Inulinase

Inulinase (EC 3.2.1.7 and EC 3.2.1.8, inulase, endoinulinase, endo-inulinase, exoinulinase, 2,1-β-D-fructan fructanohydrolase) is an enzyme with systematic name 1-β-D-fructan fructanohydrolase.[1][2] It catalyses the reaction

Endohydrolysis of (2→1)-β-D-fructosidic linkages in inulin
Inulinase
Identifiers
EC no.3.2.1.7
CAS no.9025-67-6
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
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PMCarticles
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NCBIproteins

Inulinase has 2 EC numbers of 3.2.1.7,[3] and 3.2.1.8,[2] for endo- and -exo inulinases, respectively. This classifies it as a hydrolase, specifically a glycosylase of glycosidic nature capable of hydrolyzing O- and S- glycosyl. Due to its chemical reactions, the food industry uses this enzyme to create high fructose syrup. It can be extracted from many tuber vegetables, such as Jerusalem artichoke, dahlia, and chicory.[4]

Reaction mechanism

The enzymatic reaction occurs between the inulinase and the inulin, with the assistance of water via hydrolysis. It's typically done within one step.[5] The reaction centers around the breakage of a bond.[6] The products result in fructose syrup and fructo-oligosaccharide. When these products undergo fermentation, additional products may be formed: lactic acid, bioethanol, citric acid, etc. The mechanisms involved may be considered highly efficient when compared to other enzymes.

Inulinase Production

There are several places for inulinase to be found and produced. A common way is via plants, usually tubular root vegetables (Jerusalem artichoke, dahlia, chicory), where inulinase can be extracted.[7] In this instance, fructose syrup is produced and used by the food industry.[4]

Aspergillus sp., a bacteria which can produce inulin and inulinase[7]

Another way is extraction via bacteria (fungal endophyte, Kluyveromyces marxianus, Cryptococcus aureus). Marine bacteria, yeasts, and fungi are used commonly as well.[8]

Jerusalem artichokes, native to North America[9]

Function

As a catalytic enzyme, either endo- or -exo inulinase destroys the bonds of one fructose attached to the inulin chain.[2] Various affinities depending on the environment will affect how the inulinase interacts with the inulin. It functions efficiently between 40-80 degrees Celsius, although depending on the source of the enzyme, it can go as low as 30 degrees Celsius and still work well.[10]

In regards to being tied into a larger metabolism, it helps break down inulin. When people consume inulin (a soluble fiber), the inulinase breaks down the inulin and it goes through fermentation.[11]

Structure
Crystal structure of hydrolase exo-inulinase from Aspergillus awamori[12]

Inulinase contains a N-terminal domain of glycosyl hydrolase family 32 which forms a five bladed beta propeller structure[13] and C terminal domain of glycosyl hydrolase family 32. It forms a beta sandwich module.[13]

The crystal structures are shown here on either side of the page. The two structures of endo-inulinase and exo-inulinase are mainly made of beta-sheets, with the exception of the endo-inulinase, which has one alpha helix. Being made of beta sheets allows the structures to be more stable during chemical reactions.[14]

Crystal structure of hydrolase endo-inulinase from Bifidobacterium adolescentis expressed in Escherichia coli[15]

Active and regulatory sites

Both of the inulinases have different active sites, with exo-inulinase having a funnel shape whereas endo-inulinase has a pocket shape.[16] They're complicated due to the amino acids needing to be a balance of catalytic and conserved to achieve a stable enzymatic active site. According to the most recent reference, three amino acids (serine, aspartic acid, and glutamic acid) bind via a hydrogen bond to the fructose molecule that attaches to the exo-inulinase. Whereas with three other amino acids (glutamic acid, tryptophan, and asparagine) those bind via a hydrogen bond to the ketose molecule that attaches to the endo-inulinase.[16]

The structure of the enzymes may or may not influence their function. Each version of the enzyme has about 518 amino acids within its sequence.[12][15] Due to the differences expressed in endo- and exo- inulinases, it will need to be proven or disproven the structure of these enzymes is the reason for the functionality of inulinase.

References
  1. Adams M, Richtmyer NK, Hudson CS (1943). "Some enzymes present in highly purified invertase preparations; a contribution to the study of fructofuranosidases, galactosidases, glucosidases and mannosidases". J. Am. Chem. Soc. 65 (7): 1369–1380. doi:10.1021/ja01247a029.
  2. Liu Y, Zhou SH, Cheng YR, Chi Z, Chi ZM, Liu GL (2016). "Synergistic effect between the recombinant exo-inulinase and endo-inulinase on inulin hydrolysis". Journal of Molecular Catalysis B: Enzymatic. 128: 27–38. doi:10.1016/j.molcatb.2016.03.005. ISSN 1381-1177.
  3. "Information on EC 3.2.1.7 - inulinase".
  4. Jain SC, Jain PC, Kango N (January 2012). "Production of inulinase from Kluyveromyces marxianus using dahlia tuber extract". Brazilian Journal of Microbiology. 43 (1): 62–69. doi:10.1590/S1517-83822012000100007. PMC 3768966. PMID 24031804.
  5. Choukade R, Kango N (2021). "Applications of Fungal Inulinases". Encyclopedia of Mycology. Elsevier. pp. 337–347. doi:10.1016/b978-0-12-819990-9.00016-0. ISBN 978-0-323-85180-0. S2CID 235092497.
  6. Nascimento DS, Valasques Junior G, Fernandes P, Ribeiro GC, Lima DM, Góes-Neto A, et al. (June 2012). "Production, characterization and application of inulinase from fungal endophyte CCMB 328". Anais da Academia Brasileira de Ciências. 84 (2): 443–454. doi:10.1590/S0001-37652012005000035. PMID 22634747.
  7. Hughes SR, Qureshi N, López-Núñez JC, Jones MA, Jarodsky JM, Galindo-Leva LÁ, Lindquist MR (April 2017). "Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals". World Journal of Microbiology & Biotechnology. 33 (4): 78. doi:10.1007/s11274-017-2241-6. PMID 28341907. S2CID 23678976.
  8. Chi Z, Chi Z, Zhang T, Liu G, Yue L (February 2009). "Inulinase-expressing microorganisms and applications of inulinases". Applied Microbiology and Biotechnology. 82 (2): 211–220. doi:10.1007/s00253-008-1827-1. PMID 19122997. S2CID 19215484.
  9. "Helianthus tuberosus L. GRIN-Global". npgsweb.ars-grin.gov. Retrieved 2022-10-24.
  10. Sheng J, Chi Z, Gong F, Li J (February 2008). "Purification and characterization of extracellular inulinase from a marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the purified inulinase". Applied Biochemistry and Biotechnology. 144 (2): 111–121. doi:10.1007/s12010-007-8025-y. PMID 18456943. S2CID 9987037.
  11. McRorie JW, McKeown NM (February 2017). "Understanding the Physics of Functional Fibers in the Gastrointestinal Tract: An Evidence-Based Approach to Resolving Enduring Misconceptions about Insoluble and Soluble Fiber". Journal of the Academy of Nutrition and Dietetics. 117 (2): 251–264. doi:10.1016/j.jand.2016.09.021. PMID 27863994.
  12. Nagem RA, Rojas AL, Golubev AM, Korneeva OS, Eneyskaya EV, Kulminskaya AA, et al. (November 2004). "Crystal structure of exo-inulinase from Aspergillus awamori: the enzyme fold and structural determinants of substrate recognition". Journal of Molecular Biology. 344 (2): 471–480. doi:10.1016/j.jmb.2004.09.024. PMID 15522299.
  13. Alberto F, Bignon C, Sulzenbacher G, Henrissat B, Czjzek M (April 2004). "The three-dimensional structure of invertase (beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases". The Journal of Biological Chemistry. 279 (18): 18903–10. doi:10.1074/jbc.M313911200. PMID 14973124.
  14. Cohen N, Eisenbach CD (April 2020). "Molecular Mechanics of Beta-Sheets". ACS Biomaterials Science & Engineering. 6 (4): 1940–1949. doi:10.1021/acsbiomaterials.9b01983. PMID 33455356. S2CID 213207373.
  15. Lima MZ, Muniz JR (2020-02-05). "Structure of GH32 hydrolase from Bifidobacterium adolescentis in complex with frutose". dx.doi.org. doi:10.2210/pdb6nun/pdb. S2CID 242805321. Retrieved 2022-10-24.
  16. Singh PK, Shukla P (September 2012). "Molecular modeling and docking of microbial inulinases towards perceptive enzyme-substrate interactions". Indian Journal of Microbiology. 52 (3): 373–380. doi:10.1007/s12088-012-0248-0. PMC 3460115. PMID 23997327.
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