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

Effects of initial esculin concentrations on RCC-III synthesis.

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

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

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 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

High‑<i>k</i> Polymer Nanocomposites Filled with Hyperbranched Phthalocyanine-Coated BaTiO₃ for High-Temperature and Elevated Field Applications

Two sets of thermal stable nanocomposites were fabricated by using engineering plastics poly­(ether sulfone) (PES) as a matrix and phthalocyanine molecules (CuPc) or hyperbranched phthalocyanine (HCuPc)-coated barium titanate (BT) nanoparticles as fillers for high electric field and high-temperature dielectric applications. By side-by-side comparison, the hyperbranched coating is finely addressed for enhancing the dielectric response and breakdown strength of the composites. Specifically, BT–HCuPc/PES exhibits 40% lower dielectric loss and about 110% larger breakdown strength than BT–CuPc/PES. The addition of hyperbranched phthalocyanine may enhance the compatibility and dispersion of the ceramic fillers in the polymer matrix and reduces the charge carrier between the filler and matrix. Meanwhile, high dielectric constant, high breakdown, and low dielectric loss are well-maintained in the composites filled with hyperbranched phthalocyanine-modified BT from room temperature to 150 °C. The discharged energy density of the composites (20 vol % BT–HCuPc/PES) can reach 2.0 J/cm³ at 300 MV/m, about 166% of that of the polymer matrix (1.2 J/cm³). Our findings on hyperbranched coating structure could be applicable to other ceramic–polymer composites to enhance their dielectric response

Purification, Cloning, Characterization, and N-Glycosylation Analysis of a Novel β-Fructosidase from <i>Aspergillus oryzae</i> FS4 Synthesizing Levan- and Neolevan-Type Fructooligosaccharides

<div><p>β-Fructosidases are a widespread group of enzymes that catalyze the hydrolysis of terminal fructosyl units from various substrates. These enzymes also exhibit transglycosylation activity when they function with high concentrations of sucrose, which is used to synthesize fructooligosaccharides (FOS) in the food industry. A β-fructosidase (BfrA) with high transglycosylation activity was purified from <i>Aspergillus oryzae</i> FS4 as a monomeric glycoprotein. Compared with the most extensively studied <i>Aspergillus</i> spp. fructosidases that synthesize inulin-type β-(2-1)-linked FOS, BfrA has unique transfructosylating property of synthesizing levan- and neolevan-type β-(2-6)-linked FOS. The coding sequence (<i>bfrA</i>FS4, 1.86 kb) of BfrA was amplified and expressed in <i>Escherichia coli</i> and <i>Pichia pastoris</i>. Both native and recombinant proteins showed transfructosylation and hydrolyzation activities with broad substrate specificity. These proteins could hydrolyze the following linkages: Glc α-1, 2-β Fru; Glc α-1, 3-α Fru; and Glc α-1, 5-β Fru. Compared with the unglycosylated <i>E. coli</i>-expressed BfrA (E.BfrA), the N-glycosylated native (N.BfrA) and the <i>P. pastoris</i>-expressed BfrA (P.BfrA) were highly stable at a wide pH range (pH 4 to 11), and significantly more thermostable at temperatures up to 50°C with a maximum activity at 55°C. Using sucrose as substrate, the <i>Km</i> and <i>k_cat</i> values for total activity were 37.19±5.28 mM and 1.0016±0.039×10⁴ s⁻¹ for N.BfrA. Moreover, 10 of 13 putative N-glycosylation sites were glycosylated on N.BfrA, and N-glycosylation was essential for enzyme thermal stability and optima activity. Thus, BfrA has demonstrated as a well-characterized <i>A. oryzae</i> fructosidase with unique transfructosylating capability of synthesizing levan- and neolevan-type FOS.</p></div

Separation of BfrA from <i>A. oryzae</i> SF4.

<p>(A) Native gradient PAGE analysis of purified N.BfrA. Lane 1, molecular mass standards; lane 2, partially purified fructosidase was subjected to electrophoresis on 5% to 15% native gradient polyacrylamide gels. The targeted protein band with fructosidase activity was indicated by an arrow. (B) SDS–PAGE for all steps of purification of BfrA from <i>A. oryzae</i> SF4. Lane 1, molecular mass standards; lane 2, crude enzyme; lane 3, protein fraction after dialysis; lane 4, protein fraction from DEAE–Sepharose fast flow step; lane 5, protein fraction from Superdex 200 step; lane 6, protein fraction from Native–PAGE purification. Protein bands were visualized by staining with Coomassie blue. Target protein band was indicated by an arrow.</p

TLC analysis of FOS from transglycosylation reactions.

<p>The transglycosylation reactions were catalyzed by native and recombinant BfrAs incubated with 33% sucrose at optimal reaction condition for 5 h. The FOS were separated by Bio-Gel P2 column. SD, FOS standards; Glc, glucose; Fru, fructose; Suc, sucrose; NK, neokestose; NA, transglycosylation reaction catalyzed by N.BfrA; P, transglycosylation reaction catalyzed by P.BfrA; E, transglycosylation reaction catalyzed by E.BfrA. The neokestose (NK), 6-kestose (6-K), and 6-nystose (6-N) were indicated by blue, red, and dark arrows, respectively.</p