Efficient synthesis of nicotinic acid by biomimetic mineralization-immobilized whole-cell biocatalysts

Abstract

The industrial production of nicotinic acid (vitamin B3) primarily relies on chemical synthesis, which suffers from harsh reaction conditions and severe environmental pollution. Although biocatalysis using nitrilase offers a greener alternative, the practical application of free enzymes is constrained by poor operational stability and difficulties in recovery. We developed a biomimetic hybrid immobilization platform that combines a zeolite core with poly(ethylene glycol) (PEG200) crosslinking, and a TEOS-derived silica shell to encapsulate whole cells of Corynebacterium glutamicum engineered to express a mutant nitrilase from Pseudomonas putida. This core–shell architecture is designed to balance mechanical robustness (from the silica shell) with biocompatibility and mass transfer (from PEG200 crosslinking). Systematic optimization of the immobilization conditions (6 g/L zeolite, 50 g/L cell loading, 3 g/L PEG200, 7 g/L TEOS) yielded a molar conversion rate of 88.78% for 50 g/L 3-cyanopyridine, significantly outperforming free cells (62.83%). Scanning electron microscopy (SEM) confirmed the formation of a dense yet porous core–shell structure. Compared with free cells, the immobilized biocatalyst exhibited optimal activity at 40℃ and pH 7.0, with a broader pH tolerance range (7.0–9.0). In repeated batch reactions, the immobilized cells achieved 14 reuse cycles with a loading of 700 g/L, producing 603.56 g/L nicotinic acid at a total molar conversion of 90.2%, whereas free cells were limited to 12 cycles and 86.7% conversion. This work establishes a sustainable biocatalytic platform for nicotinic acid production and demonstrates a generalizable immobilization strategy for whole-cell catalysis based on rationally designed organic–inorganic hybrid shells.

Keywords

Nicotinic acid / Nitrilase / Corynebacterium glutamicum / Whole-cell catalysis / Enzyme immobilization

References

[1] Almeida VM, Marana SR. Optimum temperature may be a misleading parameter in enzyme characterization and application. PLoS ONE, 2019, 14 e0212977

[2] Ariaeenejad S, Motamedi E, Salimi M, Tayi F, Zahouily M. Enzyme immobilization on hydrogels: an overview on methods, interactions, and divers applications. Int J Biol Macromol, 2026, 336 149325

[3] Ayala JA, Nieto M. Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein. Biochem J, 1978, 169: 371-380

[4] Besanger TR, Chen Y, Deisingh AK, Hodgson R, Jin W, Mayer S, Brook MA, Brennan JD. Screening of inhibitors using enzymes entrapped in sol−gel-derived materials. Anal Chem, 2003, 75: 2382-2391

[5] Boronovskiy SE, Kopylova VS, Nartsissov YR. Metabolism and receptor mechanisms of niacin action. Cell Tissue Biol, 2024, 18: 128-147

[6] Cheng Y, Wu Z, Zhang B, Zhang J, Shi J, Jiang Z. Permeable and robust polymer-silica hybrid armor on cell catalyst for sustainable biomanufacturing. Particuology, 2024, 92: 106-112

[7] Dong J, Wu X, Weng W, Cheng F, Zhang L, Li C, Wang L, Liang C, Cheng G. A preblocking strategy with rapid immobilization of nitrile hydratase and precise control of hydrophobic microenvironment for high regioselective amide transformation. ACS Sustain Chem Eng, 2024, 12: 11181-11194

[8] Gao H, Zhang J, Huang Z, Zhang X, Rao Z, Xu M. The maintenance of redox homeostasis to regulate efficient glutathione metabolism in Corynebacterium glutamicum. Chem Eng J, 2025, 506 160237

[9] Gong J-S, Dong T-T, Gu B-C, Li H, Dou W-F, Lu Z-M, Zhou Z-M, Shi J-S, Xu Z-H. Semirational engineering accelerates the laboratory evolution of nitrilase catalytic efficiency for nicotinic acid biosynthesis. ChemCatChem, 2017, 9: 3395-3401

[10] Guo J, Amini S, Lei Q, Ping Y, Agola JO, Wang L, Zhou L, Cao J, Franco S, Noureddine A, Miserez A, Zhu W, Brinker CJ. Robust and long-term cellular protein and enzymatic activity preservation in biomineralized mammalian cells. ACS Nano, 2022, 16: 2164-2175

[11] Hekmat A, Saboury AA, Moosavi-Movahedi AA, Ghourchian H, Ahmad F. Effects of pH on the activity and structure of choline oxidase from Alcaligenes species. Acta Biochim Pol, 2008, 55: 549-557

[12] Hobbs JK, Jiao W, Easter AD, Parker EJ, Schipper LA, Arcus VL. Change in heat capacity for enzyme catalysis determines temperature dependence of enzyme catalyzed rates. ACS Chem Biol, 2013, 8: 2388-2393

[13] Hu C, Wang Y, Xu P, Yu Z. Living materials revolutionizing biocatalysis with engineered whole-cell systems. Chem Eng J, 2025, 519 163891

[14] Ishak SNH, Saad AHM, Latip W, Rahman RNZRA, Salleh AB, Kamarudin NHA, Leow ATC, Ali MSM. Enhancing industrial biocatalyst performance and cost-efficiency through adsorption-based enzyme immobilization: a review. Int J Biol Macromol, 2025, 316 144278

[15] Ishida H, Fukuda N, Shimada M, Yamamoto K, Gaponova L, Higuchi R, Kolosiuk A, Hoshina R, Arikawa M, Fukuda Y, Islam MDS, Harumoto T, Wan Y, Itoh T, Inai Y, Takeishi A, Aonuma H, Kikuchi T, Nishigori S, Maruyama T, Ikeda K, Iriko H, Suzaki T. Artifact-free preparation of biological samples for SEM by optimized water freeze-drying. Sci Rep, 2025, 16 717

[16] Kaye AD, Coffman GD, Mashaw SA, Thomassen AS, Broocks KM, Anwar AI, Ahmadzadeh S, Shekoohi S. Niacin and stroke: the role of supplementation and emerging concepts in clinical practice, a narrative review. Curr Issue Mol Biol, 2025, 47(6400

[17] Lei Q, Guo J, Kong F, Cao J, Wang L, Zhu W, Brinker CJ. Bioinspired cell silicification: from extracellular to intracellular. J Am Chem Soc, 2021, 143: 6305-6322

[18] Li H, Yang T, Gong J-S, Xiong L, Lu Z-M, Li H, Shi J-s, Xu Z. Improving the catalytic potential and substrate tolerance of Gibberella intermedia nitrilase by whole-cell immobilization. Bioprocess Biosyst Eng, 2014, 38: 189-197

[19] Li N, Cui W, Cong P, Tang J, Guan Y, Huang C, Liu Y, Yu C, Yang R, Zhang X. Biomimetic inorganic-organic hybrid nanoparticles from magnesium-substituted amorphous calcium phosphate clusters and polyacrylic acid molecules. Bioact Mater, 2021, 6: 2303-2314

[20] Liu X, Zhang W, Zhao Z, Dai X, Yang Y, Bai Z. Protein secretion in Corynebacterium glutamicum. Crit Rev Biotechnol, 2017, 37: 541-551

[21] Liu X-X, Li Y, Bai Z-H. Chapter 12 - Corynebacterium glutamicum as a robust microbial factory for production of value-added proteins and small molecules: fundamentals and applications. In: Singh V (ed) Microbial Cell Factories Engineering for Production of Biomolecules. Academic Press, 2021;pp. 235–263. https://doi.org/10.1016/B978-0-12-821477-0.00006-4

[22] Lu X-F, Tang X-L, Xu C-H, Diao H-J, Wu Z-M, Zheng R-C, Zheng Y-G. Engineering residues on C interface to improve thermostability of nitrilase for biosynthesis of Pregabalin precursor. AIChE J, 2023, 69 e18211

[23] Lu Y, Wu X, Lou H, Li Z. Anchoring effect-induced conformation remodeling in epoxy-functionalized covalent organic frameworks for enhanced enzymatic efficiency. Langmuir, 2025, 41: 11765-11775

[24] Mateo C, Pessela BCC, Fuentes M, Torres R, Betancor L, Hidalgo A, Fernandez-Lorente G, Fernandez-Lafuente R, Guisan JM. Stabilization of multimeric enzymes via immobilization and further cross-linking with aldehyde-dextran. Methods Mol Biol, 2020, 2100: 175-187

[25] McLeod MJ, Barwell SAE, Holyoak T, Thorne RE. A structural perspective on the temperature dependent activity of enzymes. Structure, 2025, 33: 924-34.e2

[26] Michaud M, Teepakorn C, De Berardinis V, Zaparucha A, Nonglaton G, Coste P, Anxionnaz-Minvielle Z. Experimental and computational analysis of coated milli-structured bioreactor with immobilized nitrilase for continuous production of nicotinic acid. Chemical Eng J Adv, 2025, 22 100766

[27] Milessi TS, Perez CL, Zangirolami TC, Corradini FAS, Sandri JP, Foulquié-Moreno MR, Giordano RC, Thevelein JM, Giordano RLC. Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor. Biotechnol Biofuels, 2020, 13 85

[28] Patel A, Joshi M, Sharma S. Designing of a novel heterogeneous catalyst comprising 12-tungstophosphoric acid and zeolite HY for the synthesis of bio-based esters. Biomass Convers Biorefinery, 2024, 14: 11549-11567

[29] 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

[30] Rodrigues RC, Berenguer-Murcia Á, Carballares D, Morellon-Sterling R, Fernandez-Lafuente R. Stabilization of enzymes via immobilization: multipoint covalent attachment and other stabilization strategies. Biotechnol Adv, 2021, 52 107821

[31] Shakibania S, Biggs MJP, Krukiewicz K. Adjusting cell-surface interactions through a covalent immobilization of biomolecules. Adv Mater Interfaces, 2025, 12 2400774

[32] Singh RV, Kalia VC, Sambyal K, Singh B, Kumar A, Lee JK. Enzymatic approaches to nicotinic acid synthesis: recent advances and future prospects. Front Bioeng Biotechnol, 2025, 13 1585736

[33] Soto ER, Specht CA, Rus F, Lee CK, Abraham A, Levitz SM, Ostroff GR. An efficient (nano) silica - in glucan particles protein encapsulation approach for improved thermal stability. J Control Release, 2023, 357: 175-184

[34] Tang F, Tan Q, Yang Z, Xu D, Ju Q, Kim SW, Zhao Y. PSVII-20 Functional requirement of nicotinamide for growth performance, blood profiles, and gut health of finishing pigs. J Anim Sci, 2025, 103: 515-516

[35] Vejvoda V, Kaplan O, Bezouška K, Pompach P, Šulc M, Cantarella M, Benada O, Uhnáková B, Rinágelová A, Lutz-Wahl S, Fischer L, Křen V, Martínková L. Purification and characterization of a nitrilase from Fusarium solani O1. J Mol Catal B Enzym, 2008, 50: 99-106

[36] Wahab WAA. Review of research progress in immobilization and chemical modification of microbial enzymes and their application. Microb Cell Fact, 2025, 24 167

[37] Wang Q, Sun J, Zang L, Yao H, Wang L, Zhu R, Li J, Zeng S, Tang H, Wang T, Liu J, Wang B, Li B, Liu Z, Dai Z. Programmable cell aggregation by a synthetic biosilicification approach. iScience, 2025, 28 112519

[38] Wei G, Ma W, Zhang A, Cao X, Shen J, Li Y, Chen K, Ouyang P. Enhancing catalytic stability and cadaverine tolerance by whole-cell immobilization and the addition of cell protectant during cadaverine production. Appl Microbiol Biotechnol, 2018, 102: 7837-7847

[39] Yang T, Li H, Gong J, Xiong L, et al. Biotransformation of 3-cyanopyridine for producing nicotinic acid by immobilized Gibberella intermedia. Chem Ind Eng Prog 2014;33:2432–2437.

[40] Zhao J, Bo T, Wu Y, Geng Z, Zhao J, Wu K, Zheng Y, Chen T, Ma H, Wang Z. Engineering Corynebacterium glutamicum for the production of 5-aminolevulinic acid under microaerobic conditions guided by a genome-scale metabolic network. J Agric Food Chem, 2025, 73: 12809-12820

[41] Zhou L, Lei Q, Guo J, Gao Y, Shi J, Yu H, Yin W, Cao J, Xiao B, Andreo J, Ettlinger R, Jeffrey Brinker C, Wuttke S, Zhu W. Long-term whole blood DNA preservation by cost-efficient cryosilicification. Nat Commun, 2022, 13 6265

[42] Zou Y, Fan Z, Liu X, Yang Y, Zhan C, Liu C, Bai Z. Engineering MtrAB two-component system enhances protein and glutamate export in Corynebacterium glutamicum through cell wall remodeling. Bioresour Technol, 2025, 436 133024

Funding

the National Key Research and Development Program of China(2023YFA0914500)

the Frontier Technology Research and Development Plan of Jiangsu Province(BF2024012)

the Major Scientific and Technological Project for “unveiling and commanding” of Hohhot(2023-unveiling and commanding-He-1)

the National Natural Science Foundation of China(32471530, 32501354)

the Independent Research Project of the State Key Laboratory of Food Science and Resources of Jiangnan University(SKLF-ZZB-202408)

the Project for “unveiling and commanding” of Urumqi(B241011002)

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