Generation of an α‐L‐rhamnosidase library and its application for the selective derhamnosylation of natural products

Abstract

A screening of 16 different fungal strains was performed under different cultivation conditions, using L-rhamnose or L-rhamnose-containing flavonoid glycosides (rutin, hesperidin, and naringin) as specific inducers. No significant constitutive production of alpha-L-rhamnosidases was detected in noninduced cultures, while high levels of these glycosidase activities were obtained using different inducers. New species, so far unknown for the production of alpha-L-rhamnosidases, were identified. More than 30 different alpha-L-rhamnosidase samples were prepared by ammonium sulfate precipitation. Substrate specificity of this alpha-L-rhamnosidase library was tested with various L-rhamnose-containing natural compounds (flavonoids, terpenoids, and saponins). Most of the enzymatic preparations showed broad substrate specificity, and some of them were also acting on sterically hindered substrates (e.g., quercitrin). The screening of the library under different reaction conditions showed the coexistence, in the same preparation, of more than one alpha-L-rhamnosidase activities with different substrate specificity and different stability towards organic cosolvents. To exploit this enzymatic library for synthetic applications, the presence of contaminating alpha-L-arabinosidases and beta-D-glucosidases was investigated. The latter enzymes were observed in several preparations, while alpha-L-arabinosidase content was generally quite low. The selective derhamnosylation of the saponin desglucoruscin was performed on a preparative scale. The enzyme obtained by rhamnose induction of the Aspergillus niger K2 CCIM strain showed high activity towards this substrate and negligible alpha-L-arabinosidase contamination. Therefore, it was chosen as a catalyst for the selective derhamnosylation reaction, which provided the desired product in 70% yield.

Biocatalyzed Generation of Molecular Diversity: Selective Modification of the Saponin Asiaticoside

“…[8] A new screening of these 31 different enzymatic samples allowed the identification of 10 preparations, originating from 4 different fungi, that were able to hydrolyze asiaticoside. As expected, the contaminating b-d-glucosidases present in these crude samples were also active on the derhamnosylated intermediate derivative 2 (DeRha-Asia) and therefore, in all cases, the final products detected by TLC were either the fully deglycosylated aglycone (asiatic acid, 4) or the monosaccharidic derivative derhamno-degluco-asiaticoside 3 (DeRhaDeGlc-Asia, Figure 2 and Table 1).…”

Optimization of Cultivation Conditions of Penicillium tardum - the α-L-Rhamnosidase Producer

“…У першу чергу це мікроміцети, серед яких найбільша кількість продуцентів глікозидаз [6][7][8][9][10]. На сьо годні у промисловості використовуються ферментні препарати Pénicillium decumbens і Aspergillus niger, які синтезують нарингіназу і гесперидиназу відповідно (SigmaAldrich, USA).…”

“…Внесення ж більшої (15-25 %) або меншої (5 %) кількості посівного матеріалу не призводило до зростання a-L-рамнозидазної активності у досліджених штамів. Подібні результати були отримані D. Monti зі співавторами [8], які спостерігали максимальний синтез грибних a-L-рамнозидаз за концентрації інокулюму 10 %. Однак отримані дані не узгоджуються з результатами V Kumar [10], який відзна чив позитивний вплив на синтез a-L-рамнозидази A. niger MTCC-1344 15 % інокулюму.…”

“…При оптимізації середовища росту Р. tardum використовували такі концентрації дже рел вуглецю і азоту, які наводяться іншими авторами при вивченні їх впливу на синтез а-Ь-рамнозидаз [1,3,[8][9][10][11].…”

“…Найкра щим джерелом вуглецю виявилась рамноза. Це узгоджується з літературними даними [3,[7][8][9][10][11], згідно з якими такі джерела вуглецю як нарингін, рутин і рамноза індукують синтез а-Ь-рамнозидази, в той час як арабіноза, ксилоза, арабіногалактан, фруктоза, пектин, целюлоза, ксилан та арабани не впливають на синтез ферменту.…”