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Literature summary extracted from

  • Afriat-Jurnou, L.; Cohen, R.; Paluy, I.; Ben-Adiva, R.; Yadid, I.
    Directed evolution of an endoinulinase from Talaromyces purpureogenus toward efficient production of inulooligosaccharides (2018), Biotechnol. Prog., 34, 868-877 .
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.2.1.7 additional information no effect by 1 mM DTT on wild-type enzyme activity Talaromyces purpureogenus
3.2.1.7 SDS activates the wild-type enzyme by 62% at 0.1% v/v Talaromyces purpureogenus
3.2.1.7 Triton X-100 activates the wild-type enzyme by 33% at 0.1% v/v Talaromyces purpureogenus

Application

EC Number Application Comment Organism
3.2.1.7 synthesis endoinulinases are an industrial tool critical for the production of inulooligosaccharides (IOS) Talaromyces purpureogenus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.2.1.7 DNA and amino acid sequence determination and analysis, recombinant expression of MBP-tagged enzyme in Escherichia coli strain BL21 Talaromyces purpureogenus

Protein Variants

EC Number Protein Variants Comment Organism
3.2.1.7 additional information directed evolution yields variants showing up to 5fold improvements in soluble enzyme production compared to the starting point which enables high-yield production of highly purified recombinant enzyme. The distribution of the enzymatic reaction products demonstrates that after 24 h of incubation, the main product (57%) has a degree of polymerization of 3 (DP3). The MBP-fused wild-type enzyme shows increased activity compared to unaltered wild-type enzyme. The evolved endoinulinase mutant variants exhibit increased solubility and activity compared to wild-type Talaromyces purpureogenus
3.2.1.7 Y128H site-directed mutagenesis, the mutation Y128H is in close proximity to the active site, it leads to modified enzyme catalytic activity with increased kcat and kcat/KM compared to untagged wild-type enzyme, while the activity of MBP-tagged wild-type enzyme is higher Talaromyces purpureogenus
3.2.1.7 Y128H/A316T/E344K/T504M site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme and MBP-fused wild-type enzyme Talaromyces purpureogenus
3.2.1.7 Y128H/E344K/T504M site-directed mutagenesis, the mutation E344K is located on the enzyme protein surface, mutation Y128H is in close proximity to the active site. The mutant shows reduced activity compared to wild-type enzyme and MBP-fused wild-type enzyme Talaromyces purpureogenus

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.2.1.7 Al3+ 64% inhibition of wild-type enzyme at 1 mM Talaromyces purpureogenus
3.2.1.7 Cu2+ 50% inhibition of wild-type enzyme at 1 mM Talaromyces purpureogenus
3.2.1.7 EDTA 68% inhibition of wild-type enzyme at 1 mM Talaromyces purpureogenus
3.2.1.7 additional information no effect by 1 mM DTT on enzyme activity Talaromyces purpureogenus
3.2.1.7 Ni2+ 14% inhibition of wild-type enzyme at 1 mM Talaromyces purpureogenus
3.2.1.7 Zn2+ 34% inhibition of wild-type enzyme at 1 mM Talaromyces purpureogenus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.2.1.7 0.21
-
 inulin pH 5.4, 55°C, recombinant untagged wild-type enzyme Talaromyces purpureogenus
3.2.1.7 0.78
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged wild-type enzyme Talaromyces purpureogenus
3.2.1.7 2.4
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H/E344K/T504M enzyme Talaromyces purpureogenus
3.2.1.7 2.82
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H enzyme Talaromyces purpureogenus
3.2.1.7 3.12
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H/A316T/E344K/T504M enzyme Talaromyces purpureogenus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.2.1.7 Ca2+ activates the wild-type enzyme 16% at 1 mM Talaromyces purpureogenus
3.2.1.7 Mg2+ activates the wild-type enzyme 32% at 1 mM Talaromyces purpureogenus
3.2.1.7 additional information no effect by 1 mM K+ on enzyme activity Talaromyces purpureogenus

Organism

EC Number Organism UniProt Comment Textmining
3.2.1.7 Talaromyces purpureogenus O00056 i.e. Penicillium purpureogenum
-
3.2.1.7 Talaromyces purpureogenus ATCC 4713 O00056 i.e. Penicillium purpureogenum
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.2.1.7 recombinant MBP-tagged enzyme from Escherichia coli strain BL21 by amylose affinity chromatography, and gel filtration Talaromyces purpureogenus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.2.1.7 inulin + H2O
-
 Talaromyces purpureogenus ?
-
 ?
3.2.1.7 inulin + H2O
-
 Talaromyces purpureogenus ATCC 4713 ?
-
 ?
3.2.1.7 additional information the enzyme targets the internal beta-2,1 fructofuranosidic linkages of inulin to yield both GFn and Fm molecules (G for glucose, F for fructose, n and m indicate the number of fructose moieties) Talaromyces purpureogenus ?
-
 ?
3.2.1.7 additional information the enzyme targets the internal beta-2,1 fructofuranosidic linkages of inulin to yield both GFn and Fm molecules (G for glucose, F for fructose, n and m indicate the number of fructose moieties) Talaromyces purpureogenus ATCC 4713 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.2.1.7 beta-2,1-D-fructan fructanohydrolase
-
 Talaromyces purpureogenus
3.2.1.7 endo-inulinase
-
 Talaromyces purpureogenus
3.2.1.7 endoinulinase
-
 Talaromyces purpureogenus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.2.1.7 55
-
 recombinant wild-type and mutant enzymes Talaromyces purpureogenus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.2.1.7 120
-
 inulin pH 5.4, 55°C, recombinant untagged wild-type enzyme Talaromyces purpureogenus
3.2.1.7 685
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H/A316T/E344K/T504M enzyme Talaromyces purpureogenus
3.2.1.7 792
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H/E344K/T504M enzyme Talaromyces purpureogenus
3.2.1.7 850
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged wild-type enzyme Talaromyces purpureogenus
3.2.1.7 1797
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H enzyme Talaromyces purpureogenus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.2.1.7 5.4
-
 assay at Talaromyces purpureogenus

General Information

EC Number General Information Comment Organism
3.2.1.7 additional information enzyme structure homology modeling using the structure of endoinulinase from Aspergillus ficcum (PDB ID 3RWK) as template Talaromyces purpureogenus
3.2.1.7 physiological function inulinases are fructofuranosyl hydrolases that target the beta-2,1 linkage of inulin and hydrolyze it into fructose, glucose and inulooligosaccharides (IOS), IOS are functioning as dietary fibers Talaromyces purpureogenus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.2.1.7 219.55
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H/A316T/E344K/T504M enzyme Talaromyces purpureogenus
3.2.1.7 330
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H/E344K/T504M enzyme Talaromyces purpureogenus
3.2.1.7 571.43
-
 inulin pH 5.4, 55°C, recombinant untagged wild-type enzyme Talaromyces purpureogenus
3.2.1.7 637.23
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged mutant Y128H enzyme Talaromyces purpureogenus
3.2.1.7 1089.74
-
 inulin pH 5.4, 55°C, recombinant MBP-tagged wild-type enzyme Talaromyces purpureogenus