Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin – the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli . The recombinant protein was easily purified by heat precipitation at 80 o C for 30 min. This enzyme was characterized and required Mg 2+ as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 o C in the presence of 0.5 mM MgCl 2 , suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.
Amylopectin, the primary form of starch in plant leaves, seeds and tubers, features a tree-like architecture with branched glucose chains. Excess branches result in the formation of soluble phytoglycogen instead of starch granules. In higher plants and green algae, the debranching enzyme isoamylase ISA1 forms either homomultimer or hetero-multimer with ISA2 to facilitate branch trimming and starch granule formation, but the molecular basis remains largely unknown. In this study, we reconstitute the rice OsISA1-ISA2 complex in vitro and determine the cryo-EM structures of the OsISA1 homodimer, as well as the malto-oligosaccharide (MOS)-free and MOS-bound OsISA1-ISA2 heterocomplex. The OsISA1 dimer shows a tail-to-tail rod-like architecture, whereas the OsISA1-ISA2 complex mainly exhibits as a trimer, with OsISA2 flanking on the N-terminal segments of the dimeric OsISA1. Combined with comprehensive biochemical analyses, these structural data elucidate the organization of the ISA1-ISA2 heterocomplex in higher plants and demonstrate how ISA1 and ISA2 cooperate during amylopectin biosynthesis.
The debranching of α-1,6-glucose branch-points in starch by isoamylase is essential for in vitro enzymatic cascades containing α-glucan phosphorylase (αGP) that convert starch into valuable compounds. Herein, a highly active isoamylase, GK2827, from thermophilic Geobacillus kaustophilus was enzymatically characterized with a half-life of 46.6 h at 65°C. GK2827 exhibits a specific activity of 848 U/mg toward maltodextrin (dextrose equivalent 8.6), which is 31.8-fold higher than that toward pullulan. Molecular dynamics simulations revealed that a combination of favorable electrostatic interactions, stronger covalent binding, and stable van der Waals interactions enhance the binding affinity of GK2827 for maltodextrin compared to its affinity for pullulan. In an enzymatic cascade comprising αGP, α-glucosidase and cellobiose phosphorylase for cellobiose synthesis in one-pot, the inclusion of GK2827 increased the maltodextrin utilization rate from 52.6% to 80.9%. This excellent heterologously expressed isoamylase facilitates αGP-based starch phosphorylation in biomanufacturing various functional saccharides via in vitro enzymatic cascade.
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