Extracellular chitinolytic enzymes of Clostridium paraputrificum J4; separation and characterization

Extracellular chitinolytic enzymes of Clostridium paraputrificum J4; separation and characterization

J. Dušková1, G. Tiščenko1, P. Kolenko1, T. Skálová1, J. Šimůnek2, J. Dohnálek1

<aff><oid id="1">Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,

Heyrovského nám. 2, Praha 6, <cny>Czech Republic</cny></aff>,

²Academy of Sciences of the Czech Republic, Vídeňská 1083, Praha 10, Czech Republic

duskova@imc.cas.cz

Chitinases, glycosyl hydrolases, which catalyze degradation of chitin, are present in a wide range of organisms from primitive to higher types. They play an important role in physiology and metabolism of some bacteria by chitin digestion and utilization, in defense against infection by pathogenic fungi, in fungi hyphal growth and many others [1].

We focus on extracellular chitinases of a human intestinal bacterium Clostridium paraputrificum J4, especially on their isolation either from crude culture medium or production by recombinant protein expression in Escherichia coli, subsequent enzymatic characterization and structure-function analysis. Two enzymes with molecular weights 62.3 kDa (Chit62J4) and 87.0 kDa (ChitBJ4) were purified [2]. They possess chitinolytic activity toward 4-nitrophenyl-N,N′-diacetyl-β-D-chitobioside and 4-nitrophenyl-β-D-N,N′,N′′-triacetylchitotriose and a negligible activity toward 4-nitrophenyl-N-acetyl-β-D-glucosaminide. Both enzymes belong to glycoside hydrolase family 18. Chit62J4 comprises a catalytic domain similar to chitinase D from Bacilus circulans [1], two domains with unknown function and a chitin binding domain. ChitBJ4, a sequence homolog to Chitinase B from Clostridium paraputrificum M21 [3] is composed of a catalytic domain, two Ig-like domains and a chitin binding domain. Further characterizations (MALDI, DLS, proteolytic analysis, kinetic studies) are discussed, crystallization of both enzymes is under way.

This work was supported by the Grant Agency of the Czech Republic (project no. 310/09/1407 and by the Ministry of Education, Youth and Sports of the Czech Republic (project BIOPOL, no. CZ.1.07/2.3.00/30.0029)

1. Y.C. Hsieh, Y.J. Wu, T.Y. Chiang, C.Y. Kuo, K.L. Shrestha, C.F. Chao, Y.C. Huang, P. Chuankhayan, W.G. Wu, Y.K. Li, C.J. Chen, J. Biol. Chem., 285, (2010), pp. 31603-31615.

2. J. Dušková, G. Tishchenko, E. Ponomareva, J. Šimůnek, I. Koppová, T. Skálová, A. Štěpánková, J. Hašek, J. Dohnálek, Acta Biochim. Pol., 58, (2011), pp. 261-263.

3. K. Morimoto, S. Karita, T. Kimura, K. Sakka, and K. Ohmiya, J. Bacteriol., 179, (1997), pp. 7306-7314