Enhancing the endo-activity of the thermophilic chitinase to yield chitooligosaccharides with high degrees of polymerization

Abstract

Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides, which are advantageous for plant immunity, animal nutrition and health. However, thermophilic endo-chitinases are scarce and the transformation from exo- to endo-activity of chitinases is still a challenging problem. In this study, to enhance the endo-activity of the thermophilic chitinase Chi304, we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses. Four effective single-point mutants were identified among 28 designed mutations. The ratio of (GlcNAc)₃ to (GlcNAc)₂ quantity (DP_3/2) in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89, 1.65, 1.24, and 1.38 times that of Chi304, respectively. When combining to double-point mutants, the DP_3/2 proportions produced by F79A/W140R, F79A/M264L, F79A/W272R, and M264L/W272R were 2.06, 1.67, 1.82, and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity. When applied to produce chitooligosaccharides (DP ≥ 3), F79A/W140R accumulated the most (GlcNAc)₄, while M264L/W272R was the best to produce (GlcNAc)₃, which was 2.28 times that of Chi304. The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate. Overall, this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.

Keywords

Chitin / Chitinase / Endo-activity / Mutants / Functional oligosaccharides

References

[1] Adasme MF, Linnemann KL, Bolz SN, Kaiser F, Salentin S, Haupt VJ, Schroeder M. PLIP 2021: expanding the scope of the protein–ligand interaction profiler to DNA and RNA. Nucleic Acids Res, 2021, 49(W1): W530-W534.

[2] Adrangi S, Faramarzi MA. From bacteria to human: a journey into the world of chitinases. Biotechnol Adv, 2013, 31(8): 1786-1795.

[3] Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997, 25(17): 3389-3402.

[4] Baban J, Fjeld S, Sakuda S, Eijsink VG, Sørlie M. The roles of three Serratia marcescens chitinases in chitin conversion are reflected in different thermodynamic signatures of allosamidin binding. J Phys Chem B, 2010, 114(18): 6144-6149.

[5] Berini F, Katz C, Gruzdev N, Casartelli M, Tettamanti G, Marinelli F. Microbial and viral chitinases: attractive biopesticides for integrated pest management. Biotechnol Adv, 2018, 36(3): 818-838.

[6] Deng JJ, Li ZQ, Mo ZQ, Xu S, Mao HH, Shi D, Luo XC. Immunomodulatory effects of N-acetyl chitooligosaccharides on RAW264.7 macrophages. Mar Drugs, 2020

[7] Dixon EV, Claridge JK, Harvey DJ, Baruah K, Yu X, Vesiljevic S, Crispin M. Fragments of bacterial endoglycosidase s and immunoglobulin g reveal subdomains of each that contribute to deglycosylation. J Biol Chem, 2014, 289(20): 13876-13889.

[8] Fowler LR, Morain SR. Schrödinger's App. Am J Law Med, 2020, 46(2–3): 203-218.

[9] Freschlin CR, Fahlberg SA, Romero PA. Machine learning to navigate fitness landscapes for protein engineering. Curr Opin Biotechnol, 2022

[10] Hayafune M, Berisio R, Marchetti R, Silipo A, Kayama M, Desaki Y, Shibuya N. Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization. Proc Natl Acad Sci U S A, 2014, 111(3): E404-413.

[11] Horn SJ, Sikorski P, Cederkvist JB, Vaaje-Kolstad G, Sørlie M, Synstad B, Eijsink VG. Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides. Proc Natl Acad Sci U S A, 2006, 103(48): 18089-18094. 2006PNAS..10318089H

[12] Jakubec D, Skoda P, Krivak R, Novotny M, Hoksza D. PrankWeb 3: accelerated ligand-binding site predictions for experimental and modelled protein structures. Nucleic Acids Res, 2022, 50(W1): W593-w597.

[13] Jiménez-Ortega E, Kidibule PE, Fernández-Lobato M, Sanz-Aparicio J. Structure-function insights into the fungal endo-chitinase Chit33 depict its mechanism on chitinous material. Int J Mol Sci, 2022

[14] Kirsch RD, Joly E. An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res, 1998, 26(7): 1848-1850.

[15] Kobe B, Deisenhofer J. A structural basis of the interactions between leucine-rich repeats and protein ligands. Nature, 1995, 374(6518): 183-186. 1995Natur.374..183K

[16] Kozome D, Uechi K, Taira T, Fukada H, Kubota T, Ishikawa K. Structural analysis and construction of a thermostable antifungal chitinase. Appl Environ Microbiol, 2022, 88(12

[17] Li H, Greene LH. Sequence and structural analysis of the chitinase insertion domain reveals two conserved motifs involved in chitin-binding. PLoS ONE, 2010, 5(1 2010PLoSO...5.8654L

[18] Li JL, Wang SJ, Liu C, Li YX, Wei Y, Fu G, Zhang DW. Going beyond the local catalytic activity space of chitinase using a simulation-based iterative saturation mutagenesis strategy. ACS Catal, 2022, 12(16): 10235-10244.

[19] Liaqat F, Eltem R. Chitooligosaccharides and their biological activities: a comprehensive review. Carbohydr Polym, 2018, 184(1879-1344 Electronic): 243-259.

[20] Liu T, Liu Z, Song C, Hu Y, Han Z, She J, Chai J. Chitin-induced dimerization activates a plant immune receptor. Science, 2012, 336(6085): 1160-1164. 2012Sci...336.1160L

[21] Lv C, Gu T, Ma R, Yao W, Huang Y, Gu J, Zhao G. Biochemical characterization of a GH19 chitinase from Streptomyces alfalfae and its applications in crystalline chitin conversion and biocontrol. Int J Biol Macromol, 2021

[22] Madhuprakash J, El Gueddari NE, Moerschbacher BM, Podile AR. Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains. PLoS ONE, 2015, 10(1

[23] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem, 2009, 30(16): 2785-2791.

[24] Ohnuma T, Numata T, Osawa T, Mizuhara M, Vårum KM, Fukamizo T. Crystal structure and mode of action of a class V chitinase from Nicotiana tabacum. Plant Mol Biol, 2011, 75(3): 291-304.

[25] Sasaki C, Itoh Y, Takehara H, Kuhara S, Fukamizo T. Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by kinetic and molecular modeling studies. Plant Mol Biol, 2003, 52(1): 43-52.

[26] Sasaki C, Vårum KM, Itoh Y, Tamoi M, Fukamizo T. Rice chitinases: sugar recognition specificities of the individual subsites. Glycobiology, 2006, 16(12): 1242-1250.

[27] Sikorski P, Sørbotten A, Horn SJ, Eijsink VG, Vårum KM. Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan. Biochemistry, 2006, 45(31): 9566-9574.

[28] Song H, Bremer BJ, Hinds EC, Raskutti G, Romero PA. Inferring protein sequence-function relationships with large-scale positive-unlabeled learning. Cell Syst, 2021, 12(1): 92-101.e108.

[29] Su H, Gao L, Sun J, Mao X. Engineering a carbohydrate binding module to enhance chitinase catalytic efficiency on insoluble chitinous substrate. Food Chem, 2021

[30] Tian J, Wu N, Guo X, Guo J, Zhang J, Fan Y. Predicting the phenotypic effects of non-synonymous single nucleotide polymorphisms based on support vector machines. BMC Bioinform, 2007

[31] Tsurkan MV, Voronkina A, Khrunyk Y, Wysokowski M, Petrenko I, Ehrlich H. Progress in chitin analytics. Carbohydr Polym, 2021, 252(1879-1344 (Electronic)

[32] Weinstein J, Khersonsky O, Fleishman SJ. Practically useful protein-design methods combining phylogenetic and atomistic calculations. Curr Opin Struct Biol, 2020

[33] Xu P, Ni ZF, Zong MH, Ou XY, Yang JG, Lou WY. Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design. Int J Biol Macromol, 2020

[34] Yamada A, Shibuya N, Kodama O, Akatsuka T. Induction of phytoalexin formation in suspension-cultured rice cells by N-acetylchitooligosaccharides. Biosci Biotechnol Biochem, 1993, 57(3): 405-409.

[35] Zakariassen H, Aam BB, Horn SJ, Vårum KM, Sørlie M, Eijsink VG. Aromatic residues in the catalytic center of chitinase A from Serratia marcescens affect processivity, enzyme activity, and biomass converting efficiency. J Biol Chem, 2009, 284(16): 10610-10617.

[36] Zhang Y, Guan F, Xu G, Liu X, Zhang Y, Sun J, Tian J. A novel thermophilic chitinase directly mined from the marine metagenome using the deep learning tool Preoptem. Biosour Bioprocess, 2022

[37] Zheng J, Cheng G, Li Q, Jiao S, Feng C, Zhao X, Liu H. Chitin oligosaccharide modulates gut microbiota and attenuates high-fat-diet-induced metabolic syndrome in mice. Mar Drugs, 2018

Funding

the National Key R&D Program of China(2021YFC2103002)

the National Natural Science Foundation of China(32202720)

The Agricultural Science and Technology Innovation Program(CAAS-ZDRW202304)

the China Agriculture Research System of MOF and MARA(CARS-41)