Continuous cadaverine production using κ-carrageenan/polyethylenimine-immobilized cells with exceptional catalytic performance

Abstract

Cadaverine, a key precursor for nylon, is conventionally produced using free whole-cell lysine decarboxylase systems, with poor recoverability and limited reusability undermining industrial viability. Here, CadA-overexpressing E. coli were entrapped in κ-carrageenan and subsequently crosslinked with polyethylenimine (PEI) to enhance stability and operational longevity. Systematic optimization of gel parameters and CTAB permeabilization enabled robust 100-mL packed-bed reactor directly fed with lysine fermentation broth. The reactor maintained stable operation for 200 h, yielding 6.40 L of broth containing 112 g/L cadaverine at 97.4% conversion and productivity of 44.8 g/g_DCW/h. Immobilized cells reached a total catalyst productivity of 8.96 kg cadaverine per gram dry cell weight and 19-fold increase over free cells. These advantages position κ-carrageenan/PEI immobilization as a practical and economical strategy for continuous cadaverine production.

Keywords

Lysine decarboxylase / Cadaverine / Polyethylenimine / Immobilization / Packed-bed reactor

References

[1] Abutu D, Aderemi B, Ameh A, Yussof HW, Agi A. Nano-enhanced biocarriers: ferric oxide-modified chitosan and calcium alginate beads for improved fermentation efficiency and reusability in a bubble column bioreactor. Biotechnol Lett. 2025, 47: 1-24.

[2] Bhatia SK, Kim YH, Kim HJ, Seo HM, Kim JH, Song HSet al. . Biotransformation of lysine into cadaverine using barium alginate-immobilized Escherichia coli overexpressing CadA. Bioprocess Biosyst Eng. 2015, 38: 2315-22.

[3] Bié J, Sepodes B, Fernandes PC, Ribeiro MH. Enzyme immobilization and co-immobilization: main framework, advances and some applications. Processes. 2022, 10. 494

[4] Bonaccorso A, Zingale E, Carbone C, Musumeci T, Pignatello R. A current overview of polyions and polyelectrolyte complexes for pharmaceutical applications with special emphasis to brain purposes. J Pharm Invest. 2025.

[5] Cavalcante ALG, Dari DN, Da Silva Aires FI, De Castro EC, Dos Santos KM, Dos Santos JCS. Advancements in enzyme immobilization on magnetic nanomaterials: toward sustainable industrial applications. RSC Adv. 2024, 14: 17946-88.

[6] Cha J, Cho J, Kwon I. Self-sufficient reusable biocatalytic system outfitted with multiple oxidoreductases and flexible polypeptide-based cofactor swing arms. ACS Sustain Chem Eng. 2023, 11: 3710-9.

[7] Chamorro AF, Lerma TA, Palencia M. CTAB surfactant promotes rapid, efficient, and simultaneous removal of cationic and anionic dyes through adsorption on glycerol/citrate polyester. Water. 2024, 16. 1860

[8] Chen QS, Yuan X, Peng F, Lou WY. Immobilization of engineered E. coli cells for asymmetric reduction of methyl acetoacetate to methyl-(R)-3-hydroxybutyrate. Bioresour Bioprocess. 2022, 9. 19

[9] Colin C, Akpo E, Perrin A, Cornu D, Cambedouzou J. Encapsulation in alginates hydrogels and controlled release: an overview. Molecules. 2024, 29. 2515

[10] Darwish SS, Hassan ME, Ahmed HE, El Fadl MA, El Bhery MB. Evaluation of effectiveness of covalently immobilized α-amylase and lipase in cleaning of historical textiles. Biointerface Res Appl Chem. 2021, 11: 9952-9962.

[11] De Schouwer F, Claes L, Claes N, Bals S, Degrève J, De Vos DE. Pd-catalyzed decarboxylation of glutamic acid and pyroglutamic acid to bio-based 2-pyrrolidone. Green Chem. 2015, 17: 2263-2270.

[12] Dookeran ZA, Nielsen DR. <article-title update="added"> Systematic engineering of Synechococcus elongatus UTEX 2973 for photosynthetic production of <scp>l</scp> -lysine, cadaverine, and glutarate. ACS Synth Biol. 2021, 10: 3561-75.

[13] Feng J, Han Y, Xu S, Liao Y, Wang YT, Xu Set al. . Engineering RuBisCO-based shunt for improved cadaverine production in Escherichia coli. Bioresour Technol. 2024, 398. 130529

[14] Gao SY, Zhang A, Ma D, Zhang K, Wang J, Wang Xet al. . Enhancing pH stability of lysine decarboxylase via rational engineering and its application in cadaverine industrial production. Biochem Eng J. 2022, 186. 108548

[15] Hammond C. Intensification studies of heterogeneous catalysts: probing and overcoming catalyst deactivation during liquid phase operation. Green Chem. 2017, 19: 2711-28.

[16] Hashimoto M, Eda Y, Osanai Y, Iwai T, Aoki S. A novel decarboxylation of α-amino acids. A facile method of decarboxylation by the use of 2-cyclohexen-1-one as a catalyst. Chem Lett. 1986, 15: 893-6.

[17] Hoffer BW, Moulijn JA. Hydrogenation of dinitriles on Raney-type Ni catalysts: kinetic and mechanistic aspects. Appl Catal A Gen. 2009, 352: 193-201.

[18] Huang YH, Ji XL, Ma ZL, Łężyk M, Xue YJ, Zhao H. Green chemical and biological synthesis of cadaverine: recent development and challenges. RSC Adv. 2021, 11: 23922-42.

[19] Imam HT, Marr PC, Marr AC. Enzyme entrapment, biocatalyst immobilization without covalent attachment. Green Chem. 2021, 23: 4980-5005.

[20] Jabeen F, Ahmad R, Mir S, Awwad NS, Ibrahium HA. Carrageenan: structure, properties and applications with special emphasis on food science. RSC Adv. 2025, 15: 22035-62.

[21] Jang YS, Kim B, Shin JH, Choi YJ, Choi S, Song CWet al. . Bio‐based production of C2–C6 platform chemicals. Biotechnol Bioeng. 2012, 109: 2437-59.

[22] Jiang Y, Zheng JX, Wang MN, Xu WQ, Wang YQ, Wen Let al. . Pros and cons in various immobilization techniques and carriers for enzymes. Appl Biochem Biotechnol. 2024, 196: 5633-55.

[23] Jing WJ, Hou FH, Wu XM, Zheng MQ, Zheng Y, Lu FPet al. . A critical review on immobilized sucrose isomerase and cells for producing isomaltulose. Foods. 2024, 13. 1228

[24] Kang SB, Choi JI. Enhanced cadaverine production by recombinant Corynebacterium glutamicum with a heterologous DR1558 regulator at low pH condition. Process Biochem. 2021, 111: 63-70.

[25] Kim HT, Baritugo KA, Oh YH, Hyun SM, Khang TU, Kang KH, Jung SH, Song BK, Park K, Kim IK. Metabolic engineering of Corynebacterium glutamicum for the high-level production of cadaverine that can be used for the synthesis of biopolyamide 510. ACS Sustainable Chem Eng. 2018, 6: 5296-5305.

[26] Kind S, Kreye S, Wittmann C. Metabolic engineering of cellular transport for overproduction of the platform chemical 1, 5-diaminopentane in Corynebacterium glutamicum. Metab Eng. 2011, 13: 617-27.

[27] Lapponi MJ, Méndez MB, Trelles JA, Rivero CW. Cell immobilization strategies for biotransformations. Curr Opin Green Sustain Chem. 2022, 33. 100565

[28] Leong YK, Chen CH, Huang SF, Lin HY, Li SF, Ng ISet al. . High-level l-lysine bioconversion into cadaverine with enhanced productivity using engineered Escherichia coli whole-cell biocatalyst. Biochem Eng J. 2020, 157. 107547

[29] Li M, Li DX, Huang YY, Liu M, Wang HX, Tang Qet al. . Improving the secretion of cadaverine in Corynebacterium glutamicum by cadaverine–lysine antiporter. J Ind Microbiol Biotechnol. 2014, 41: 701-9.

[30] Li JL, Liu YL, Hu LT, Xu RR, Zhang WJ, Hu Set al. . Construction of immobilized enzyme cascades for the biosynthesis of nucleotide sugars UDP-N-acetylglucosamine and UDP-glucuronic acid. Syst Microbiol Biomanuf. 2024, 4: 895-905.

[31] Li CQ, Zhao YY, Li GH, Deng Y. Polyamine biosynthesis-driven CO₂ capture for sustainable applications. Chem Eng J. 2025.

[32] Liang XX, Zhong HJ, Ding HY, Yu B, Ma X, Liu XYet al. . Polyvinyl alcohol (PVA)-based hydrogels: recent progress in fabrication, properties, and multifunctional applications. Polymers. 2024, 16. 2755

[33] Liu CP, Gao C, Song LF, Li XM, Chen XL, Wu J, Song W, Wei WQ, Liu LM. Fine-tuning pyridoxal 5′-phosphate synthesis in Escherichia coli for cadaverine production in minimal culture media. ACS Synth Biol. 2024, 13: 1820-1830.

[34] Liu MM, Lv Y, Niu X, Liao Y, Xu S, Wang Xet al. . Effective coproduction of bio-based polyamide 54 monomers cadaverine and succinic acid by dynamic regulatory and ORP strategy. Chem Eng J. 2025.

[35] Lv YP, Liu SM, Wei L, Zhang L, Qi HS. Engineering a hybrid system of Corynebacterium glutamicum and co-immobilized enzymes for efficient cadaverine production from glycerol. Green Chem. 2025, 27: 7263-80.

[36] Ma WC, Cao WJ, Zhang H, Chen KQ, Li Y, Ouyang PK. Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB. Biotechnol Lett. 2015, 37: 799-806.

[37] Ma H, Liu X, Nobbs AH, Mishra A, Patil AJ, Mann S. Protocell flow reactors for enzyme and whole‐cell mediated biocatalysis. Adv Mater. 2024, 36. 2404607

[38] Maghraby YR, El-Shabasy RM, Ibrahim AH, Azzazy HMES. Enzyme immobilization technologies and industrial applications. ACS Omega. 2023, 8: 5184-96.

[39] Menegatti T, Žnidaršič-Plazl P. Hydrogel-based enzyme and cofactor co-immobilization for efficient continuous transamination in a microbioreactor. Front Bioeng Biotechnol. 2021, 9. 752064

[40] Mi JL, Liu SM, Du Y, Qi HS, Zhang L. Cofactor self-sufficient by co-immobilization of pyridoxal 5′-phosphate and lysine decarboxylase for cadaverine production. Bioresour Technol Rep. 2022, 17. 100939

[41] Oh YH, Choi JW, Kim EY, Song BK, Jeong KJ, Park Ket al. . Construction of synthetic promoter-based expression cassettes for the production of cadaverine in recombinant Corynebacterium glutamicum. Appl Biochem Biotechnol. 2015, 176: 2065-75.

[42] Osire T, Yang TW, Xu MJ, Zhang X, Long MF, A Ngon NKet al. . Integrated gene engineering synergistically improved substrate-product transport, cofactor generation and gene translation for cadaverine biosynthesis in E. coli. Int J Biol Macromol. 2021, 169: 8-17.

[43] Patti S, Magrini Alunno I, Pedroni S, Riva S, Ferrandi EE, Monti D. Advances and challenges in the development of immobilized enzymes for batch and flow biocatalyzed processes. ChemSusChem. 2025, 18. e202402007

[44] Qian ZG, Xia XX, Lee SY. Metabolic engineering of Escherichia coli for the production of cadaverine: a five carbon diamine. Biotechnol Bioeng. 2011, 108: 93-103.

[45] Reus B, Damian M, Mutti FG. Advances in cofactor immobilization for enhanced continuous-flow biocatalysis. J Flow Chem. 2024, 14: 219-38.

[46] Rodrigues De Melo R, Alnoch RC, Sousa ASD, Harumi Sato H, Ruller R, Mateo C. Cross-linking with polyethylenimine confers better functional characteristics to an immobilized β-glucosidase from Exiguobacterium antarcticum B7. Catalysts. 2019, 9. 223

[47] Rui JQ, You SP, Zheng YX, Wang CY, Gao YT, Zhang Wet al. . High-efficiency and low-cost production of cadaverine from a permeabilized-cell bioconversion by a Lysine-induced engineered Escherichia coli. Bioresour Technol. 2020, 302. 122844

[48] Sahu RB, Singh P. Fermentative production of bioethanol using immobilized beads of Aspergillus terreus MZ769058. Syst Microbiol Biomanuf. 2024, 4: 1273-83.

[49] Shin JH, Kim HU, Kim DI, Lee SY. Production of bulk chemicals via novel metabolic pathways in microorganisms. Biotechnol Adv. 2013, 31: 925-35.

[50] Struszczyk Świta K, Kaczmarek MB, Antczak T, Marchut Mikołajczyk O. Continuous production of chitooligosaccharides in a column reactor by the PUF-immobilized whole cell enzymes of Mucor circinelloides IBT-83. Microb Cell Fact. 2024, 23. 258

[51] Thompson MP, Peñafiel I, Cosgrove SC, Turner NJ. Biocatalysis using immobilized enzymes in continuous flow for the synthesis of fine chemicals. Org Process Res Dev. 2018, 23: 9-18.

[52] Ting WW, Ng IS. Metabolic manipulation through CRISPRi and gene deletion to enhance cadaverine production in Escherichia coli. J Biosci Bioeng. 2020, 130: 553-62.

[53] Ting WW, Huang CY, Wu PY, Huang SF, Lin HY, Li SF, Chang JS, Ng IS. Whole-cell biocatalyst for cadaverine production using stable, constitutive and high expression of lysine decarboxylase in recombinant Escherichia coli W3110. Enzyme Microb Technol. 2021, 148. 109811

[54] Trawczynska I. Use of the chemical permeabilization process in yeast cells: production of high-activity whole cell biocatalysts. Biotechnologia. 2020, 101: 239-52.

[55] Vaidyanathan S, Orr BG, Banaszak Holl MM. Detergent induction of HEK 293A cell membrane permeability measured under quiescent and superfusion conditions using whole cell patch clamp. J Phys Chem B. 2014, 118: 2112-23.

[56] Verduyckt J, Van Hoof M, De Schouwer F, Wolberg M, Kurttepeli M, Eloy P, Gaigneaux EM, Bals S, Kirschhock CE, De Vos DE. PdPb-catalyzed decarboxylation of proline to pyrrolidine: highly selective formation of a biobased amine in water. ACS Catal. 2016, 6: 7303-7310.

[57] Wang X, Guo X, Wang J, Li H, He F, Xu Set al. . Ameliorating end-product inhibition to improve cadaverine production in engineered Escherichia coli and its application in the synthesis of bio-based diisocyanates. Synth Syst Biotechnol. 2021, 6: 243-53.

[58] Wang JD, Wang S, Zhao SY, Sun PJ, Zhang Z, Xu QY. Productivity enhancement in L-lysine fermentation using oxygen-enhanced bioreactor and oxygen vector. Front Bioeng Biotechnol. 2023, 11. 1181963

[59] Wei GG, Ma WC, Zhang A, Cao X, Shen JS, Li Yet al. . Enhancing catalytic stability and cadaverine tolerance by whole-cell immobilization and the addition of cell protectant during cadaverine production. Appl Microbiol Biotechnol. 2018, 102: 7837-47.

[60] Wu JX, Li YF, Chen XP, Li N, He WH, Feng YJet al. . Improved membrane permeability with cetyltrimethylammonium bromide (CTAB) addition for enhanced bidirectional transport of substrate and electron shuttles. Sci Total Environ. 2022, 822. 153443

[61] Xu X, Wan JC, Sun J, Wu LN, Lei JP. Tailored metal‐organic framework‐based enzyme hybrids: immobilization strategies, improved performance, and biological applications. Small Sci. 2025, 5. 2500222

[62] Yu LJ, Li NQ, Liu CL, Liu XC. Immobilized cell and preparation method thereof. United States Patent No. US 10,619,148 B2. 2020.

[63] Zhang SL, Bai J, Kong WX, Song HL, Liu YT, Liu GHet al. . Dendritic mesoporous silica nanoparticles for enzyme immobilization. Green Chemical Engineering. 2024, 5: 173-86.

[64] Zhao CH, Zheng TR, Feng YH, Wang X, Zhang LZ, Hu QTet al. . Engineered Halomonas spp. for production of l-lysine and cadaverine. Bioresour Technol. 2022, 349. 126865

Funding

Advanced Technology Research and Development Program of Jiangsu Province(BF2025072)

National Natural Science Foundation of China(22377040)

Jiangsu Basic Research Center for Syn-thetic Biology(BK20233003)

Fundamental Research Funds for the Central Univer-sities(JUSRP202416001)

Postgraduate Research & Practice Innovation Program of Jiangsu Province(KYCX24_2581)

RIGHTS & PERMISSIONS

Jiangnan University