Cloning, expression, and characterization of thermal-stable and pH-stable agarase from mangrove sediments

AgaM1, a -agarase belonging to glycoside hydrolases family 16 (GH16), was cloned from the environmental DNA of mangrove sediments. The gene agaM1 is 2136bp in length and encodes a protein of 712 amino acids. The properties of recombinant AgaM1 (rAgaM1) were studied using prokaryotic expression. The optimum temperature and pH were 50 degrees C and 7.0, respectively, and rAgaM1 exhibited a high adaptability to wide ranges of temperature and pH. A relatively high activity was retained at from 30 to 60 degrees C and from pH 6.0 to 9.0. Thermal stability was showed more than 70% relative activity after pre-incubation at 40 degrees C for 60h. Outstanding pH stability were observed for rAgaM1 from pH 5.0 to 10.0 after pre-incubation for 60h. Thin-layer chromatography revealed neoagarotetraose (NA4) and neoagarohexaose (NA6) were the end-products of rAgaM1-degraded agarose. Besides, rAgaM1 were found with a K-m of 1.82mgml(-1) and a V-m of 357.14Umg(-1) for agarose. The K-m was smaller than those of most agarases reported previously. This discrepancy revealed the high affinity of rAgaM1 to agarose. Overall, the results indicated the potential of rAgaM1 in future industrial application.