Identification of oligosaccharides in <i>H</i>. <i>gigas</i> whole-body extracts.

<p>Oligosaccharides were extracted with DDW from 3 crushed <i>H</i>. <i>gigas</i> and were separated by TLC and stained with H₂SO₄ for oligosaccharides (A, left) or the Glucose CII Kit for glucose (A, right). Glucose (G), maltose (M), and maltotriose (M3) were used as standard. The maltose and cellobiose contents were measured by the increase in the glucose content after α- or β-glucosidase treatment (B).</p

Purification of HGcel.

<p>HGcel was purified from crushed <i>H</i>. <i>gigas</i> by anion-exchange column chromatography as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042727#s4" target="_blank">Methods</a> section. HGcel migrated as a single 59-kDa band on an SDS-PAGE gel (5–20% gradient) (A). The effects of pH (B) and temperature on the enzyme's activity and stability are expressed relative to their maximum respective values (C). HGcel converted cellulose to glucose (Glu) and cellobiose (C2). In these reactions, 200 mU of HGcel was added to 500 µl of 5% cellulose solutions (pH 5.6) (D). One of the 5% (w/v) cellulose suspensions was added to HGcel (+E). Another was not added and was used as a reference (R). The kinetics of the glucose production from cellulose are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042727#s4" target="_blank">Methods</a> section of this paper (E). The products of the HGcel reaction with cellobiose (C2), cellotriose (C3), cellotetraose (C4), and cellopentaose (C5) were analyzed using TLC. Addition of HGcel (12 mU) is indicated by a ‘+’. HGcel was more efficient at hydrolyzing cello-oligosaccharides larger than cellotriose (F). HGcel (12 mU) was allowed to react with <i>p</i>-nitro phenyl cello-oligosaccharides at 35°C for 1 h. <i>p</i>-nitro phenyl binds to the reducing ends of cello-oligosaccharides. The release of glucose or <i>p</i>-nitro phenol in the reactions is shown (G). The effect of hydrostatic pressure (100 MPa) on the enzymatic activity is expressed as the percentage of its activity at atmospheric pressure (0.1 MPa) (H). The enzymatic reaction was performed using 10 mU of HGcel with 1% CMC solution in airtight plastic tubes at 2°C. The enzymatic activities were measured after 8 h and 16 h of incubation. The kinetics of sawdust digestion by HGcel were measured by determining the production of glucose in a reaction containing HGcel (380 mU) and either sawdust or CMC at 35°C (I).</p

<i>H</i>. <i>gigas</i> possesses polysaccharide hydrolase activities.

<p>We captured deep-sea animals using baited traps containing a slice of mackerel. One baited trap contained approximately 50 individuals (A). Digestive enzyme activities were assessed by halo formation in agar plates containing starch azure (amylase), CMC and trypan blue (cellulase), glucomannan (mannanase), and xylan (xylanase). The halos produced by the amylase and cellulase activities were visualized directly, while the halos resulting from mannanase and xylanase were detected after staining with 0.5% Congo red followed by washing with DDW (B). The kinetics of the reactions were determined by TLC as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042727#s4" target="_blank">Methods</a> section of this paper. Reactions with crushed <i>H</i>. <i>gigas</i> with 0.5% (w/v) starch (C), 0.2% (w/v) glucomannan (E) or 1% (w/v) CMC (D) were conducted in 100 mM sodium acetate buffer (pH 5.6) at 30°C. The pH dependencies of the amylase, mannanase, and cellulase activities were measured with protein extracts (F). The enzyme reactions were conducted in 100 mM sodium acetate buffer (pH 4.4–5.6) or 100 mM sodium phosphate buffer (pH 6.2–6.8). The relative activities are shown.</p

Crystal structure of the catalytic domain of a GH16 β-agarase from a deep-sea bacterium, <i>Microbulbifer thermotolerans</i> JAMB-A94

<div><p>A deep-sea bacterium, <i>Microbulbifer thermotolerans</i> JAMB-A94, has a β-agarase (<i>Mt</i>AgaA) belonging to the glycoside hydrolase family (GH) 16. The optimal temperature of this bacterium for growth is 43–49 °C, and <i>Mt</i>AgaA is stable at 60 °C, which is one of the most thermostable enzymes among GH16 β-agarases. Here, we determined the catalytic domain structure of <i>Mt</i>AgaA. <i>Mt</i>AgaA consists of a β-jelly roll fold, as observed in other GH16 enzymes. The structure of <i>Mt</i>AgaA was most similar to two β-agarases from <i>Zobellia galactanivorans, Zg</i>AgaA, and <i>Zg</i>AgaB. Although the catalytic cleft structure of <i>Mt</i>AgaA was similar to <i>Zg</i>AgaA and <i>Zg</i>AgaB, residues at subsite −4 of <i>Mt</i>AgaA were not conserved between them. Also, an α-helix, designated as α4′, was uniquely located near the catalytic cleft of <i>Mt</i>AgaA. A comparison of the structures of the three enzymes suggested that multiple factors, including increased numbers of arginine and proline residues, could contribute to the thermostability of <i>Mt</i>AgaA.</p></div