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Endo-β-1,4-mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide (GGM5; 6(III) ,6(IV) -α-d-galactosyl mannopentaose) to GGM3 and M2, whereas TfMan hydrolyzed GGM5 to GGM4 and M1. To determine the region involved in the substrate specificity, we constructed chimeric enzymes of StMan and TfMan and evaluated their substrate specificities. Moreover, the crystal structure of the catalytic domain of StMan (StMandC) and the complex structure of the inactive mutant StE273AdC with M6 were solved at resolutions of 1.60 and 1.50 Å, respectively. Structural comparisons of StMandC and the catalytic domain of TfMan lead to the identification of a subsite around -1 in StMandC that could accommodate a galactose branch. These findings demonstrate that the two loops (loop7 and loop8) are responsible for substrate recognition in GH5 actinomycete mannanases. In particular, Trp281 in loop7 of StMan, which is located in a narrow and deep cleft, plays an important role in its affinity toward linear substrates. Asp310 in loop8 of StMan specifically bound to the galactosyl unit in the -1 subsite.

In nature, permafrost regions and deep ocean sediments contain a large amount of gas hydrate. As a basic property of the sediments, the particle size of the porous media is a critical factor affecting hydrate production. In this study, methane hydrate formed and dissociated in the sediments with different particle sizes, including the particle sizes of 14–20 mesh, 35–60 mesh, 80–120 mesh, and 400–500 mesh. The experimental results showed that two stages were included during the hydrate formation process. In the first stage, the hydrate was mainly formed in the upper of the sediments, which hindered the further contact of gas/water and resulted in the decrease of the hydrate formation rate in the second stage. As the particle size of the porous media decreased, the induction time for the hydrate nucleation decreased and the hydrate formation rate increased. In the porous media with 400–500 mesh, the hydrate started forming while the gas was injected into the hydrate simulator. It was found that the hydrate formation rate in the sediments was limited by the mass transport rate of gas and water. In the constant pressure stage (CPS) of the hydrate dissociation, the maximum value of the hydrate dissociation rate was obtained in the porous media with 35–60 mesh. It was found for the first time that the change characteristics of the average hydrate dissociation rate with the medium particle size of the porous media were similar to those of the effective thermal conductivity with the medium particle size of the porous media. This demonstrated that the heat transfer rate of the sediments determined the hydrate dissociation rate, and the influences of the capillary force and the hydrate distribution on the hydrate dissociation were minor. The experimental results also suggested that the coarse-dominated sediments are more advantageous for gas hydrate production.

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