Reshaping the binding channel of a novel GH113 family β-mannanase from Paenibacillus cineris (PcMan113) for enhanced activity

Abstract

Endo-β-mannanases are important enzymes for degrading lignocellulosic biomass to generate mannan, which has significant health effects as a prebiotic that promotes the development of gut microbiota. Here, a novel endo-β-mannanase belonging to glycoside hydrolase (GH) family 113 from Paenibacillus cineris (PcMan113) was cloned, expressed and characterized, as one of only a few reported GH113 family β-mannanases. Compared to other functionally and structurally characterized GH113 mannanases, recombinant PcMan113 showed a broader substrate spectrum and a better performance. Based on a structural homology model, the highly active mutant PcMT3 (F110E/N246Y) was obtained, with 4.60- and 5.53-fold increases of enzyme activity (towards KG) and catalytic efficiency (k_cat/K_m, against M5) compared with the WT enzyme, respectively. Furthermore, molecular dynamics (MD) simulations were conducted to precisely explore the differences of catalytic activity between WT and PcMT3, which revealed that PcMT3 has a less flexible conformation, as well as an enlarged substrate-binding channel with decreased steric hindrance and increased binding energy in substrate recognition. In conclusion, we obtained a highly active variant of PcMan113 with potential for commercial application in the manufacture of manno-oligosaccharides.

Keywords

Endo-β-mannanase / GH113 family / Structural analysis / Binding channel / Molecular dynamics

References

[1] Abraham M, Murtola T, Schulz R, Páll S, Smith J, Hess B, Lindahl E. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 2015, 1–2: 19-25.

[2] Akino T, Nakamura N, Horikoshi K. Characterization of three BETA-mannanases of an alkalophilic Bacillus sp. Agric Biol Chem, 1988, 52: 773-779.

[3] Cao LL, Liu WB, Wang LJ. Developing a green and edible film from cassia gum: the effects of glycerol and sorbitol. J Clean Prod, 2018, 175: 276-282.

[4] Couturier M, Roussel A, Rosengren A, Leone P, Stålbrand H, Berrin JG. Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserine reveal differences upon manno-oligosaccharide catalysis. J Biol Chem, 2013, 288: 14624-14635.

[5] David A, Chauhan PS, Kumar A, Angural S, Kumar D, Puri N, Gupta N. Coproduction of protease and mannanase from Bacillus nealsonii PN-11 in solid state fermentation and their combined application as detergent additives. Int J Biol Macromol, 2018, 108: 1176-1184.

[6] Eom GT, Oh JY, Park JH, Jegal J, Song JK. Secretory production of enzymatically active endo-β-1,4-mannanase from Bacillus subtilis by ABC exporter in Escherichia coli. Process Biochem, 2016, 51(8): 999-1005.

[7] Ghasemian M, Jahanian R. Dietary mannan-oligosaccharides supplementation could affect performance, immunocompetence, serum lipid metabolites, intestinal bacterial populations, and ileal nutrient digestibility in aged laying hens. Anim Feed Sci Technol, 2016, 213: 81-89.

[8] Giannenas I, Doukas D, Karamoutsios A, Tzora A, Bonos E, Skoufos I, Tsinas A, Christaki E, Tontis D, Florou-Paneri P. Effects of Enterococcus faecium, mannan oligosaccharide, benzoic acid and their mixture on growth performance, intestinal microbiota, intestinal morphology and blood lymphocyte subpopulations of fattening pigs. Anim Feed Sci Technol, 2016, 220: 159-167.

[9] Hall BG. Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol, 2013, 30(5): 1229-1235.

[10] Hogg D, Pell G, Dupree P, Goubet F. The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation. Biochem J, 2003, 371: 1027-1043.

[11] Humphrey W, Dalke A, Schulten K. VMD-visual molecular dynamics. J Mol Graphics, 1996, 14: 33-38.

[12] Jin Y, Petricevic M, John A, Raich L, Jenkins H, Portela De Souza L, Cuskin F, Gilbert HJ, Rovira C, Goddard-Borger ED, Williams SJ, Davies GJ. A β-1,4-mannanase with a lysozyme-like fold and a novel molecular catalytic mechanism. ACS Cent Sci, 2016, 2: 896-903.

[13] Koziara KB, Stroet M, Malde AK, Mark AE. Testing and validation of the Automated Topology Builder (ATB) version 2.0: prediction of hydration free enthalpies. J Comput Aided Mol Des, 2014, 28: 221-233.

[14] Kumagai Y, Yamashita K, Tagami T, Uraji M, Wan K, Okuyama M, Yao M, Kimura A, Hatanaka T. The loop structure of Actinomyceteglycoside hydrolase family 5 mannanases governs substrate recognition. FEBS J, 2015, 282: 4001-4014.

[15] La Rosa SL, Leth ML, Michalak L, Hansen ME, Pudlo NA, Glowacki R, Westereng B. The human gut Firmicutev Roseburia intestinalisis a primary degrader of dietary β-mannans. Nat Commun, 2019, 10: 1-14.

[16] Le Nours J, Anderson L, Stoll SL, Leggio L. The structure and characterization of a modular endo-β-1,4-mannanase from Cellulomonas fimi. Biochemistry, 2005, 44: 12700-21270.

[17] Li YX, Yi P, Liu J, Yan QJ, Jiang ZQ. High-level expression of an engineered Β-mannanase (mRmMan5A) in Pichia pastoris for manno-oligosaccharide production using steam explosion pretreated palm kernel cake. Bioresour Technol, 2017, 256: 30-37.

[18] Liu W, Ma C, Liu W, Zheng Y, Chen C-C, Liang A, Luo G, Li MJ, Song Y, Guo R-T, Zhang T. Functional and structural investigation of a novel β-mannanase baman113a from bacillus sp. N16–5. Int J Biol Macromol, 2021, 182: 899-909.

[19] Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res, 2014, 42: D490-D495.

[20] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin-phenol reagent. J Biol Chem, 1951, 193: 265-275.

[21] Malgas S, van Dyk JS, Pletschke BI. A review of the enzymatic hydrolysis of mannans and synergistic interactions betweenβ-mannanase, β-mannosidase and α-galactosidase. World J Microbiol Biotechnol, 2015, 31: 1167-1175.

[22] Mandelli F, de Morais MAB, de Lima EA, Oliveira PGF, Murakami MT. Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-β-1,4-mannanase. J Biol Chem, 2020, 295: 5012-5021.

[23] Mano MC, Neri-Numa IA, Da Silva JB, Paulino BN, Pessoa MG, Pastore GM. Oligosaccharide biotechnology: an approach of prebiotic revolution on the industry. Appl Microbiol Biot, 2018, 102: 17-37.

[24] Moreira LR, Filho EX. An overview of mannan structure and mannan-degrading enzyme systems. Appl Microbiol Biot, 2008, 79: 165-178.

[25] Pérez-Burillo S, Pastoriza S, Fernández-Arteaga A, Luzón G, Jiménez-Hernández N, D’Auria G, Rufián-Henares JÁ. Spent coffee grounds extract, rich in mannooligosaccharides, promotes a healthier gut microbial community in a dose-dependent manner. J Agric Food Chem, 2019, 67: 2500-2509.

[26] Pongsapipatana N, Damrongteerapap P, Chantorn S, Sintuprapa W, Keawsompong S, Nitisinprasert S. Molecular cloning of kman coding for mannanase from Klebsiella oxytoca KUB-CW2-3 and its hybrid mannanase characters. Enzym Microb Technol, 2016, 89: 39-51.

[27] Rai PR, Tiwary AK, Rana V. Superior disintegrating properties of calcium cross-linked cassia fistula gum derivatives for fast dissolving tablets. Carbohyd Polym, 2012, 87: 1098-1104.

[28] Saha BC. Hemicellulose Bioconversion. J Ind Microbiol Biot, 2003, 30: 279-291.

[29] Šali A, Blundell T. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol, 1993, 234: 779-815.

[30] Shimizu M, Kaneko Y, Ishihara S, Mochizuki M, Sakai K, Yamada M, Murata S, Itoh E, Yamamoto T, Sugimura Y. Novel β-1,4-mannanase belonging to a new glycoside hydrolase family in Aspergillus nidulans. J Biol Chem, 2015, 290: 27914-32792.

[31] Song YJ, Sun WY, Fan Y, Xue YF. Galactomannan degrading enzymes from the mannan utilization gene cluster of alkaliphilic Bacillus sp. N16–5 and their synergy on galactomannan degradation. J Agric Food Chem, 2018, 66: 11055-11063.

[32] Srivastava M, Kapoor VP. Seed galactomannans: an overview. Chem Biodiversity, 2005, 2: 295-317.

[33] Srivastava PK, Kapoor M. Cost-effective endo-mannanase from Bacillus sp. CFR1601 and its application in generation of oligosaccharides from guar gum and as detergent additive. Prep Biochem Biotechnol, 2014, 44: 392-417.

[34] Srivastava PK, Kapoor M. Production, properties and applications of endo-β-1,4-mannanases. Biotechnol Adv, 2017, 35: 1-19.

[35] Srivastava PK, Kapoor M. Production, properties, and applications of endo-β-mannanases. Biotechnol Adv, 2017, 35: 1-9.

[36] Torrecillas S, Montero D, Izquierdo M. Improved health and growth offish fed mannan-oligosaccharides: potential mode of action. Fish Shellfish Immunol, 2014, 36: 525-544.

[37] Xia W, Lu H, Xia M, Cui Y, Bai Y, Qian L, Shi P, Luo H, Yao B. A novel glycoside hydrolase family 113 endo-β-1,4-mannanase from Alicyclobacillus sp. strain A4 and insight into the substrate recognition and catalytic mechanism of this family. Appl Environ Microbiol, 2016, 82: 2718-2727.

[38] Yamabhai M, Sak-Ubol S, Srila W, Haltrich D. Mannan biotechnology: from biofuels to health. Crit Rev Biotechnol, 2016, 36: 32-42.

[39] Yang H, Shi PJ, Lu HQ, Wang HM, Luo HY, Huang HQ, Yang PL, Yao B. A thermophilic β-mannanase from Neosartorya fischeri P1 with broad pH stability and significant hydrolysis ability of various mannan polymers. Food Chem, 2015, 173: 283-289.

[40] Yang J, Chen Q, Zhou B, Wang X, Liu S. Mannooligosaccharide preparation by the hydrolysis of konjac flour with a thermostable endo mannanase from Talaromyces cellulolyticus. J Appl Microbiol, 2019, 127: 520-532.

[41] You X, Qin Z, Yan QJ, Yang SQ, Li YX, Jiang ZQ. Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 beta-1,4-mannanase from Amphibacillus xylanus. J Biol Chem, 2018, 293: 11746-11757.

[42] Zhang YL, Ju JS, Peng H, Gao F, Zhou C, Zeng Y, Xue YF, Li Y, Henrissat B, Gao GF, Ma YH. Biochemical and structural characterization of the intracellular mannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A. J Biol Chem, 2008, 283: 31551-31558.

[43] Zhao D, Zhang X, Cui DZ, Zhao M. Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes. PLoS ONE, 2012, 7: e38817.

[44] Zheng C, Li F, Hao Z, Liu T. Effects of adding mannan oligosaccharides on digestibility and metabolism of nutrients, ruminal fermentation parameters, immunity, and antioxidant capacity of sheep. J Anim Sci, 2018, 96: 284-292.