Carbohydrate-Binding Module and Linker Allow Cold Adaptation and Salt Tolerance of Maltopentaose-Forming Amylase From Marine Bacterium Saccharophagus degradans 2-40T

Abstract

Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.

Starch-Binding Domain Modulates the Specificity of Maltopentaose Production at Moderate Temperatures

“…The amplified genes were assembled into the Bacillus subtilis WB600 expression vector pST by In-Fusion cloning (Takara, Tokyo, Japan). The expression and purification of the recombinant proteins were described previously. , …”

“…The expression and purification of the recombinant proteins were described previously. 21,25 2.2. Enzyme Assay.…”

“…We then truncated the putative SBD (V446−F542) and linker (I428− K445) from SdMFA to construct SdMFA-CD (Figure 2A). SdMFA-CD was expressed in B. subtilis WB600 and purified by the combination of phenyl-superose and gel filtration chromatography 25 (Figure S1). Repeating the assay for the enzymatic hydrolysis showed that SdMFA-CD lost its activity toward raw starch (Table S2).…”

The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing

“…A more deeply recessed anionic patch is located immediately adjacent to the cleft and occupied by a glycerol molecule in the structure of RsgI9 S1C‐CTD . Although its function remains unknown, similar anionic pockets are present in carbohydrate‐binding proteins and often interact with metal cations (e.g., Ca 2+ ) 65–67 …”

“…Although its function remains unknown, similar anionic pockets are present in carbohydrate-binding proteins and often interact with metal cations (e.g., Ca 2+ ). [65][66][67] Despite exhibiting structural homology with members of the Deg/HtrA protease family, the S1C domain in RsgI9 is enzymatically inactive in vitro. In marked contrast to the trypsin control, neither the intact ectodomain nor the bi-domain unit exhibits proteolytic activity when assayed using azocasein as a substrate (Figure 4A).…”