Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

Abstract

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A (540)=4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A (540)=0.24 and 0.12, respectively) than the control (A (540)=0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40 °C and pH 7.0. The K(m), V(max), and K(cat) values for agarose were 2.18 mg/ml (approximately 1.82 × 10(-5) M), 39.06 U/mg of protein, and 9.5 × 10(3)/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.

Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type -Agarase-Producing Neoagarobiose

“…Among these hydrolytic enzymes, the GH16 family agarase (DagA) was identified in S. coelicolor A3(2) as a ␤-agarase that can hydrolyze agar into neoagarotetraose or neoagarohexaose as the final product (31). From genomic sequencing data for S. coelicolor A3(2), we found that Sco3487, annotated as a putative hydrolase, was a good agarase candidate belonging to the GH50 family.…”

“…The reaction was stopped by cooling the test tube in an ice-cold water bath, and the absorbance at 540 nm was recorded. One unit of enzyme activity was defined as the amount of activity producing an A 540 of 0.001 after a 15-min incubation (31).…”

“…We previously reported that DagA agarase hydrolyzes agarose by endocleavage to produce neoagarotetraose and neoagarohexaose, indicating that it cannot recognize or hydrolyze neoagarotetraose and neoagarohexaose substrates (31). When we prepared a DagA-hydrolyzed reaction mixture with Sco3487 addition, neoagarotetraose and neoagarohexaose were completely hydrolyzed into neoagarobiose, which was more effective than treatment of agarose with Sco3487 only (Fig.…”

“…The agarase gene (dagA) of S. coelicolor, which encodes an extracellular agarase, has been cloned and overexpressed in Streptomyces lividans (5). We recently reported that DagA (Sco3471, 32 kDa), which belongs to the GH16 family, is an endo-type ␤-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as major products (31). From the genomic sequence data of S. coelicolor A3(2) (3), we found 1 putative hydrolase (Sco3487) that has significant homology to the GH50 family of agarases.…”

Identification and biochemical characterization of a novel endo-type β-agarase AgaW from Cohnella sp. strain LGH

“…The agarolytic activity was measured by the 3,5-dinitrosalicylic acid (DNS) method as previously reported (Chi et al 2014;Temuujin et al 2011), with slight modification. Briefly, 10 μl of the enzyme solution was mixed with 390 μl of 10 mM potassium phosphate buffer (pH 7.0) plus 0.5 % agarose and incubated at 50°C for 10 min.…”