DBPapers
DOI: 10.5593/SGEM2016/B61/S25.091

PRODUCING RECOMBINANT ß-MANNANASE OF B. SUBTILIS AND RESEARCHING PHYSICAL AND CHEMICAL PROPERTIES OF THE ENZYME

O. Korneeva, E. Anohina
Wednesday 7 September 2016 by Libadmin2016

References: 16th International Multidisciplinary Scientific GeoConference SGEM 2016, www.sgem.org, SGEM2016 Conference Proceedings, ISBN 978-619-7105-68-1 / ISSN 1314-2704, June 28 - July 6, 2016, Book6 Vol. 1, 693-700 pp

ABSTRACT

The enzyme β-mannanase (ЕС 3.2.1.78) cleaves mannans of hemicellulose fraction of plant cell walls, which are resistant to the gastrointestinal tract of animals and poultry. One of the important directions of the use of β-mannanases is the production of mannose-containing hydrolysates from vegetable raw materials. The products of mannans hydrolysis - mannose and manno-oligosaccharides – exhibit bifidogenic and immunostimulatory effects. As a result of genetic modification of the strain B. subtilis performed by us there has been received an active producer of β- mannanase. The encoding part of the gene of the β-mannanase B. subtilis 168 was amplified by PCR and cloned into the vector pET28b (+) which contains hexahistidine sequence (6His-Tag), and then after induced gene expression in E. coli cells, the recombinant protein was purified by affinity chromatography on a Ni2+-NTA agarose. The purified protein has a molecular weight of 40 kDa and a specific activity of 774 U/mg, the highest activity of β-mannanase was observed at 40° C and pH 7.0. The investigation of pH and temperature stability of the enzyme showed that at pH 7.0 and a temperature of 30° C β-mannanase maintained about 70% activity during the 6 hours of incubation. The maximum degree of enzymatic hydrolysis of the galactomannan of locust bean gum, being 85%, was observed at the optimum temperature and pH and with the enzyme dosage of 9 U/g of substrate for 3 h. The identification of hydrolysis products showed the presence of mannose, mannotriose, mannotetrose and mannopentose in the derived hydrolysates that indirectly indicates the affiliation of β- mannanase B. subtilis to the enzymes of endo-effect.

Keywords: recombinant β-mannanase, mannose containing hydrolysates