for alginate metabolism in Agrobacterium tumefaciens strain C58: molecular identification of Atu 3025 as an exotype family PL-15 Alginate lyase. Res Microbiol 157:642–649. https://doi.org/10.1016/j.resmic.2006.02.006
Reddy BHN, Rauta PR, Lakshmi VV, Sreenivasa S (2018) Development, formulation, and evaluation of sodium alginate-g-poly (acryl amide-co-acrylic acid/cloiste-30b)/agnps hydrogel composites and their applications in paclitaxel drug delivery and anticancer activity. Int J Appl. Pharm 10:141–150. https://doi.org/10.22159/IJAP.2018V10I3.25062
Sawant SS, Salunke BK, Kim BS (2015) A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity. Enzyme Microb Technol 77:8–13. https://doi.org/10.1016/j.enzmictec.2015.05.003
Smidsrod O, Haug A, Larsen BR (1966) The influence of pH on the rate of hydrolysis of acidic polysaccharides. Acta Chem Scand 20:1026–1034. https://doi.org/10.3891/acta.chem.scand.20-1026
Suraj B, Panneerselvam T, Ponnusamy P, Suthendran K, Parasuraman P, Sankarganesh A, Murugesan S, Uma Priya M, Lokeshkumar R, Selvaraj K (2019) Optimization of bioactive compounds extraction assisted by microwave parameters from Kappaphycusalvarezii using RSM and ANFIS modeling. J Food Meas Charact 13:2773–2789. https://doi.org/10.1007/s11694-019-00198-1
Swift SM, Hudgens JW, Heselpoth RD, Bales PM, Nelson DC (2014) Characterization of AlgMsp, an alginate lyase from Microbulbifer sp. 6532A. PLoS ONE 9(11):e112939. https://doi.org/10.1371/journal.pone.0112939
Takagi T, Sugeno M (1993) Readings in fuzzy sets for intelligent systems. Elsevier, Amsterdam, pp 387–403
Tomohiro H, Minoru N, Tetsuo Y, Kenji S, Masahiro T, Tadayuki I, Akira K, Kousaku M (1994) Production of bacterial alginate-specific lyase by recombinant Bacillus subtilis. J Ferment Bioeng 78:79–83. https://doi.org/10.1016/0922-338X(94)90183-X
Vanavil B, Perumalsamy M, Seshagiri Rao A (2013) Biosurfactant production from novel air isolate NITT6L: screening, characterization, and optimization of media. J Microbiol Biotechnol 23:1229–1243. https://doi.org/10.4014/jmb.1212.12031
Wang M, Chen L, Zhang Z, Wang X, Qin S, Yan P (2017) Screening of alginate lyase-excreting microorganisms from the surface of brown algae. AMB Express 7:74. https://doi.org/10.1186/s13568-017-0361-x
Wijngaard HH, Brunton N (2010) The optimization of solid–liquid extraction of antioxidants from apple pomace by response surface methodology. J Food Engg 96(1):134–140. https://doi.org/10.1016/j.jfoodeng.2009.07.010
Wong TY, Preston LA, Schiller NL (2000) Alginate Lyase: review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications. Annu Rev Microbiol 54:289–340. https://doi.org/10.1146/annurev.micro.54.1.289
**ng M, Cao Q, Wang Y, **ao H, Zhao J, Zhang Q, Ji A, Song S (2020) Advances in research on the bioactivity of alginate oligosaccharides. Mar Drugs 18:144. https://doi.org/10.3390/md18030144
Yamasaki M, Ogura K, Hashimoto W, Mikami B, Murata K (2005) A structural basis for depolymerization of alginate by polysaccharide lyase family-7. J Mol Biol 352:11–21. https://doi.org/10.1016/j.jmb.2005.06.075
Yang M, Li N, Yang S, Yu Y, Han Z, Li L, Mou H (2019) Study on expression and action mode of recombinant Alginate lyases based on conserved domains reconstruction. Appl Microbiol Biotechnol 103:807–817. https://doi.org/10.1007/s00253-018-9502-7
Zhu B, Yin H (2015) Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered 6:125–131. https://doi.org/10.1080/21655979.2015.1030543
Zhu Y, Wu L, Chen Y, Ni H, **ao A, Cai H (2016) Characterization of an extracellular bifunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates. Microbiol Res 182:49–58. https://doi.org/10.1016/j.micres.2015.09.004
Zhu X, Li X, Shi H, Zhou J, Tan Z, Yuan M, Yao P, Liu X (2018) Characterization of a novel alginate lyase from marine bacterium Vibrio furnissii H1. Mar Drugs 16:30. https://doi.org/10.3390/md16010030
Acknowledgements
The authors acknowledge the support provided by the management of the Kalasalingam Academy of Research and Education in terms of research facilities to carry out this study.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Author information
Authors and Affiliations
Contributions
P. Ramya, K. Sundar, and B. Vanavil contributed to the study conception and design. P. Ramya performed the experiments. K. Selvaraj and K. Suthendran contributed to the framework of ANFIS modelling. P. Ramya and B. Vanavil analyzed the results. The manuscript was written by P. Ramya with input from all authors. B. Vanavil and K. Sundar provided a critical review of the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethical approval
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Handling Editor: Raja Sudhakaran
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Ramya, P., Selvaraj, K., Suthendran, K. et al. Optimization of alginate lyase production using Enterobacter tabaci RAU2C isolated from marine environment by RSM and ANFIS modelling. Aquacult Int 31, 3207–3237 (2023). https://doi.org/10.1007/s10499-023-01302-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10499-023-01302-5