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The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α-L-rhamnosidase from Alternaria sp. L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2'-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α-L-rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

A strain, Alternaria alternata SK37.001, which produces an intracellular α-l-rhamnosidase, was newly isolated from citrus orchard soil. The molecular mass of the enzyme was 66 kDa, as evaluated by SDS-PAGE and 135 kDa, as determined by gel filtration, which indicated that the enzyme is a dimer. The enzyme had a specific activity of 21.7 U mg -1 after step-by-step purification. The optimal pH and temperature were 5.5 and 60 °C, respectively. The enzyme was relatively stable at a pH of 4.0-8.0 and a temperature between 30 and 50 °C compared with other pH levels and temperatures investigated. The enzyme activity was accelerated by Ba 2+ and Al 3+ but inhibited by Ni 2+ , Cu 2+ and Co 2+ , especially Ni 2+ . The kinetic parameters of K m and V max were 4.84 mM and 53.1 μmol mg -1  min -1 , respectively. The α-l-rhamnosidase could hydrolyze quercitrin, naringin and neohesperidin, hesperidin and rutin rhamnose-containing glycosides but could not hydrolyze ginsenoside Rg2 or saiko-saponin C., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp 252 , Asp 257 , Asp 264 , Glu 530 , Arg 548 , His 553 , and Trp 555 ) and may form the hydrophobic pocket in stabilizing donor (Trp 261 , Tyr 302 , Tyr 316 , and Trp 369 ) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp 257 . Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

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 Alternaria sp. L1 was expressed in Pichia pastroris 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.

Background: There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G., Results: The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media., Conclusions: The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

An α-l-rhamnosidase gene (rhaL1) containing an open reading frame of 2046-bp encoding a 681-amino acid protein (RhaL1) was cloned from Alternaria sp. L1 for naringin hydrolysis on the cell surface of Saccharomyces cerevisiae EBY-100. RhaL1 anchored to the yeast cell surface showed maximum enzyme activity at pH 6.0-6.5 and 70°C and was stable at pH 2.5-12.0 below 60°C. When the yeast cells were employed to hydrolyze naringin in grapefruit juice, about 85% naringin was hydrolyzed at 60°C in 10min. The yeast cells were harvested and recycled for the next batch. The hydrolysis rate of the naringin was maintained at over 80% for 10 batches. These results demonstrate the stability of the RhaL1-expressing yeast cells and effective in hydrolysis of naringin in juice. Thus, the system could have promise for industrial bitterness reduction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

During cultivation in a minimal medium with glucose Alternaria tenuissima and Aspergillus vesicolor produce constitutively alpha- and beta-glucanases. Fractions of beta-1,3-glucanases exhibiting affinity for laminarin were separated by means of gel filtration chromatography. Two neutral beta-1,3-glucanases with affinity for yeast glucan were isolated by affinity chromatography and further characterized.