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Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species

Author:
Volkov, Pavel V., Gusakov, Alexander V., Rubtsova, Ekaterina A., Rozhkova, Alexandra M., Matys, Veronica Yu., Nemashkalov, Vitaly A., Sinitsyn, Arkady P.
Source:
Biochimie 2019 v.157 pp. 123-130
ISSN:
0300-9084
Subject:
Penicillium, Talaromyces funiculosus, Talaromyces verruculosus, chromatography, dextran, dextranase, genes, glucose, glycosylation, hydrolysis, isomaltose, matrix-assisted laser desorption-ionization mass spectrometry, melting point, molecular weight, pH, polyacrylamide gel electrophoresis, protein secretion
Abstract:
The dexA gene encoding Penicillium funiculosum dextranase (GenBank accession MH581385) belonging to family 49 of glycoside hydrolases (GH49) was cloned and heterologously expressed in two recipient strains, P. canescens RN3-11-7 and P. verruculosum B1-537. Crude enzyme preparations with the recombinant dextranase content of 8–36% of the total secreted protein were obtained on the basis of new Penicillium strains. Both recombinant forms of the dextranase were isolated in a homogeneous state using chromatographic techniques. The purified enzymes displayed very similar properties, that is, pI 4.55, activity optima at pH 4.5–5.0 and 55–60 °C and a melting temperature of 60.7–60.9 °C. They were characterized by similar specific activities (1020–1340 U/mg) against dextrans with a mean molecular mass of 20, 70 and 500 kDa, as well as similar kinetic parameters in the hydrolysis of 70 kDa dextran (Km = 1.10–1.11 g/L, kcat = 640–680 s−1). However, the recombinant dextranases expressed in P. canescens and P. verruculosum had different molecular masses according to the data of SDS-PAGE (∼63 and ∼60 kDa, respectively); this was the result of different N-glycosylation patterns as MALDI-TOF mass spectrometry analysis showed. The main products of dextran hydrolysis at its initial phase were isomaltooligosaccharides, while after the prolonged time (24 h) the reaction system contained isomaltose and glucose as the major products and minor amounts of other oligosaccharides.
Agid:
6236129