Methodological Approach to Determine the Sequence of Enzymes for Plant Tissue Polyglycan Complex Fragmentation

Background. Maximum stepwise enzymatic decomposition of cell wall matrix biopolymer complex potentially allows obtaining a complex of components with valuable physicochemical properties which are widely used in food industry and medicine. At the same time, the goal of deep processing and maximum raw material conversion is achieved. Optimal form of active agents is a homoenzyme one with the narrowest possible spectrum of target activities. However, in most casei, even homoenzymes have side effects. As a result, the properties of the final products obtained using these enzymes may differ from those required. Therefore, the development of a methodological approach to passive inactivation of non-target activities of enzymes by determining a rational sequence of their application is relevant. 
Materials and methods. The object of research was a set of data on the spectrum and magnitude of target activities of enzymes which have potential use for polyglycans obtaining from cell wall matrix during sequential processing of sugar beet pulp. In this research it was used an exclusion iterative combinatorial approach based on a comprehensive analysis of the target characteristics for each enzyme in order to identify criteria that allow for the unambiguous ranking of variants within each iteration, at the same time excluding those ones that do not meet the specified conditions. 
Results. The pool of enzymes is considered as an abstract set, where target and parasitic activities are grouped according to the component composition within the cell wall matrix. Based on this, an activity matrix was formed for the entire pool of enzymes. The number of rows with a non-zero value within each target activity and the number of columns with a non-zero value within each element of the enzyme set were defined as criteria. Based on the analysis of the numerical values for criteria within the each iteration, a rank is assigned to each one. The boundary conditions were set. The elements of the set that do not satisfy the boundary conditions were discarded. The implementation algorithm of the methodological approach has an iterative form using combinatorial methods. The approach was tested on a complex of enzymes for sugar beet pulp polyglycan matrix decomposition. 
Conclusions. As a result of this research a system of criteria, methodological approach and algorithm for determining the sequence of homoenzymes application for stepwise obtaining the biologically active components of plant raw material polyglycan complex have been developed. This system was based on passive inactivation of non-target activities. Presumably, the developed criteria, methodological approach and algorithm for its implementation are universal and applicable to the analysis of homoenzyme complexes for its use in deep processing. The developed methodological approach is an integral part of the decision-making tree for development the technologies of industrial production of plant polyglycans with the guaranteed physicochemical characteristics.

1. Pérez García, M., Zhang, Y., Hayes, J., Salazar, A., Zabotina O.A., & Hong M. (2011). Structure and Interactions of Plant Cell-Wall Polysaccharides by Two- and Three-Dimensional Magic-Angle-Spinning Solid-State NMR. Biochemistry, 50(6):989-1000. https://doi.org/10.1021/bi101795q

2. Yoo, H.D., Kim, D., & Paek, S.H. (2012). Plant cell wall polysaccharides as potential resources for the development of novel prebiotics. Biomol Ther (Seoul), 20(4):371-9. https://doi.org/10.4062/biomolther.2012.20.4.371.

3. Held, M.A., Jiang, N., Basu, D., Showalter, A.M., & Faik, A. (2014). Plant Cell Wall Polysaccharides: Structure and Biosynthesis. In: Ramawat K., Mérillon J.M. (eds) Polysaccharides. Springer, Cham. pp 1-47. https://doi.org/10.1007/978-3-319-03751-6_73-1.

4. Voiniciuc, C., Pauly, M., & Usadel, B. (2018). Monitoring Polysaccharide Dynamics in the Plant Cell Wall. Plant Physiol., 2018, 176(4):2590-2600. https://doi.org/10.1104%2Fpp.17.01776.

5. Amos, R.A., & Mohnen, D. (2019). Critical Review of Plant Cell Wall Matrix Polysaccharide Glycosyltransferase Activities Verified by Heterologous Protein Expression. Front. Plant Sci., 10:915. https://doi.org/10.3389/fpls.2019.00915

6. Siemińska-Kuczer, A., Szymańska-Chargot, M., & Zdunek, A. (2022). Recent advances in interactions between polyphenols and plant cell wall polysaccharides as studied using an adsorption technique. Food Chemistry, 373B:131487. https://doi.org/10.1016/j.foodchem.2021.131487.

7. Du, J., Anderson, C.T., & Xiao, C. (2022). Dynamics of pectic homogalacturonan in cellular morphogenesis and adhesion, wall integrity sensing and plant development. Nat. Plants, 8:332340. https://doi.org/10.1038/s41477-022-01120-2.

8. Panchev, I.N., Slavov, A., Nikolova, Kr., & Kovacheva, D. (2010). On the water-sorption properties of pectin. Food Hydrocolloids, 24(8):763e769. https://doi.org/10.1016/j.foodhyd.2010.04.002

9. Venzon, S.S., Canteri, M.H., Granato, D., Junior, B.D., Maciel, G.M., Stafussa, A.P., & Haminiuk, C.W. (2015). Physicochemical properties of modified citrus pectins extracted from orange pomace. J. Food Sci. Technol., 52(7):4102-12. https://doi.org/10.1007/s13197-014-1419-2.

10. de Moura, F.A., Macagnan, F.T., Dos Santos, L.R., Bizzani, M., de Oliveira Petkowicz, C.L., & da Silva, L.P. (2017). Characterization and physicochemical properties of pectins extracted from agroindustrial by-products. J. Food Sci. Technol., 2017, 54(10):3111-3117. https://doi.org/10.1007/s13197-017-2747-9.

11. Bok-Badura, J., Jakóbik-Kolon, A., Karoń, K., & Mitko, K. (2018). Sorption studies of heavy metal ions on pectin-nano-titanium dioxide composite adsorbent. Separation Science and Technology, 53(7):1034-1044, https://doi.org/10.1080/01496395.2017.1329840.

12. Wang, R., Li, Y., Shuai, X., Chen, J., Liang, R., & Liu, C. (2021). Development of Pectin-Based Aerogels with Several Excellent Properties for the Adsorption of Pb2+. Foods, 10(12):3127. https://doi.org/10.3390/foods10123127.

13. Semenycheva, L.L., Kuleshova, N.V., Mitin, A.V., Belaya, T.A., & Mochkina, D.V. (2020). Molecular weight characteristics and sorption properties of pectin extracted from different substrates. Proceedings of Universities. Applied Chemistry and Biotechnology, 10(4):728-737. https://doi.org/10.21285/2227-2925-2020-10-4-728-737.

14. Einhorn-Stoll, U., Kastner, H., & Senge, B. (2012). Comparison of Molecular Parameters; Material Properties and Gelling behaviour of Commercial Citrus Pectins. In: Williams P.A., Phillips G.O. (eds) Gums and Stabilisers for the Food Industry 16. The Royal Society of Chemistry, pp.199-206. ISBN 978-1-84973-358-8. https://doi.org/10.1039/9781849734554-00199.

15. Yuliarti, O., Low Sok-Hoon, A., & Yee, S. (2017). Chong Influence of pH, pectin and Ca concentration on gelation properties of low-methoxyl pectin extracted from Cyclea barbata Miers. Food Structure, 11:16-23. https://doi.org/10.1016/j.foostr.2016.10.005.

16. Gawkowska, D., Cybulska, J., & Zdunek, A. (2018). Structure-Related Gelling of Pectins and Linking with Other Natural Compounds: A Review. Polymers (Basel), 10(7):762. https://doi.org/10.3390/polym10070762.

17. Lee, B.H., Jung, H.T., Kim, H.S., & Yoo, S.H. (2021). Structural and gelling properties of very low methoxyl pectin produced by an alkali-treatment. Korean J. Food Sci. Technol., 2021, 53(2):121-125. https://doi.org/10.9721/KJFST.2021.53.2.121.

18. Kazantsev, E.V., Kondratev, N.B., Rudenko, O.S., Petrova, N.A., & Belova, I.A. (2022). Formation of a foamy structure of confectionery pastille products. Food systems, 5(1):64-69. (In Russ.) https://doi.org/10.21323/2618-9771-2022-5-1-64-69.

19. Nakauma, M., Funami, T., Noda, S., Ishihara, S., Al-Assaf, S., Nishinari, K., & Phillips, G.O. (2008). Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids, 22(7):1254-1267. https://doi.org/10.1016/j.foodhyd.2007.09.004.

20. Duan, X., Yang, Z., Yang, J., Liu, F., Xu, X., & Pan, S. (2021). Structural and Emulsifying Properties of Citric Acid Extracted Satsuma Mandarin Peel Pectin. Foods, 10:2459. https://doi.org/10.3390/foods10102459.

21. Bindereif, B., Karbstein, H.P., Zahn, K., & van der Schaaf, U.S. (2022). Effect of Conformation of Sugar Beet Pectin on the Interfacial and Emulsifying Properties. Foods, 11:214. https://doi.org/10.3390/foods11020214.

22. Prandi, B., Baldassarre, S., Babbar, N. et al. (2018). Pectin oligosaccharides from sugar beet pulp: molecular characterization and potential prebiotic activity. Food Funct., 9:1557-1569. https://doi.org/10.1039/C7FO01182B.

23. Ishisono, K., Mano, T., Yabe, T., & Kitaguchi, K. (2019). Dietary Fiber Pectin Ameliorates Experimental Colitis in a Neutral Sugar Side Chain-Dependent Manner. Front. Immunol., 10:2979. https://doi.org/10.3389/fimmu.2019.02979.

24. Larsen, N., Bussolo de Souza, C., Krych, L., Barbosa Cahú, T., Wiese, M., Kot, W., Hansen, K.M., Blennow, A., Venema, K., & Jespersen, L. (2019). Potential of Pectins to Beneficially Modulate the Gut Microbiota Depends on Their Structural Properties. Front. Microbiol., 10:223. https://doi.org/10.3389/fmicb.2019.00223.

25. Sundar Raj, A.A., Rubila, S., Jayabalan, R,. & Ranganathan, T.V. (2012). A Review on Pectin: Chemistry Due to General Properties of Pectin and Its Pharmaceutical Uses. Omics International, 1(12):1-4. https://doi.org/10.4172/scientificreports.550.

26. Kumar, R.V., Srivastava, D., Singh, V. et al. (2020). Characterization, biological evaluation and molecular docking of mulberry fruit pectin. Sci Rep, 10:21789. https://doi.org/10.1038/s41598-020-78086-8.

27. Zhang, W., Xu, P., & Zhang, H. (2015). Pectin in cancer therapy: A review. Trends in Food Science & Technology, 44(2):258-271. https://doi.org/10.1016/j.tifs.2015.04.001.

28. Delphi, L., & Sepehri, H. (2016). Apple pectin: A natural source for cancer suppression in 4T1 breast cancer cells in vitro and express p53 in mouse bearing 4T1 cancer tumors, in vivo. Biomed Pharmacother., 2016, 84:637-644. https://doi.org/10.1016/j.biopha.2016.09.080.

29. Wang, L., Zhao, L., Gong, F.l. et al. (2022). Modified citrus pectin inhibits breast cancer development in mice by targeting tumor-associated macrophage survival and polarization in hypoxic microenvironment. Acta Pharmacol. Sin., 43:1556-1567. https://doi.org/10.1038/s41401-021-00748-8.

30. Kondratenko, V.V., Kondratenko, T.Yu., Petrov, A.N., & Belozerov, G.A.. (2020). Assessing protopectin transformation potential of plant tissue using a zoned criterion space. Foods and Raw Materials, 8(2):348-361. http://doi.org/10.21603/2308-4057-2020-2-348-361.

31. Kondratenko, V.V., Kondratenko, T.Yu., & Petrov, A.N. (2021). Directed homoenzymatic fragmentation of the plant protopectin complex: Assessment criteria. Foods and Raw Materials, 9(2):254-261. https://doi.org/10.21603/2308-4057-2021-2-254-261.

32. Marry, M., McCann, M.C., Kolpak, F., White, A.R., Stacey, N.J., & Roberts, K. (2000). Extraction of pectic polysaccharides from sugar-beet cell walls. J. Sci. Food Agric, 80:17-28. https://doi.org/10.1002/(SICI)1097-0010(20000101)80:1<17::AID-JSFA491>3.0.CO;2-4.

33. Yapo, B.M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of extraction conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts. Food Chemistry, 100(4):1356-1364. https://doi.org/10.1016/j.foodchem.2005.12.012.

34. Elizaryev, A., Kostryukova, N., Vdovina, I., Riianova, E., Melnikova, A., & Sadykova, A. (2020). Low-waste production of pectin from beet pulp. E3S Web Conf., 203:04012. https://doi.org/10.1051/e3sconf/202020304012.

35. Jung, S.K., Parisutham, V., Jeong, S.H., & Lee, S.K. (2012). Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels. Journal of Biomedicine and Biotechnology, 2012:Article ID 405842. https://doi.org/10.1155/2012/405842.

36. Dominiak, M., Søndergaard, K.M., Wichmann, J., Vidal-Melgosa, S., Willats, W.G.T., Meyer, A.S., & Mikkelsen, J.D. (2014). Application of enzymes for efficient extraction, modification, and development of functional properties of lime pectin. Food Hydrocolloids, 40:273-282. https://doi.org/10.1016/j.foodhyd.2014.03.009.

37. Marjamaa, K., & Kruus, K. (2018). Enzyme biotechnology in degradation and modification of plant cell wall polymers. Physiol. Plantarum, 164:106-118. https://doi.org/10.1111/ppl.12800.

38. Mota, Th.R., de Oliveira, D.M., Marchiosi, R., Ferrarese-Filho, O., & dos Santos, W.D. (2018). Plant cell wall composition and enzymatic deconstruction. AIMS Bioengineering, 5(1):63-77. https://doi.org/10.3934/bioeng.2018.1.63.

39. Carpita, N.C., & McCann, M.C. (2020). Redesigning plant cell walls for the biomass-based bioeconomy. J. Biol. Chem., 295(44):15144-15157. https://doi.org/10.1074/jbc.rev120.014561.

40. Abou-Elseoud, W.S., Hassan, E.A., & Hassan, M.L. (2021). Extraction of pectin from sugar beet pulp by enzymatic and ultrasound-assisted treatments. Carbohydrate Polymer Technologies and Applications, 2:100042. https://doi.org/10.1016/j.carpta.2021.100042.

41. Hennessey-Ramos, L., Murillo-Arango, W., Vasco-Correa, J., & Paz Astudillo, I.C. (2021). Enzymatic Extraction and Characterization of Pectin from Cocoa Pod Husks (Theobroma cacao L.) Using Celluclast® 1.5 L. Molecules, 26:1473. https://doi.org/10.3390/molecules26051473.

42. Bonnin, E., Garnier, C., & Ralet, M.C. (2014). Pectin-modifying enzymes and pectin-derived materials: applications and impacts. Appl. Microbiol. Biotechnol., 98:519-532. https://doi.org/10.1007/s00253-013-5388-6.

43. Gudmundsson, M. (2014). Structure and functional studies of plant cell wall degrading enzymes. Diss. (sammanfattning/summary) [Doctoral thesis] Uppsala: Sveriges lantbruksuniv., Acta Universitatis Agriculturae Sueciae, 2014:83. 96 p. ISSN 1652-6880. ISBN 978-91-576-8114-0. http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-e-2233.

44. Xia, J.L., & Li, P.J. (2019). Pectic enzymes. In: Melton L., Shahidi F., Varelis P. (eds) Encyclopedia of Food Chemistry. Academic Press, Oxford, UK. pp.270-276. https://doi.org/10.1016/B978-0-08-100596-5.21645-4.

45. Giovannoni, M., & Gramegna, G., Benedetti, M., Mattei B. (2020). Industrial Use of Cell Wall Degrading Enzymes: The Fine Line Between Production Strategy and Economic Feasibility. Front. Bioeng. Biotechnol., 8:358. https://doi.org/10.3389/fbioe.2020.00356.