Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the MichaelisMenten constant of the cofactor NADH, K M NADH ) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners (E. malaccana and E. radiata) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata, in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases ( positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on finescale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation.

Background Exploring a simple and versatile technique for direct immobilization of target enzymes from cell lysate without prior purification is urgently needed. Thus, a novel all-in-one strategy for purification and immobilization of β-1,3-xylanase was proposed, the target enzymes were covalently immobilized on silica nanoparticles via elastin-like polypeptides (ELPs)-based biomimetic silicification and SpyTag/SpyCatcher spontaneous reaction. Thus, the functional carriers that did not require the time-consuming surface modification step were quickly and efficiently prepared. These carriers could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification. Results The ELPs-SpyCatcher hardly leaked from the carriers (0.5%), and the immobilization yield of enzyme was up to 96%. Immobilized enzyme retained 85.6% of the initial activity and showed 88.6% of the activity recovery. Compared with free ones, the immobilized β-1,3-xylanase showed improved thermal stability, elevated storage stability and good pH tolerance. It also retained more than 70.6% of initial activity after 12 reaction cycles, demonstrating its excellent reusability. Conclusions The results clearly highlighted the effectiveness of the novel enzyme immobilization method proposed here due to the improvement of overall performance of immobilized enzyme in respect to free form for the hydrolysis of macromolecular substrates. Thus, it may have great potential in the conversion of algae biomass as well as other related fields.

The identification of microbial markers adequate to delineate the disease‐related microbiome alterations from the complex human gut microbiota is of great interest. Here, we develop a framework combining neural network (NN) and random forest, resulting in 40 marker species and 90 marker genes identified from the metagenomic data set (185 healthy and 183 type 2 diabetes [T2D] samples), respectively. In terms of these markers, the NN model obtained higher accuracy in classifying the T2D‐related samples than other methods; the interaction network analyses identified the key species and functional modules; the regression analysis determined that fasting blood glucose is the most significant factor (p < 0.05) in the T2D‐related alteration of the human gut microbiome. We also observed that those marker species varied little across the case and control samples greatly shift in the different stages of the T2D development, suggestive of their important roles in the T2D‐related microbiome alteration. Our study provides a new way of identifying the disease‐related biomarkers and analyzing the role they may play in the development of the disease.