Enzymatic Synthesis of Rhamnose Containing Chemicals by Reverse Hydrolysis

<div><p>Rhamnose containing chemicals (RCCs) are widely occurred in plants and bacteria and are known to possess important bioactivities. However, few of them were available using the enzymatic synthesis method because of the scarcity of the α-L-rhamnosidases with wide acceptor specificity. In this work, an α-L-rhamnosidase from <i>Alternaria</i> sp. L1 was expressed in <i>Pichia pastroris</i> strain GS115. The recombinant enzyme was purified and used to synthesize novel RCCs through reverse hydrolysis in the presence of rhamnose as donor and mannitol, fructose or esculin as acceptors. The effects of initial substrate concentrations, reaction time, and temperature on RCC yields were investigated in detail when using mannitol as the acceptor. The mannitol derivative achieved a maximal yield of 36.1% by incubation of the enzyme with 0.4 M L-rhamnose and 0.2 M mannitol in pH 6.5 buffers at 55°C for 48 h. In identical conditions except for the initial acceptor concentrations, the maximal yields of fructose and esculin derivatives reached 11.9% and 17.9% respectively. The structures of the three derivatives were identified to be α-L-rhamnopyranosyl-(1→6')-D-mannitol, α-L-rhamnopyranosyl-(1→1')-β-D-fructopyranose, and 6,7-dihydroxycoumarin α-L-rhamnopyranosyl-(1→6')-β-D-glucopyranoside by ESI-MS and NMR spectroscopy. The high glycosylation efficiency as well as the broad acceptor specificity of this enzyme makes it a powerful tool for the synthesis of novel rhamnosyl glycosides.</p></div

RCCs syntheses by the recombinant RhaL1 via reverse hydrolysis.

<p>The recombinant enzyme synthesized novel RCCs in the presence of L-rhamnose as donor and mannitol, fructose or esculin as acceptors.</p

Effects of reaction conditions on RCC-I synthesis.

<p>(a) initial L-rhamnose concentrations; (b) temperature and reaction time; (c) initial mannitol concentrations. Data points represent the means ± S.D. of three replicates.</p

Effects of initial esculin concentrations on RCC-III synthesis.

<p>Data points represent the means ± S.D. of three replicates.</p

Effects of temperature (a) and pH (b) on activity (Δ) and stability (■) of recombinant RhaL1.

<p>Data points represent the means ± S.D. of three replicates.</p

SDS-PAGE of the purified recombinant RhaL1 and PNGase F-treated RhaL1.

<p>Lane 1, PNGase F-treated RhaL1; Lane 2, purified recombinant RhaL1; M, molecular mass markers.</p