Maltopentaose (Maltopentose)

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

Maltooligosaccharide-forming amylases (MFAs) hydrolyze starch into maltooligosaccharides with a defined degree of polymerization. However, the enzymatic mechanism underlying the product specificity remains partially understood. Here, we show that Saccharophagus degradans MFA (SdMFA) contains a noncatalytic starch-binding domain (SBD), which belongs to the carbohydrate-binding module family 20 and enables modulation of the product specificity. Removal of SBD from SdMFA resulted in a 3.5-fold lower production of the target maltopentaose. Conversely, appending SBD to another MFA from Bacillus megaterium improved the specificity for maltopentaose. SdMFA exhibited a higher level of exo-action and greater product specificity when reacting with amylopectin than with amylose. Our structural analysis and molecular dynamics simulation suggested that SBD could promote the recognition of nonreducing ends of substrates and delivery of the substrate chain to a groove end toward the active site in the catalytic domain. Furthermore, we demonstrate that a moderate temperature could mediate SBD to interact with the substrate with loose affinity, which facilitates the substrate to slide toward the active site. Together, our study reveals the structural and conditional bases for the specificity of MFAs, providing generalizable strategies to engineer MFAs and optimize the biosynthesis of maltooligosaccharides.

Fusion of maltooligosaccharide-forming amylases from two origins for the improvement of maltopentaose synthesis

Maltopentaose-forming amylases are promising enzymes for their ability to hydrolyze starch and produce functional maltooligosaccharides. Two maltopentaose-forming amylase genes from Bacillus megaterium (BmMFA) and Saccharophagus degradans (SdMFA) were expressed heterologously and their characteristics were analyzed. BmMFA has substantial thermostability and SdMFA owns superior product specificity. The carbohydrate-binding module of SdMFA was fused with BmMFA and the fused protein showed ideal enzymatic properties and displayed potential for industrial production of maltopentaose. Under the optimized conditions, the final product containing 47.41% maltopentaose was obtained with a conversion rate of 92.67% from starch. This study provides a novel strategy for the directed modification of MFAses through protein fusion approach.

Maltopentaose-conjugated CTA for RAFT polymerization generating nanostructured bioresource-block copolymer

We now describe the synthesis of a new family of oligosaccharide-conjugated functional molecules, which act as chain transfer agents (CTAs) for the reversible addition-fragmentation chain transfer (RAFT) polymerization. The synthesis was started from the catalyst-free direct N-glycosyl reaction of 5-azidopentylamine onto maltopentaose (Mal5) in dry methanol at room temperature and subsequent N-protected reaction with acetic anhydride, producing a stable oligosaccharide-building block, such as Mal5 with an azidopentyl group (Mal5-N3). The azido group was hydrogenated using platinum dioxide (PtO2) as a catalyst to give Mal5 with aminopentyl group (Mal5-NH2), which was then reacted with CTA molecules bearing activated ester moieties. These reactions produced Mal5-modified macro-CTAs (Mal5-CTAs, 1), which were used for the RAFT polymerizations of styrene (St) and methyl methacrylate (MMA) in DMF. The polymerizations were performed using the [M]0/[1]0 values ranging from 50 to 600, affording the Mal5-hybrid amphiphilic block copolymers (BCPs), such as Mal5-polystyrene (2) and Mal5-poly(methyl methacrylate) (3), with a quantitative end-functionality and the controlled molecular weights between 4310 and 20 300 g mol(-1). The small-angle X-ray scattering (SAXS) measurements were accomplished for 2 and 3 to ensure their abilities to form phase separated structures in their bulk states with the increasing temperatures from 30 to 190 °C. The featured results were observed for 2 (?Mal5 = 0.14) and 3 (?Mal5 = 0.16) at temperatures above 100 °C, where ?Mal5 denotes the volume fraction of the Mal5 unit in the BCP sample. For both BCP samples, the primary scattering peaks q* were clearly observed together with the higher-ordered scattering peaks √2q* and √3q*. Thus, these Mal5-hybrid amphiphilic BCP samples have a body centered cubic (BCC) phase morphology. The domain spacing (d) values of the BCC morphology for 2 (?Mal5 = 0.14) and 3 (?Mal5 = 0.16) were 10.4 and 9.55 nm, respectively, which were determined using Bragg's relation (d = 2π/q*). The present RAFT agents were shown to eventually provide the phase separated structural polymeric materials in which 5.4 nm bioresource-spherical domains were periodically arrayed at the interval of about 10 nm.

alpha-Amylase determination using maltopentaose as substrate

The rationale of choosing a NADP-coupled continuous method, with the substrate maltopentaose, as a method for the determination of alpha-amylase (EC 3.2.1.1) activity is investigated. The method presented is investigated with respect to all reaction parameters, including possible influence of protein, and shows zero order reaction kinetics after a 5-6 minute lag phase. The blank reaction from maltopentaose substrate is constant and is 13% of the upper limit of the reference interval for serum. The course of the blank reaction can be used to check that the maltopentaose is of adequate purity for use in the assay. Km for maltopentaose is 0.48 mmol/l. There is no interference from endogenous glucose when the total NADP turnover is less than 0.25 mmol/l. Data for sensitivity, linearity and long term precision over an eighteen month period are given, together with reference intervals for serum and for urine. The method is recommended for consideration as a reference method.

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

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 2-40 ^T . 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.