Purification and Characterization of Thermostable Agarase from Bacillus sp. BI-3, a Thermophilic Bacterium Isolated from Hot Spring

Abstract

An extracellular agarase was purified from Bacillus sp. BI-3, a thermophilic agar-degrading bacterium isolated from a hot spring in Indonesia. The purified agarase revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 58 kDa. The optimum pH and temperature of the agarase were 6.4 and 70°C, respectively. The activity of the agarase was stable at high temperatures, and more than 50% activity was retained at 80°C for 15 min. Furthermore, the enzyme was stable in the pH range of 5.8-8.0, and more than 60% of the residual activity was retained. Significant activation of the agarase was observed in the presence of K(+), Na(+), Ca(2+), Mg(2+), and Sr(2+); on the other hand, Ba(2+), Zn(2+), Cu(2+), Mn(2+), Co(2+), Fe(2+), and EDTA inhibited or inactivated the enzyme activity. The components of the hydrolytic product analyzed by thin-layer chromatography showed that the agarase mainly produced neoagarobiose. This study is the first to present evidence of agarolytic activity in aerobic thermophilic bacteria.

Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

“…5a ). The optimum pH for Amy63 amylase activity was 6.0, which is more acidic than the optimal pH of any other reported α-amylase 18 19 . This activity remained stable in the pH range from 5.0 to 11.0 ( Fig.…”

“…Because temperature and pH are decisive parameters for enzyme activity 18 , we characterized the effects of altering temperature and pH on the amylase, agarase and carrageenase activities and stability of Amy63 by using DNS methods, as described in the Methods. The optimal temperature for Amy63 amylase activity was 60 °C, and this activity was stable up to 55 °C ( Fig.…”

Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4 from Paenibacillus agarexedens

“…The products of LMP agarose, HMP agarose, and agar hydrolysis by rAgaB-4 were detected using thin layer chromatography (TLC) performed on silica gel 60 plates (Merck Millipore) as previously described (Li et al 2014 ) with some modifications. Hydrolysis reactions were conducted at 40 °C for 24 h in 50 mM sodium phosphate buffer (pH 6) containing 1% (w/v) LMP agarose, HMP agarose and agar with 1.54 μg of purified rAgaB-4, respectively.…”

“…Yen (Sie et al 2009 ) from city drain water; and Bacillus sp. BI-3 (Li et al 2014 ) from hot spring. Based on the amino acid sequence similarity, known β-agarases are classified into the four GH families of GH16, GH50, GH86, and GH118 (Lombard et al 2014 ).…”

Biochemical Characterization of a Novel GH86 ��-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7

“…Alteromonas [11], Pseudomonas [12], Gayadomonas [3], Vibrio [13,14], Pseudoalteromonas [15], Zobellia [16], Catenovulum [17], Agarivorans [18], Cohnella [19], Bacillus [20], Thalassomonas [21], Streptomyces [22,23], Stenotrophomonas [24], Simiduia [25], and Flammeovirga [26]. They have been classified into a number of glycoside hydrolase (GH) families, including GH16 [27], GH39 [3], GH50 [26], GH86 [28], and GH118 [18].…”