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Literature summary for 3.2.1.7 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

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

Application

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

Cloned(Commentary)

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

Protein Variants

Protein Variants Comment Organism
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
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
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
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

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

KM Value [mM]

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

Metals/Ions

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

Organism

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

Purification (Commentary)

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
inulin + H2O
-
Talaromyces purpureogenus ?
-
?
inulin + H2O
-
Talaromyces purpureogenus ATCC 4713 ?
-
?
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 ?
-
?
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

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

General Information Comment Organism
additional information enzyme structure homology modeling using the structure of endoinulinase from Aspergillus ficcum (PDB ID 3RWK) as template Talaromyces purpureogenus
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]

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