Recombinant Chymotrypsin-Like Peptidase from Tenebrio molitor with a Non-Canonical Substrate-Binding Site

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Resumo

We characterized an alkaline chymotrypsin-like serine peptidase from the yellow mealworm Tenebrio molitor with a non-canonical substrate-binding subsite for its possible application as a component (an additive) in various biological products. The enzyme was obtained as a recombinant preparation. Purification was carried out using affinity chromatography on Ni2+-NTA agarose. The specificity constants (kcat/KM) for the chymotrypsin substrates, Glp-AAF-pNA, Suc-AAPF-pNA, and Ac-Y-pNA were 7, 4.2 and 0.9 (µM∙min)–1, respectively. Optimum of the proteolytic activity was observed at pH 9.0. The enzyme was stable at the alkaline pH range, and in the presence of BSA also in the acidic region. Peptidase was inhibited by synthetic inhibitors such as PMSF, TPCK, chymostatin, while EDTA, E-64, and pepstatin had no effect on the enzyme activity. The purified enzyme showed high stability over time in the presence of BSA. The short life cycle of the insect and the production of a large number of peptidases in the midgut with high catalytic activity and stability can make T. molitor an excellent alternative source of industrially important enzymes for application as components (additives) in various biological products (e. g., stain removers, detergents, etc.).

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Sobre autores

V. Tereshchenkova

Lomonosov Moscow State University, Faculty of Chemistry

Autor responsável pela correspondência
Email: elp@belozersky.msu.ru
Rússia, Moscow

N. Zhiganov

Lomonosov Moscow State University, Faculty of Biology

Email: elp@belozersky.msu.ru
Rússia, Moscow

A. Gubaeva

Lomonosov Moscow State University, Faculty of Chemistry

Email: elp@belozersky.msu.ru
Rússia, Moscow

F. Akentyev

National Research Center “Kurchatov Institute”; “Kurchatov Genome Center”, National Research Center “Kurchatov Institute”

Email: elp@belozersky.msu.ru
Rússia, Moscow; Moscow

Ya. Dunaevsky

Lomonosov Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology

Email: elp@belozersky.msu.ru
Rússia, Moscow

D. Kozlov

National Research Center “Kurchatov Institute”

Email: elp@belozersky.msu.ru
Rússia, Moscow

M. Belozersky

Lomonosov Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology

Email: elp@belozersky.msu.ru
Rússia, Moscow

E. Elpidina

Lomonosov Moscow State University, A.N. Belozersky Research Institute of Physico-Chemical Biology

Email: elp@belozersky.msu.ru
Rússia, Moscow

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2. Fig. 1. Sequence of rProSerP38. R| – place of processing by trypsin (cleavage of propeptide). Amino acid residues of the active center (HDS) are marked in red, and substrate-binding subsite S1 (GGD) – in blue.

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3. Fig. 2. Dependence of the activity of the processed preparation of the recombinant propeptidase rProSerP38 T. molitor on the processing time. Error bars reflect the confidence interval.

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4. Fig. 3. Stability of the mature form of the recombinant T. molitor peptidase rSerP38. Error bars represent the confidence interval.

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5. Fig. 4. Stability of processed recombinant peptidase rProSerP38 of T. molitor. Error bars represent confidence intervals.

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6. Fig. 5. Purification of the processed preparation of the recombinant propeptidase rProSerP38 T. molitor by metal-chelate affinity chromatography on Ni2+-NTA agarose: 1 – enzyme activity on the substrate Suc-AAPF-pNA, 2 – imidazole concentration gradient (mM).

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7. Fig. 6. Result of post-electrophoretic testing of activity for the fluorogenic substrate Suc-AAPF-AMC: 1 – rProSerP38 preparation before processing; 2 – rProSerP38 peptidase preparation processed by trypsin; 3 – pooled eluate fractions 16–18 before desalting; 4 – pooled fractions 16–18 after desalting and concentration.

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8. Fig. 7. Stability of processed and purified T. molitor rProSerP38 peptidase preparation. Error bars represent confidence intervals.

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9. Fig. 8. Stability of the mature form of the recombinant T. molitor peptidase rSerP38 in the presence of BSA.

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10. Fig. 9. Effect of pH on the activity of recombinant peptidase rSerP38 T. molitor.

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11. Fig. 10. Effect of pH on the stability of recombinant peptidase rSerP38 T. molitor (7 nM): a) without BSA, incubation for 30 min; b) in the presence of BSA (0.62 μM).

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12. Fig. 11. Activity of recombinant peptidase rSerP38 T. molitor towards various substrates.

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13. Fig. 12. Dependence of the rate of the enzymatic reaction of hydrolysis of substrates by rSerP38 peptidase: a) Glp-AAF-pNA (0.1–1.0 μM); b) Suc-AAPF-pNA (0.1–1.0 mM); c) Ac-Y-pNA (0.1–3.0 mM).

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