Safe Sialidase Production by the Saprophyte Oerskovia paurometabola: Gene Sequence and Enzyme Purification

Abstract

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed β-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.

Keywords: Oerskovia paurometabola; enzyme purification; gene sequencing; sialidase.

Figure 1

Elution profile of the anion-exchange chromatography of the sialidase from O129 on a DEAE cellulose column. The active peak was eluted with 0.1 M PBS at pH 7.5.

Figure 2

Elution profile of the sialidase from O. paurometabola O129 on a Q-Sepharose column in an FPLC system. The active peak is indicated by the arrow.

Figure 3

Sialidase of O. paurometabola O129 (marked by arrow), shown by SDS-PAGE in a 10% separating polyacrylamide gel. The enzyme was stained either by silver staining (a,b) or by the use of the Pierce™ Glycoprotein Staining Kit (Thermo Scientific Inc., Waltham, MA, USA), (c). Lanes and samples: (a): 1, molecular weight marker; 2–4, crude enzyme obtained after (NH₄)₂SO₄ precipitation, dialysis, and purification through a DEAE cellulose column; (b): 1, molecular weight marker; 2, entirely purified sialidase of O. paurometabola O129; (c): 1, molecular weight marker; 2, purified sialidase after glycoprotein staining. The characteristic magenta color, which the purified protein acquired as a result of Schiff base staining, reveals the very likely presence of a glycosyl moiety. As a molecular weight marker, we used the Perfect^TM Tricolor Protein Ladder (EURx, Gdansk, Poland). The determination of the molecular weight of the purified sialidase was performed by the estimation of the relative mobility factor (Rf). The decimal log value corresponding to this Rf is approximately 70 kDa.

Figure 4

Substrate specificity of the sialidase isolated from O. paurometabola O129.

Figure 5

Effect of various inducers on the enzyme activity. The control sample was taken from a culture in a semisynthetic medium without inducer.

Figure 6

Thermostability of the sialidase from O. paurometabola O129. Each measurement point reflects the number of minutes during which the enzyme was subjected to the action of the corresponding temperature, after which it was cooled, and its activity was tested. Results are presented as percent residual activity relative to the activity of an unheated control enzyme sample.

Figure 7

Amino acid sequence alignment of the novel O. paurometabola O129 sialidase (NCBI GenBank accession number OP429228) with the deduced amino acid sequences of the enzymes of O. paurometabola DSM 14281 (WP_204809048.1), O. gallyi strain Sa2CUA8 (WP_191790498.1), O. jenensis (WP_205305555.1), O. turbata (WP_030151436.1), O. enterophila (WP_068625153.1), and M. viridifaciens (WP_089006911.1). Full identity is outlined in dark green, partial identity, in green and light green, and lack of identity, in white. The Y/FRIP motif is enclosed in a red rectangle; the Asp-boxes are enclosed in yellow rectangles. The predicted conserved catalytic residues are marked with inverted triangles—red for the arginine triad R25, R227, R287, white for the glutamate E211 and E330, black for the aspartate D49 and tyrosine Y314.

Figure 8

3D model of the sialidase of O. paurometabola O129 obtained by SWISS-MODEL Workspace [27]. (a) “Ball and stick” presentation of the chain; (b) “Space-full” model.

Figure 9

Amino acid sequence alignment of the novel O. paurometabola O129 sialidase (NCBI GenBank accession number OP429228) with the deduced amino acid sequences of the enzymes of V. cholerae (AWB74302.1), C. perfringens (APZ74275.1), A. viscosus (VEI16517.1), Str. pneumoniae (WP_129559156.1), and Tannerella forsythia (CQ24155.1). Full identity is outlined in dark violet, partial identity, in light violet, and lack of identity, in white. The Y/FRIP motif is enclosed in a red rectangle; the Asp-boxes are enclosed in yellow rectangles. The predicted conserved catalytic residues are marked with inverted triangles—red for the arginine triad, white for glutamate, and black for aspartate and tyrosine.

Figure 10

Signal peptide of O. paurometabola O129 sialidase as predicted with high probability by SignalP 5.0 software. The cleavage site LAA is marked by a green peak.