Identification and characterization of nitrilase NitC from Baijiu fermentation and evaluation in cyanide degradation

Abstract

Cyanide, a toxic compound from plant metabolism, might be accumulated in the plant-based food fermentation. It is important to degrade cyanide in these food fermentations. This study identified a potential cyanide-degradation enzyme gene (nitC) in Bacillus amyloliquefaciens CCTCC M 20242168 with cyanide-degrading activity in Baijiu fermentation. The nitC gene was expressed in Escherichia coli BL21 (DE3), and the purified enzyme successfully degraded cyanide into ammonia and formic acid. The purified nitrilase showed the highest activity (23.7 U/g) at the optimal pH of 6.5 and temperature of 35 °C. Substrate specificity analysis revealed high catalytic activity toward both cyanide and benzonitrile. Nitrilase NitC also exhibited high enzymatic activity under acidic conditions and its observed tolerance towards 1%–10% ethanol, suggesting its potential application in cyanide bioremediation, particularly under acidic conditions.

Keywords

Fermented foodgrains / Nitrilase / Food safety / Cyanide degradation / Acid-tolerant enzyme / Bacillus amyloliquefaciens

References

[1] Nielsen KA, Olsen CE, Pontoppidan K, Moller BL. Leucine-derived cyano glucosides in barley. Plant Physiol, 2002, 129(3): 1066-1075

[2] Vetter J. Plant cyanogenic glycosides. Toxicon, 2000

[3] Kaul P, Banerjee A, Banerjee UC. Polaina J, MacCabe AP. Nitrile Hydrolases. Industrial enzymes: structure, function and applications, 2007, Dordrecht, Springer53147

[4] Shen T, Wu Q, Xu Y. Biodegradation of cyanide with Saccharomyces cerevisiae in Baijiu fermentation. Food Control, 2021, 127: 1-7

[5] Tokpohozin SE, Fischer S, Becker T. Assessment of malting and mash bio-acidification on the turnover of sorghum cyanogenic glucoside and protein hydrolysis improvement. LWT, 2018, 90: 303-309

[6] Narayanan NN, Ihemere U, Ellery C, Sayre RT. Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels. PLoS ONE, 2011, 6(7): 1-11

[7] Betancourt-Buitrago LA, Hernandez-Ramirez A, Colina-Marquez JA, Bustillo-Lecompte CF, Rehmann L, Machuca-Martinez F. Recent developments in the photocatalytic treatment of cyanide wastewater: an approach to remediation and recovery of metals. Processes, 2019

[8] Ikediobi CO, Olugboji O, Okoh PN. Cyanide profile of component parts of sorghum (Sorghum bicolor L. Moench) sprouts. Food Chem, 1988, 27(3): 167-175

[9] Alvillo-Rivera A, Garrido-Hoyos S, Buitrón G, Thangarasu-Sarasvathi P, Rosano-Ortega G. Biological treatment for the degradation of cyanide: a review. J Mater Res Technol, 2021, 12: 1418-1433

[10] Gupta N, Balomajumder C, Agarwal VK. Enzymatic mechanism and biochemistry for cyanide degradation: a review. J Hazard Mater, 2010, 176(1-3): 1-13

[11] Park JM, Trevor Sewell B, Benedik MJ. Cyanide bioremediation: the potential of engineered nitrilases. Appl Microbiol Biotechnol, 2017, 101(8): 3029-3042

[12] Ebbs S. Biological degradation of cyanide compounds. Curr Opin Biotechnol, 2004, 15(3): 231-236

[13] Jandhyala DM, Willson RC, Sewell BT, Benedik MJ. Comparison of cyanide-degrading nitrilases. Appl Microbiol Biotechnol, 2005, 68(3): 327-335

[14] Raybuck SA. Microbes and microbial enzymes for cyanide degradation. Biodegradation, 1992, 3(1): 3-18

[15] Luque-Almagro VíctorM, Huertas MJ, Martínez-Luque M, Moreno-Vivián C, Roldán MD, García-Gil LJ, et al.. Bacterial degradation of cyanide and its metal complexes under alkaline conditions. Appl Environ Microbiol, 2005, 71(2): 940-947

[16] Sharma M, Akhter Y, Chatterjee S. A review on remediation of cyanide containing industrial wastes using biological systems with special reference to enzymatic degradation. World J Microb Biot, 2019, 35(5): 70

[17] O'Reilly C, Turner PD. The nitrilase family of CN hydrolysing enzymes—a comparative study. J Appl Microbiol, 2003, 95(61161-1174

[18] Vaishnav A, Kumar R, Singh HB, Sarma BK. Extending the benefits of PGPR to bioremediation of nitrile pollution in crop lands for enhancing crop productivity. Sci Total Environ, 2022, 826(1–11): 154170

[19] Rangel-González MG, Solís-Domínguez FA, Herrera-Martínez A, Carrillo-González R, López-Luna J, Del Carmen Angeles González-Chávez M, et al.. Cyanide biodegradation: a scoping review. Int J Environ Sci Technol, 2024

[20] Xia Y, Zhao J, Saeed M, Hussain N, Chen X, Guo Z, et al.. Molecular modification strategies of nitrilase for its potential application in agriculture. J Agric Food Chem, 2024, 72(2715106-15121

[21] Arevalo SJ, Sifuentes DZ, Portocarrero AC, Reátegui MB, Pimentel CM, Martins LF, et al.. Genomic characterization of Bacillus safensis isolated from mine tailings in Peru and evaluation of its cyanide-degrading enzyme CynD. Appl Environ Microbiol, 2022, 88(14): 1-14

[22] Wang Z, Chen Y, Yan M, Li K, Okoye CO, Fang Z, et al.. Research progress on the degradation mechanism and modification of keratinase. Appl Microbiol Biotechnol, 2023, 107(4): 1003-1017

[23] Luchan G. The effect of glutamate decarboxylase system on acid resistance and its regulatory mechanisms as well as application in Lactobacillus brevis, 2019Jiangnan University

[24] Sonbol SA, Ferreira AJ, Siam R. Red Sea Atlantis II brine pool nitrilase with unique thermostability profile and heavy metal tolerance. BMC Biotechnol, 2016, 16: 14

[25] Ma Z, Nang SC, Liu Z, Zhu J, Mu K, Xu L, et al.. Membrane lipid homeostasis dually regulates conformational transition of phosphoethanolamine transferase EptA. Nat Commun, 2024, 15(1): 1-14

[26] Datta S, Christena LR, Rajaram YR. Enzyme immobilization: an overview on techniques and support materials. 3 Biotech, 2013, 3(31-9

[27] Fawcett JK, Scott JE. A rapid and precise method for the determination of urea. JCP, 1960, 13(2): 156-159

[28] Moosavizadeh A, Motallebi M, Jahromi ZM, Mekuto L. Cloning and heterologous expression of Fusarium oxysporum nitrilase gene in Escherichia coli and evaluation in cyanide degradation. Enzyme Microb Technol, 2024, 174: 110389

[29] Sonbol SA, Ferreira AJS, Siam R. Red Sea Atlantis II brine pool nitrilase with unique thermostability profile and heavy metal tolerance. BMC Biotechnol, 2016, 16(1): 14

[30] Dennett GV, Blamey JM. A new thermophilic nitrilase from an antarctic hyperthermophilic microorganism. Front Bioeng Biotechnol, 2016, 4(51-9

[31] Barclay M, Day JC, Thompson IP, Knowles CJ, Bailey MJ. Substrate-regulated cyanide hydratase (chy) gene expression in Fusarium solani: the potential of a transcription-based assay for monitoring the biotransformation of cyanide complexes. Environ Microbiol, 2002, 4(3183-189

[32] Sexton AC, Howlett BJ. Characterisation of a cyanide hydratase gene in the phytopathogenic fungus Leptosphaeria maculans. Mol Gen Genet MGG, 2000, 263(3): 463-470

[33] Mouchlis VD, Bucher D, McCammon JA, Dennis EA. Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates. Proc Natl Acad Sci USA, 2015, 112(6): E516-E525

[34] Khan FI, Lan D, Durrani R, Huan W, Zhao Z, Wang Y. The lid domain in lipases: structural and functional determinant of enzymatic properties. Front Bioeng Biotechnol, 2017, 5(16): 1-13

[35] Peters GH, Bywater RP. Influence of a lipid interface on protein dynamics in a fungal lipase. Biophys J, 2001, 81(6): 3052-3065

[36] Wiktor M, Weichert D, Howe N, Huang CY, Olieric V, Boland C, et al.. Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis. Nat Commun, 2017, 8: 15952

[37] Liu X, Wu DA-O, Abid AA-O, Liu Y, Zhou J, Zhang Q. Determination of paddy soil ammonia nitrogen using rapid detection kit coupled with microplate reader. Toxics, 2022, 10(12): 1-11

[38] Jones LB, Wang X, Gullapalli JS, Kunz DA. Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764. Biochem Biophys Rep, 2021, 25: 1-8

[39] Zhang L, Yin B, Wang C, Jiang S, Wang H, Yuan YA, et al.. Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803. J Struct Biol, 2014, 188(293-101

[40] Williamson DS, Dent KC, Weber BW, Varsani A, Frederick J, Thuku RN, et al.. Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8. Appl Microbiol Biotechnol, 2010, 88(1): 143-153

[41] Raczynska JE, Vorgias CE, Antranikian G, Rypniewski W. Crystallographic analysis of a thermoactive nitrilase. J Struct Biol, 2011, 173(2): 294-302

Funding

National Natural Science Foundation of China(32172175)

Priority Academic Program Development of Jiangsu Higher Education Institutions(Priority Academic Program Development of Jiangsu Higher Education Institutions)

the 111 Project(111–2-06)

Support Program for Longyuan Youth and Fundamental Research Funds for the Universities of Gansu Province(JUSRP202404014)

RIGHTS & PERMISSIONS

Jiangnan University