In Vitro Synthesis and Crystallization of beta-1,4-Mannan

摘要

In vitro polymerization of beta-mannans is a challenging reaction due to the steric hindrance confered by the configuration of mannosyl residues and the thermodynamic instability of the beta-anomer. Whatever the approach used to date-whether chemical, or enzymatic with glycosynthases and mannosyltransferases pure beta-1,4-mannans have never been synthesized in vitro. This has limited attempts to investigate their role in the production of plant and algal cell walls, in which they are highly abundant. It has also impeded the exploitation of their properties as biosourced materials. In this paper, we demonstrate that TM1225, a thermoactive glycoside phosphorylase from the hyperthermophile species Thermotoga maritima, is a powerful biocatalytic tool for the ecofriendly synthesis of pure beta-1,4-mannan. The recombinant production of this enzyme and its biochemical characterization allowed us to prove that it catalyzes the reversible phosphorolysis of beta-1,4-mannosides, and determine its role in the metabolism of the algal mannans on which T. maritima feeds in submarine sediments. Furthermore, after optimizing the reaction conditions, we exploited the synthetic ability of TM1225 to produce beta-1,4-mannan in vitro. At 60 degrees C and from D-mannose 1-phosphate and mannohexaose, the enzyme synthesized mannoside chains with a degree of polymerization up to 16, which precipitated into lamellar single crystals. The X-ray powder diffraction and base-plane electron diffraction patterns of the lamellar crystals unambiguously show that the synthesized product belongs to the mannan I family previously observed in planta in pure linear mannans, such as those of the ivory nut. The in vitro formation of these mannan I crystals is likely determined by the high reaction temperature and the narrow chain length distribution of the insoluble chains.