Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2018, Vol. 53, ¹ 1, p. 189-200.
(how to cite this article)

doi: 10.15389/agrobiology.2018.1.189eng

UDC 573.6.086.83:577.21:57.052

Acknowledgements:
Experiments were carried out using equipment of Analytic Center for Nano and Biotechnology of Peter the Great St. Petersburg Polytechnic University
Supported financially by Russian Science Foundation (grant ¹ 16-16-10043), the methods of deacetylated chitooligosaccharides purification were developed using financial support from Russian Foundation for Basic Research (grant ¹ 15-29-05858-ofi-m)

 

PRODUCTION OF TERMINALLY N-DEACETYLATED OLIGOMERS
OF CHITOSANE USING RECOMBINANT CHITOOLIGOSACHARIDE
DEACETYLASE NodB OF BACTERIA Mesorhizobium loti EXPRESSED
IN Escherichia coli

I.V. Leppyanen1, V.V. Dolgikh2, T.O. Artamonova3, S.A. Lopatin4,
M.A. Khodorkovskii3, I.A. Tikhonovich1, E.A. Dolgikh1

1All-Russian Research Institute for Agricultural Microbiology, Federal Agency for Scientific Organizations, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia,
e-mail dol2helen@yahoo.com (✉ corresponding author), leppyanen_irina@rambler.ru, arriam2008@yandex.ru;
2All-Russian Research Institute of Plant Protection, Federal Agency for Scientific Organizations,3, sh. Podbel’skogo, St. Petersburg, 196608 Russia,
e-mail dol1slav@yahoo.com;
3Peter the Great St. Petersburg Polytechnic University, 29, ul. Politechnicheskaya,
St. Petersburg, 195251 Russia, e-mail artamonova@nanobio.spbstu.ru, khodorkovskii@mail.ru;
4Research Center of Biotechnology RAS, Federal Agency for Scientific Organizations, 33, str. 2, Leninskii prosp., Moscow, 119071 Russia, e-mail lopatin@biengi.ac.ru

ORCID:
Leppyanen I.V. orcid.org/0000-0002-2158-0855
Khodorkovskii M.A. orcid.org/0000-0001-7909-0683
Dolgikh V.V. orcid.org/0000-0002-2362-2633
Tikhonovich I.A. orcid.org/0000-0001-8968-854x
Artamonova T.O. orcid.org/0000-0002-0069-0561
Dolgikh E.A. orcid.org/0000-0002-5375-0943
Lopatin S.A. orcid.org/0000-0002-2018-7859

Received December 7, 2016

 

Chitin and chitosan oligomers affect the growth and development of plants and are able to induce plant resistance to infection with phytopathogens, which determines the interest in the preparation and use of these compounds. The influence of chitosan oligomers on the plant directly depends on the degree of deacetylation, but it is difficultly to obtain compounds with necessary structure using hydrolysis of the polymer or chemical synthesis. Such problems can be solved in the process of biosynthesis of chitooligosaccharides, when enzymes with specific activity are used. The selectivity of the chitooligosaccharide deacetylase (EC 3.5.1.-) of rhizobia to carry out the mono-deacetylation of the chitooligosaccharides at the terminal position of the molecule causes interest in studying the possibility to use this enzyme for the synthesis of such compounds. In current work we have developed approaches for the synthesis of mono-deacetylated chitopentaose (tetra-N-acetylchitopentaose) using Mesorhizobium loti CIAM1026 enzyme chitooligosaccharide deacetylase. Heterologous expression of the nodB gene encoding the M. loti chitooligosaccharide deacetylase in Escherichia coli XL1-Blue MRF' and SHuffle express strains using the modified pOPE101mod-nodB vector with deleted pelB sequence resulted in soluble enzyme preparation. The amount of soluble enzyme was higher in SHuffle express strain, which was specially developed for correct formation of disulfide bonds in synthesized proteins. Studying the properties of the enzyme purified on Ni-NTA agarose showed its ability to deacetylate penta-N-acetylchitopentaose at the terminal position. Mass spectrometric analysis confirmed the use of practically the entire substrate for the preparation of deacetylated tetra-N-acetylchitopentaose. Methods for the separation and purification of deacetylated chitooligosaccharides by ion exchange chromatography followed by desalination have been developed. Synthesis of terminally N-deacetylated chitosan oligomers may be a necessary step in the preparation of their conjugates with biologically active compounds.

Keywords: chitin and chitosan oligomers, Mesorhizobium loti chitooligosaccharide deacetylase, pOPE101-215(Yol) and pRSETb vectors, biosynthesis, Escherichia coli SHuffle express and XL1-Blue MRF’.

 

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