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Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: Purification and characterization of a trypsin-like proteinase

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Abstract

A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and K m value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0–9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N- terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50–72% identity with other insect trypsin-like proteinases, and 44–50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

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Abbreviations

Bz:

benzoyl

FPLC:

fast performance liquid chromatography

For:

formyl

PMSF:

phenylmethylsulfonyl fluoride

pNA:

p-nitroanilide

TLCK:

Nα-p-tosyl-L-lysine chloromethyl ketone

TmT1:

trypsin-like proteinase from T. molitor larvae

E-64:

L-trans-epoxysuccinyl-L-leucine amido (4-guanidine)butane

Z:

N-benzoyloxycarbonyl

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Authors and Affiliations

  1. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071, Moscow, Russia

    T. A. Tsybina

  2. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992, Moscow, Russia

    T. A. Tsybina, Y. E. Dunaevsky, M. A. Belozersky & E. N. Elpidina

  3. Department of Entomology, Faculty of Biology, Lomonosov Moscow State University, 119992, Moscow, Russia

    D. P. Zhuzhikov

  4. Grain Marketing and Production Research Center, USDA Agricultural Research Service, 1515 College Ave, Manhattan, KS, 66502, USA

    B. Oppert

Authors
  1. T. A. Tsybina
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  2. Y. E. Dunaevsky
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  3. M. A. Belozersky
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  4. D. P. Zhuzhikov
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  5. B. Oppert
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  6. E. N. Elpidina
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Correspondence to E. N. Elpidina.

Additional information

Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 370–377.

Original Russian Text Copyright © 2005 by Tsybina, Dunaevsky, Belozersky, Zhuzhikov, Oppert, Elpidina.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM04-310, February 20, 2005.

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Tsybina, T.A., Dunaevsky, Y.E., Belozersky, M.A. et al. Digestive proteinases of yellow mealworm (Tenebrio molitor) larvae: Purification and characterization of a trypsin-like proteinase. Biochemistry (Moscow) 70, 300–305 (2005). https://doi.org/10.1007/s10541-005-0115-2

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  • DOI: https://doi.org/10.1007/s10541-005-0115-2

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