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Biochemistry (Moscow)

, Volume 80, Issue 4, pp 473–482 | Cite as

Properties of enzyme preparations and homogeneous enzymes — Endoglucanases EG2 Penicillium verruculosum and LAM Myceliophthora thermophila

  • D. A. MerzlovEmail author
  • I. N. Zorov
  • G. S. Dotsenko
  • Yu. A. Denisenko
  • A. M. Rozhkova
  • A. D. Satrutdinov
  • E. A. Rubtsova
  • E. G. Kondratieva
  • A. P. Sinitsyn
Article

Abstract

The genes of endoglucanases EG2 (36.2 kDa) Penicillium verruculosum and LAM (30.8 kDa) Myceliophthora thermophila were cloned in P. verruculosum recombinant strain. New enzyme preparations with highly stable activity against β-glucan and laminarin were obtained and investigated, homogeneous enzymes EG2 (EC 3.2.1.4) and LAM (EC 3.2.1.6) being purified and characterized. For β-glucan, the EG2 K m value was found to be 10 times higher than that for LAM; however, EG2 demonstrated greater processivity due to its higher k cat. The pH and temperature optima of EG2 and LAM activity against barley β-glucan overlapped and were 4.3–4.9 and 61–67°C, respectively, and EG2 appeared to be more stable than LAM. Oligosaccharides with degree of polymerization 2–10 were formed by hydrolysis of β-glucan and laminarin by the studied enzymes. The recombinant enzyme preparations were faster and more effective in decreasing the reduced viscosity of wholegrain barley extract than some commercial enzyme preparations. Thus, the new enzyme preparations seem to be rather perspective as feed additives for degradation of non-starch polysaccharides in grain animal feed.

Key words

endoglucanase β-glucan β-glucan endodepolymerases enzyme preparations Penicillium verruculosum Myceliophthora thermophila 

Abbreviations

EP

enzyme preparations

NSP

non-starch polysaccharides

RS

reducing sugars

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References

  1. 1.
    Bedford, M., and Partridge, G. (2010) Enzymes in Farm Animal Nutrition, CABI, UK.CrossRefGoogle Scholar
  2. 2.
    Bacic, A., Fincher, G., and Stone, B. (2009) Chemistry, Biochemistry and Biology of (1–3)-β-Glucans and Related Polysaccharides, Academic Press, NY.Google Scholar
  3. 3.
    Holtekjolen, A. K., Uhlen, A. K., Brathen, E., Sahlstrom, S., and Knutsen, S. H. (2006) Contents of starch and non-starch polysaccharides in barley varieties of different origin, Food Chem., 94, 348–358.CrossRefGoogle Scholar
  4. 4.
    Aslanidis, C., and de Jong, P. J. (1990) Ligation-independent cloning of PCR products (LIC-PCR), Nucleic Acids Res., 18, 6069–6074.CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Sinitsyn, A. P., Rozhkova, A. M., Sinitsyna, O. A., Fedorova, E. A., Okunev, O. N., Bekkarevich, A. O., Sokolova, L. M., Mastys, V. Yu., Koshelev, A. V., Vinetskiy, Yu. P., Chernoglazov, V. M., and Zorov, I. N. (2010) Genetic construct providing expression of the target homologous and heterologous genes in cells of mycelial fungus Penicillium verruculosum used as a host, method for production of fungus Penicillium verruculosum strain and method for production of enzyme preparation, State Register of Inventions of the Russian Federation, RU2378372 (C2).Google Scholar
  6. 6.
    Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, NY.Google Scholar
  7. 7.
    Peterson, G. L. (1979) Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr, and Randall, Anal. Biochem., 100, 201–220.CrossRefPubMedGoogle Scholar
  8. 8.
    Dauson, R., Elliot, D., Elliot, U., and Johnes, K. (1991) Biochemist’s Handbook [Russian translation], Mir, Moscow.Google Scholar
  9. 9.
    Sinitsyn, A. P., Chernoglazov, V. M., and Gusakov, A. V. (1990) Methods for Study and Properties of Cellulolytic Enzymes [in Russian], VINITI, Moscow.Google Scholar
  10. 10.
    Sinitsyn, A. P., Gusakov, A. V., and Chernoglazov, V. M. (1995) Bioconversion of Lignocellulose Materials [in Russian], MSU Publishers, Moscow.Google Scholar
  11. 11.
    James, P. (2001) Proteome Research: Mass Spectrometry — Principles and Practice, Springer Verlag, Heidelberg.CrossRefGoogle Scholar
  12. 12.
    Gusakov, A. V., Semenova, M. V., and Sinitsyn, A. P. (2010) Mass spectrometry in the study of extracellular enzymes produced by filamentous fungi, J. Anal. Chem., 65, 1446–1461.CrossRefGoogle Scholar
  13. 13.
    Vlasenko, E., Schulein, M., Cherry, J., and Xu, F. (2010) Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases, Bioresour. Technol., 101, 2405–2411.CrossRefPubMedGoogle Scholar
  14. 14.
    Morozova, V. V., Gusakov, A. V., Andrianov, R. M., Pravilnikov, A. G., Osipov, D. O., and Sinitsyn, A. P. (2010) Cellulases of Penicillium verruculosum, Biotechnol. J., 5, 871–880.CrossRefPubMedGoogle Scholar
  15. 15.
    Collins, H. M., Burton, R. A., Topping, D. L., Liao, M. L., Bacic, A., and Fincher, G. B. (2010) Variability in fine structures of noncellulosic cell wall polysaccharides from cereal grains: potential importance in human health and nutrition, Cereal Chem., 87, 272–282.CrossRefGoogle Scholar
  16. 16.
    Tymczyszyn, E. E., Santos, M. I., Costa, M. C., Illanes, A., and Gomez-Zavaglia, A. (2014) in Carbohydrates Applications in Medicine (Gil, M. H., ed.) Research Signpost, Kerala, pp. 127–154.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • D. A. Merzlov
    • 1
    Email author
  • I. N. Zorov
    • 1
    • 2
  • G. S. Dotsenko
    • 2
  • Yu. A. Denisenko
    • 2
  • A. M. Rozhkova
    • 2
  • A. D. Satrutdinov
    • 2
  • E. A. Rubtsova
    • 2
  • E. G. Kondratieva
    • 2
  • A. P. Sinitsyn
    • 1
    • 2
  1. 1.Chemical FacultyLomonosov Moscow State UniversityMoscowRussia
  2. 2.Bach Institute of BiochemistryRussian Academy of SciencesMoscowRussia

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