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Naringinase is an important enzyme for commercial purposes due to its dual activity as both α-l-rhamnosidase and β-d-glucosidase. The traditional method for screening microbes that produce naringinase involves growing them on naringin agar, but this method has limitations and result in false positive results. This is because the growth on the naringin agar plate could be due to the presence of other organisms that produce rhamnosidase or other glucosidases, or those that use agar as a carbon source, rather than actual naringinase producers. To address these limitations, a double screen plate assay was developed using synthetic substrates, to separately test for β-d-glucosidase and α-l-rhamnosidase activity. The presence of a yellow zone of p-nitrophenol indicates the action of these enzymes, and the intensity of the yellow colour zone indicates the potential for naringinase production. This new screening method is a significant improvement in identifying real naringinase producers and represents progress towards a more reliable screening assay. [ABSTRACT FROM AUTHOR]

Despite the rapid advancement of glycosidase biotechnology, ginsenoside-transforming rhamnosidases remain underexplored due to a lack of research. In this study, we aimed to bridge this gap by evaluating eight putative rhamnosidases for their ability to transform ginsenosides. Among them, a novel rhamnosidase (C118) from Bifidobacterium was identified as being efficient at hydrolyzing ginsenoside Re. This enzyme was expressed well in Escherichia coli and exhibited optimal activity at pH of 6.0 and 45 °C. Protein structural predictions revealed that the potential active hydrophobic area near an active pocket may influence the ginsenoside-transforming activities compared to non-active screened rhamnosidases. This enzyme's thermal stability exceeded that of the only previously known ginsenoside-transforming rhamnosidase, BD890. Additionally, the kcat/Km value of C118 was 1.45 times higher than that of BD890. Using recombinant C118 from E. coli, all ginsenoside Re in a PPT-type ginsenoside mixture (2.25 mg/mL) was converted after 12 h of reaction. To the best of our knowledge, this is the most efficient ginsenoside Re-transforming α-L-rhamnosidase reported to date, enhancing our understanding of rhamnosidase–substrate interactions and potentially improving the efficiency and specificity of the conversion process. These findings offer promising implications for the production of pharmacologically active ginsenosides in the pharmaceutical, cosmetic, and functional food industries. [ABSTRACT FROM AUTHOR]

Targeted saturation mutagenesis at the residues located at the substrate-binding pocket for generating focused libraries has emerged as the technique of choice for enzyme engineering, but choosing the optimal residue number of the randomization site and the reduced amino acid alphabet to minimize the labor-determining screening effort remains a challenge. Herein, we propose the six-codon combinatorial mutagenesis (SCCM) strategy by using the BMT degeneracy codons encoding six amino acids with different chemical properties as the building blocks for the randomization of the amnio acid motif. SCCM requires only a small library of 646 clones for 95% coverage at the three-residue motif compared to conventional NNK degeneracy codons encoding all 20 canonical amino acids and requiring the screening of nearly 100,000 clones. SCCM generates a suitable number of mutant libraries, providing a new strategy for reducing the screening workload of saturated combination mutations in enzyme engineering. Using this approach, the α-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron had been successfully engineered for improving the hydrolytic activity on natural flavonoid diglycoside hesperidin via targeted directed evolution at the motifs positioning the entrance of the substrate-binding pocket. The results indicate that the conversion rates of the four mutants on hesperidin were increased by more than 30% compared with the wild type using whole-cell biotransformation. Moreover, the catalytic efficiency kcat/KM value of the mutant TM1-6-F5 was 1.4-fold higher than that of the wild type. [ABSTRACT FROM AUTHOR]

The α-l-rhamnosidase (rha1) gene was homologously expressed in Aspergillus niger strains CCTCC 206047 and CCTCC 206047ΔpyrG, using hygromycin B and auxotrophic as selection markers. The engineered A. niger strains RHA001-1 and RHA003-1 were screened, yielding α-l-rhamnosidase activities of 20.81 ± 0.56 U/mL and 15.35 ± 0.87 U/mL, respectively. The copy numbers of the rha1 gene in strains RHA001-1 and RHA003-1 were found to be 18 and 14, respectively. Correlation analysis between copy number and enzyme activity in the A. niger strains revealed that α-l-rhamnosidase activity increased with the copy number of the rha1 gene. Recombinant α-l-rhamnosidase was utilized for the enzymatic debittering of Ougan juice, and its process conditions were optimized. Furthermore, the primary bitter substance neohesperidin (2.22 g/L) in Ougan juice was converted into hesperetin 7-O-glucoside (1.47 g/L) and hesperidin (0.143 g/L). This study presents a novel approach for the production of food-grade α-l-rhamnosidase and establishes a technical foundation for its application in the beverage industry. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

α-L-rhamnosidase (Rha) is ubiquitous in nature and has high feasibility in the food and biotechnology industries. A green and environmentally friendly method was used to improve the activity of Rha. Here, we show that the effects of ultrasound treatment on the Rha. Ultrasonic treatment at 80 W for 10 min yielded the highest enzyme activity. Treatment increased enzyme activity by 26.3% and half-life by 124 min. Further, treatment increased the catalytic efficiency of Rha and increased the substrate conversion rate by 33.88%. These results demonstrate that ultrasound increases the catalytic activity and stability of Rha. Thus, ultrasonic treatment of Rha is cost-effective on the industrial scale. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosidases with desirable activity and thermostability profiles could be used for the biocatalytic production of the flavonoid glucoside isoquercetin from natural rutin for functional food. Herein, to improve the catalytic activity of GH78 α-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron VPI-5482, a list of residues located at the conserved general acid motif were selected for targeted mutagenesis by the sequence alignment of BtRha78A with homologous α-L-rhamnosidases. Ala-scanning mutagenesis and site-directed mutagenesis based on sequence alignment were performed, and the relative activity on rutin was evaluated. Furthermore, the reaction time curves and enzyme kinetics of better mutants were determined. The results indicate that the conversion rates of mutants V338A, V338I, S340A, and G341A were increased by 21.3%, 20.1%, 13.2%, and 1.6%, respectively, compared with the wild type when using whole-cell biotransformation. Moreover, the catalytic efficiency kcat/KM value of mutant V338A was 1.3-fold higher than that of the wild type. The best mutant, V338A, was employed for the enzymatic preparation of isoquercetin via the biotransformation of rutin at a concentration of 2 mM, and 1.80 g of isoquercetin was obtained. The identification of the best mutant V338A lays the foundation for the efficient preparation of isoquercetin via the biotransformation of rutin, which in turn provides theoretical guidance for its large-scale production. [ABSTRACT FROM AUTHOR]

The food enzyme α‐l‐rhamnosidase (α‐l‐rhamnoside rhamnohydrolase; EC 3.2.1.40) is produced with Penicillium adametzii strain AE‐HP by Amano Enzymes Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in two food manufacturing processes. Subsequently, the applicant has requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of four food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was calculated to be up to 0.022 mg TOS/kg body weight (bw) per day in European populations. Using the no observed adverse effect level reported in the previous opinion (300 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 13,636. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Henan University of Technology Natural Science Edition is the property of Henan University of Technology Journal Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The subsequent biochemical and structural investigations of the purified recombinant α‐l‐rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild‐type was higher toward hesperidin and narirutin, where the l‐rhamnose residue was α‐1,6‐linked to β‐d‐glucoside, than toward neohesperidin and naringin with an α‐1,2‐linkage to β‐d‐glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that Km values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. kcat values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (kcat/Km) toward hesperidin and narirutin was a result of a considerably high kcat value, while Km values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α)6‐barrel with the active site located in a deep cleft and two β‐sheet domains were also present in the N‐ and C‐terminal sites of the catalytic domain. Additionally, five asparagine‐attached N‐acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased. [ABSTRACT FROM AUTHOR]

The search for effectors capable of influencing the catalytic activity of enzymes is an important area of modern enzymology. The aim of the study was to investigate the ability of 6 coordination compounds with malatogermanate/stannate anions and 1,10-phenanthroline cations of 3d metals to modify α-L-rhamnosidase activity of Penicillium tardum, Penicillium restrictum and Eupenicillium еrubescens strains. α-L-Rhamnosidase activity was determined by the Davis method using naringin as a substrate. It was demonstrated­ that [Ni(phen)3]2[{Sn(HMal)2(Mal)}Cl]•14H2O) in 0.1% concentration had the most pronounced activating effect on α-L-rhamnosidase activity of all strains studied. Noncompetitive inhibition of α-L-rhamnosidase in E. еrubescens by [Cu(phen)3]2[{Sn(HMal)2(Mal)}Cl]•10H2O was shown. The obtained results expand the idea of glycosidases possible activators and inhibitors and indicate the perspective of their use in modern biotechnological processes.

One of the important problems of current biotechnology is the usage of enzymes of microbial origin for destruction of poorly soluble compounds and synthesis of new drugs. In recent years a great deal of researchers’ attention has been given to such technologically promising carbohydrases as O-glycosylhydrolases catalyzing the hydrolysis of O-glycoside links in glycosides, oligo- and polysaccharides, glycolipids, and other glycoconjugates. Aim. The review provides data on the position of α-L-rhamnosidases in the modern hierarchical classification of glycosidases and presents data available in the literature on the features of the enzyme structure in various microorganisms. Methods. The publications from the following databases were analyzed: PubMed (https://pubmed.nsbi.nlm.nih.gov/), the Carbohydrate-Active enZYmes (http://www.cazy.org/), the BRENDA Enzyme Database (https://www.brenda-enzymes.org/). Results. Data on the physicochemical, catalytic, and kinetic properties of α-L-rhamnosidases in microorganisms of different taxonomic groups have been systematized. The peculiarities of the substrate specificity of the enzyme depending on the nature of the protein and the growing conditions of the producer are characterized. Conclusions. Functional properties and specificity action of microbial α-L-rhamnosidases suggest their broad-range applicability for food and animal feed processing, as well as obtaining biologically active compounds for the pharmaceutical industry and medicine.

The food enzyme α‐l‐rhamnosidase (α‐l‐rhamnoside rhamnohydrolase; EC 3.2.1.40) is produced with the non‐genetically modified Penicillium adametzii strain AE‐HP by Amano Enzymes Inc. The food enzyme is considered free from viable cells of the production organism. It is intended to be used in the processing of fruits and vegetables for the production of juices and other fruit products. The dietary exposure to the food enzyme‐TOS was estimated to be up to 0.022 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 300 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure of at least 13,636. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Abstract Background Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and β-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and β-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. Results The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. Conclusions Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.

α-l-rhamnosidase [EC 3.2.1.40] belongs to glycoside hydrolase (GH) families (GH13, GH78, and GH106 families) in the carbohydrate-active enzymes (CAZy) database, which specifically hydrolyzes the non-reducing end of α-l-rhamnose. Αccording to the sites of catalytic hydrolysis, α-l-rhamnosidase can be divided into α-1, 2-rhamnosidase, α-1, 3-rhamnosidase, α-1, 4-rhamnosidase and α-1, 6-rhamnosidase. α-l-rhamnosidase is an important enzyme for various biotechnological applications, especially in food, beverage, and pharmaceutical industries. α-l-rhamnosidase has a wide range of sources and is commonly found in animals, plants, and microorganisms, and its microbial source includes a variety of bacteria, molds and yeasts (such as Lactobacillus sp., Aspergillus sp., Pichia angusta and Saccharomyces cerevisiae). In recent years, a series of advances have been achieved in various aspects of α-validates the above-described-rhamnosidase research. A number of α-l-rhamnosidases have been successfully recombinant expressed in prokaryotic systems as well as eukaryotic systems which involve Pichia pastoris, Saccharomyces cerevisiae and Aspergillus niger, and the catalytic properties of the recombinant enzymes have been improved by enzyme modification techniques. In this review, the sources and production methods, general and catalytic properties and biotechnological applications of α-l-rhamnosidase in different fields are summarized and discussed, concluding with the directions for further in-depth research on α-l-rhamnosidase. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosidase (α-L-rhamnoside-rhamnohydrolase EC 3.2.1.40) showing specificity for terminal α-1,2-, α-1,4- and α-1,6-linked rhamnose residues, which oft en present in glycoconjugates and synthetic glycosides, can be successfully used in biotechnology for the hydrolysis of rhamnopyranoside residues present in some bioflavonoids, glycoproteins, glycolipids, and other glycoconjugates. Previously, we have shown that a significant part of the coordination compounds of various metals act as effectors of the activity of α-L-rhamnosidases. The aim of this investigation was to study the effect of a number of newly synthesized coordination compounds of Ge(IV) and Ba(II), (Co(II), Ni(II), Cu(II), Zn(II) with gluconic acid on the activity of Penicillium tardum and Eupenicillium erubescens α-L-rhamnosidases. Methods. The objects of the study were Penicillium tardum and Eupenicillium erubescens α-L-rhamnosidases. α-L-Rhamnosidase activity was determined by the Davis method using naringin as a substrate. Coordination compounds Ge(IV) and Ba(II), Co(II), Ni(II), Cu(II),and Zn(II) with gluconic acid were used as enzyme activity modifiers. The synthesized complexes correspond to the formulas [M(H2O)6][Ge2(OH)2(C6H8O7)2]⋅nH2O (M = Ba(1), n = 2; Co 2, n = 4; Ni(3), n = 4; Cu(4), n = 4; Zn(5), n = 3). Results. The effect of coordination compounds 1-(5) on the activity of α-L-rhamnosidase in two strains of Penicillium tardum and Eupenicillium erubescens was studied depending on the exposure time and concentration of the effector. It was shown that compound (3) at a concentration of 0.01% (1 h incubation) led to a slight (by 5%) increase in the activity of P. tardum α-L-rhamnosidase. Compound 1 at a concentration of 0.1% led to a decrease in the activity of P. tardum α-L-rhamnosidase by 29% during the first hour, and after 24 h of incubation, a decrease in the inhibitory effect to 15% was noted. Compounds 2 and (4) activated the enzyme by 9-39% at 1h exposure. At a concentration of 0.1% and exposure time of 1 h, compound 1 increased the activity of E. erubescens α-L-rhamnosidase by 80%, while at a decrease in concentration to 0.01%, the activity increased only by 29%. In general, it should be noted that in most cases, an increase in the duration of incubation up to 24 h led to a decrease in the level of activation (or inhibition) and a return to the control values of enzyme activity. Conclusions. The variety of effects of metal coordination compounds on the activity of enzymes, depending on the nature of the cation and the origin of the enzyme, has been established. The involvement of Ba(II) had the greatest activating effect on the activity of E. erubescens α-L-rhamnosidase compared to other metals. [ABSTRACT FROM AUTHOR]

Industrial application of icariin and baohuoside I has been hindered by the short supply to a great extent. In this work, a novel GH78 α-l-rhamnosidase AmRha catalyzed the bioconversion of low-value epimedin C in crude Epimedium Folium flavonoids (EFs) to icariin and baohuoside I was developed. Firstly, the high-level expression of AmRha in Komagataella phaffii GS115 attained an enzyme activity of 571.04 U/mL. The purified recombinant AmRha could hydrolyze α-1,2-rhamnoside bond between two rhamnoses (α-Rha(2 → 1)α-Rha) in epimedin C to produce icariin with a molar conversion rate of 92.3%, in vitro. Furtherly, the biotransformation of epimedin C to icariin by the recombinant Komagataella phaffii GS115 cells was also investigated, which elevated the EFs concentration by fivefold. In addition, biotransformation of epimedins A-C and icariin in the raw EFs to baohuoside I was fulfilled by a collaboration of AmRha and β-glucosidase/β-xylosidase Dth3. The results obtained here provide a new insight into the preparation of high-value products icariin and baohuoside I from cheap raw EFs. [ABSTRACT FROM AUTHOR]

Abstract αL-rhamnosidase (EC 3.2.1.40) has been widely used in food processing and pharmaceutical preparation. The recombinant α-L-rhamnosidase N12-Rha from Aspergillus niger JMU-TS528 had significantly higher catalytic activity on α-1,6 glycosidic bond than α-1,2 glycosidic bond, and had no activity on α-1,3 glycosidic bond. The activities of hydrolyzed hesperidin and naringin were 7240 U/mL and 945 U/mL, respectively, which are 10.63 times that of native α-L-rhamnosidase. The activity could maintain more than 80% at pH 3–6 and 40–60℃. Quantum chemistry calculations showed that charge difference of the C-O atoms of the α-1,2, α-1,3 and α-1,6 bonds indicated that α-1,6 bond is most easily broken and α-1,3 bond is the most stable. Molecular dynamics simulations revealed that the key residue Trp359 that may affect substrate specificity and the main catalytic sites of N12-Rha are located in the (α/α)6-barrel domain.

The intensive development of biotechnology in the last decade is largely determined by the growing requirement needs of both medicine and various industries for products of microbial synthesis, including glycosidases, in particular a-L- rhamnosidases. lheir wide use to solve current biological-medical and chemical-technological problems stimulates re- searchers to search for compounds capable of influencing their catalytic activity. Therefore, the purpose of this work was to isolate and purify a-L-rhamnosidase from a new producer of Penicillium restrictum and to investigate multi-ligand germanium-3d-metal complexes with citric acid, phenanthroline, and bipyridine as effectors of its activity. Methods. Ihe object of the study was a-L-rhamnosidase of restrictum. Its purification was carried out by gel filtration and ion exchange chromatography on TSK-gels and Sepharose 6B. The activity of a-L-rhamnosidase was determined us- ing the Davis method with naringin as a substrate. As modifiers of enzyme activity, purposefully synthesized multi- ligand germanium-3d-metal complexes with citric acid, phenanthroline, and bipyridine (INi(bipy)3[Ge(HCit)2] •3H 2 O (1); [Ni(phen)3 ] [Ge(HCit)2]•2H2O (2); [{Cu(bipy)2}2Ge(μ-Cit)2] •12H20 (3); [{Cu(phen)2]2Ge-(Cit)2]•13H2O (4); [Zn(bipy)3][Ge(HCit)2].•12H20 (5); [Zn(phen)3][Ge(HCit)2]•3H2O (6)), were used. Results. From the supernatant of culture fluid of P. restrictum, a-L-rhamnosidase was isolated and purified 23.1 times with a yield of 0.09%. the specific activity of the enzyme was 27.8 units/mL. The enzyme was homogeneous according to gel filtration on Sepharose 6B and had a molecular mass of 50 kDa. It was established that the considered coordination compounds are able to regulate the catalytic activity of a-L-rhamnosidase of P. restrictum. All of them manifest themselves either as a tivators or as inert substances, no inhibition was observed. In addition, the dependence of the degree of enzyme activation by the compounds on their concentration is traced and corresponds to the following series: at a concentration of 0.01% —1 > 6 =-- 5 > 3 >2 =.-- 4 and at a concentration of 0.1 % —1 > 4 > 2 > 5 = 6.3. The catalytic activity is also significantly affect-ed by the time of exposure to the compounds: at a concentration of 0.01% for lh, the activity of the enzyme at the control level was observed for all compounds, whereas at a concentration of 0.1% for 24 h,the activity increased sharply in the presence of compounds 1(300%), 6 (153%), and 2 (134%). The action of the others was at the control level. Conclusions. The obtained data on new complex metal compounds with an activating effect on microbial a-L-rhamnosidases. It has been established that compounds whose structural organization ensures the synergism of the action of all components are the most promising enzyme effectors in a series of coordination compounds of biologically active metals and ligands. [ABSTRACT FROM AUTHOR]

αL-rhamnosidase (EC 3.2.1.40) has been widely used in food processing and pharmaceutical preparation. The recombinant α-L-rhamnosidase N12-Rha from Aspergillus niger JMU-TS528 had significantly higher catalytic activity on α-1,6 glycosidic bond than α-1,2 glycosidic bond, and had no activity on α-1,3 glycosidic bond. The activities of hydrolyzed hesperidin and naringin were 7240 U/mL and 945 U/mL, respectively, which are 10.63 times that of native α-L-rhamnosidase. The activity could maintain more than 80% at pH 3–6 and 40–60℃. Quantum chemistry calculations showed that charge difference of the C-O atoms of the α-1,2, α-1,3 and α-1,6 bonds indicated that α-1,6 bond is most easily broken and α-1,3 bond is the most stable. Molecular dynamics simulations revealed that the key residue Trp359 that may affect substrate specificity and the main catalytic sites of N12-Rha are located in the (α/α)6-barrel domain. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Enzymatic deramnosylation of flavonoids is a convenient tool for improving the quality of citrus juices. α-L-rhamnosidase with a specific activity of 33.1 units/mg was isolated and characterized from the culture liquid of Penicillium tardum. The molecular weight of the enzyme was 95 kDa according to the data of gel filtration on Sepharose 6B and gel electrophoresis in SDS-PAGE. The pH optimum of the enzyme activity was 5.0, and the thermo optimum was 60 °C. Enzyme showed high stability in the temperature range of 45–50 and at 60–70 °C. It retained 80 to 50% of the initial activity for 90 min. The half-life of α-L-rhamnosidase at 70 °C increased twofold in the presence of 20–40% glycerol and 2.3-fold in the presence of 4 M sorbitol. The enzyme was completely inhibited in the presence of 10−3 M Ag+ and Cd2+ and approximately by 90% in the presence of Fe2+, Fe3+, and Al3+ ions. More than 60%, the enzyme activity was inhibited by Hg2+, Co2+, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide. Activating effect of Ca2+ ions was also noted. Km and Vmax for the hydrolysis of p-nitrophenyl-α-L-rhamnopyranoside and naringin were 0.7 mM and 38.3 µM/min/mg and 1.34 mM and 43.7 µM/min/mg, respectively. Penicillium tardum α-L-rhamnosidase hydrolyzed naringin, neohesperidin, hesperidin, rutin, and narirutin at high rate, which allowed us to consider it as an effective tool for transformation of bioflavonoids in food industry. [ABSTRACT FROM AUTHOR]

In recent years, the particular interest of researchers is focused on such enzymes as α-L-rhamnosidase and α-galactosidase. These enzymes are considered useful for various applications. α-L Rhamnosidases may be applied for debittering of citrus fruit juices, due to the less bitter taste of the derhamnosylated flavonones, for rhamnose production, and for the determination of the anomeric configuration in polysaccharides, glycosides and glycolipids. These enzymes may enhance wine aroma and flavonoid bioavailability, or assist in the synthesis of pharmaceuticals. α-Galactosidase finds application in many areas. It is widely used in the food industry to improve the quality of soy products by hydrolyzing indigestible galactosides such as raffinose and stachyose, in the processing of raw materials in order to increase the yield of sugar from molasses, and for the biotransformation of human blood erythrocytes of group B (III) in universal donor erythrocytes, as well as in enzyme therapy of some congenital disorders of sphingolipid metabolism. Earlier, as a result of screening microorganisms of different taxonomic groups, we has selected active α-L-rhamnosidase and α-galactosidase producers. One way to increase their activity is using various effector compounds capable of modifying the enzyme activity. The study of the influence of various effectors is one of the priority areas of modern research in biochemistry, biocoordination chemistry, and biotechnology. Recent advantages in the area of biocoordination chemistry revealed high activating properties of double heterometallic mixed-ligand coordination compounds with germanium(IV)/tin(IV) tartaric complex anions and 1,10-phenanthroline/2,2'-bipyridine d-metallic cations. The aim is to estimate the enzyme-effector activity of five similar tartratostannates for the α-L-rhamnosidases of Cryptococcus albidus, Eupenicillium erubescens, and α-galactosidase of Penicillium restrictum. Methods. The activity of α-Galactosidase was determined using p-nitrophenyl-α-D-galactopyranoside («Sigma», USA) as a substrate. The activity of α-L-rhamnosidase was determined using the Davis method. As modifiers of enzyme activity, [Fe(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (1), [Co(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·8H2O (2), [Ni(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (3), [Cu(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·2H2O (4), and [Zn(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·6H2O (5) were used. Results. The study of the effect of complexes 1--5, which are supramolecular salts consisting of the same tartrate stannate anion (electrophilic agent) and a 1,10-phenanthroline d-metal cation (nucleophilic agent), on the Cryptococcus albidus, Eupenicillium erubescens α-L-rhamnosidases, and Penicillium restrictum α-galactosidase showed that the compounds tested had a different influence on the enzymes' activity. The catalytic activity of α-L-rhamnosidase is significantly influenced by all complexes. The effectiveness of compounds 1--5 for P. restrictum α-galactosidase was less pronounced in comparison with C. albidus and E. erubescens α-L-rhamnosidases. It was mostly at the control level. There was observed a certain pattern in the influence of complexes on α-L-rhamnosidases of Cryptococcus albidus and Eupenicillium erubescens. Compounds 2 and 5 turned out to be the most effective and activated enzymes by 500-900%. Conclusions. Compound 2 [Co(phen)3]2[Sn2(μ-Tart)2(Н2Tart)2]·8H2O is promising for further use as an effector of the α-L-rhamnosidase activity. [ABSTRACT FROM AUTHOR]

Enzymatically modified isoquercitrin (EMIQ) is a glyco-chemically modified flavonoid exhibiting notably high biological activity, such as antioxidant, anti-inflammatory and anti-allergic properties. However, the utilization of expensive substrates such as isoquercitrin and cyclodextrin in the conventional approach has hindered the industrial-scale production of EMIQ due to high cost and low yields. Hence, the development of a cost-effective and efficient method is crucial for the biological synthesis of EMIQ. In this study, a natural cascade catalytic reaction system was constructed with α -L-rhamnosidase and amylosucrase using the inexpensive substrates rutin and sucrose. Additionally, a novel approach integrating gradient temperature regulation into biological cascade reactions was implemented. Under the optimal conditions, the rutin conversion reached a remarkable 95.39% at 24 h. Meanwhile, the productivity of quercetin-3- O -tetraglucoside with the best bioavailability reached an impressive 41.69%. This study presents promising prospects for future mass production of EMIQ directly prepared from rutin and sucrose. [Display omitted] • A novel cascade strategy was constructed to produce EMIQ from inexpensive substrates. • Amylosucrase with sucrose as a substrate was used to further reduce the production cost. • Gradient temperature control was innovatively integrated into the biological cascade reaction. • The conversion rate of IQ-G3 with the best bioavailability reached an impressive 41.69%. • The industrial production of EMIQ with rutin and sucrose as materials has good potential. [ABSTRACT FROM AUTHOR]

The GH78 α-L-rhamnosidase from Aspergillus tubingensis (AT-Rha) was proved to be a new clade of Aspergillus α-L-rhamnosidases in the previous study. A putative α-L-rhamnosidase from A. kawachii IFO 4308 (AK-Rha) has 92 % identity in amino acid sequence with AT-Rha. In this study, AK-Rha was expressed in P. pastoris and characterized. Similar to AT-rRha, the recombinant AK-Rha (AK-rRha) showed a narrow substrate specificity to naringin. Interestingly, the enzyme activity of AK-rRha was 0.816 U/mg toward naringin, significantly lower than 125.142 U/mg of AT-rRha. Their large differences in catalytic efficiency was mainly due to their differences in k cat values between AK-rRha (0.67 s−1) and AT-rRha (4.89 × 104 s−1). The molecular dynamics simulation exhibited that the overall conformation of AK-Rha was rigid and that of AT-Rha was flexible; the Loop Y-L located above the catalytic domain formed different steric hindrances to naringin, and interacted with the flavonoid matrices at different strengths. The polar solvation energy analysis implied that the glycosidic bond was more easily hydrolysed in AT-Rha. The comparative study verified that the main feature of AK-Rha and AT-Rha represented Aspergillus α-L-rhamnosidase was the narrow substrate specificity toward naringin, and provided an insight of the relationships between their catalytic abilities and structures. • A putative α-L-rhamnosidase AK-Rha has 92 % identity with AT-Rha. • AK-rRha enzyme activity was 0.65 % of AT-rRha enzyme activity. • k cat value of AK-rRha was 3 orders of magnitude lower than AT-rRha. • Overall and local conformation flexibility was essential to high enzyme activity. • Steric hindrance effect of Loop Y-L was closely related to enzyme activity. [ABSTRACT FROM AUTHOR]

There has been ongoing interest in improving the efficiency of glycoside hydrolase for synthesizing glycoside compounds through protein engineering, given the potential applications of glycoside compounds. In this study, a strategy of modifying the substrate access tunnel was proposed to enhance the efficiency of reverse hydrolysis catalyzed by Aspergillus niger α-L-rhamnosidase. Analysis of the tunnel dynamics identified Tyr299 as a key modifiable residue in the substrate access tunnel. The location of Tyr299 was near the enzyme surface and at the outermost end of the substrate access tunnel, suggested its role in substrate recognition and throughput. Based on the properties of side chains, six mutants were designed and expressed by Pichia pastoris. Compared to WT, the reverse hydrolysis efficiencies of mutants Y299P and Y299W were increased by 21.3 % and 11.1 %, respectively. The calculation results of binding free energy showed that the binding free energy was inversely proportional to the reverse hydrolysis efficiency. Further, when binding free energy levels were comparable, the mutants with shorter side chains displayed a higher reverse hydrolysis efficiency. These results proved that substrate access tunnel modification was an effective method to improve the reverse hydrolysis efficacy of α-L-rhamnosidase and also provided new insights for modifying other glycoside hydrolases. • Enhancing reverse hydrolysis efficiency of α-L-rhamnosidase by altering specific site in the substrate tunnel. • The reverse hydrolysis efficiencies of Y299P and Y299W were raised by 21.3 % and 11.1 %, respectively, compared with the WT. • Low binding free energy and short side chain of the mutants contributed to the efficiency of the reverse hydrolysis. [ABSTRACT FROM AUTHOR]

Aspergillus niger has been used for homologous and heterologous expressions of many protein products. In this study, the α-L-rhamnosidase from A. niger (Rha-N1, GenBank XP_001389086.1) was homologously expressed in A. niger 3.350 by Agrobacterium tumefaciens-mediated transformation. The enzyme activity of Rha-N1 was 0.658 U/mL, which was obtained by cultivation of engineered A. niger in a 5-L bioreactor. Rha-N1 was purified by affinity chromatography and characterized. The optimum temperature and optimum pH for Rha-N1 were 60 °C and 4.5, respectively. Enzyme activity was promoted by Al3+, Li+, Mg2+, and Ba2+ and was inhibited by Mn2+, Fe3+, Ca2+, Cu2+, and organic solvents. The result indicated that rutin was the most suitable substrate for Rha-N1 by comparison with the other two flavonoid substrates hesperidin and naringin. The transformed products of isoquercitrin, hesperetin-7-O-glucoside, and prunin were identified by LC–MS and 1H-NMR. [ABSTRACT FROM AUTHOR]

A general and flexible approach toward the development of α‐l‐rhamnosidase (α‐l‐Rha‐ase) inhibitors is described. Five enantiopure poly‐substituted pyrrolidine‐based scaffolds bearing the C1‐aminomethyl moiety were designed and synthesized from five‐membered cyclic nitrones. Each structurally diversified amide library of these scaffolds was rapidly generated via combinatorial parallel synthesis and applied for in‐situ inhibition study against α‐l‐Rha‐ase, allowing us to efficiently identify new inhibition hits. Surprisingly, all promising inhibitors are derived from the same scaffold 3. Among them, the most potent and selective inhibitor is pyrrolidine 19 with Ki=0.24 μM, approximately 24‐fold more potent than the reference compound DAA (Ki=5.7 μM). It is the first study to comprehensively prepare pyrrolidine‐based scaffolds and libraries for inhibition study against α‐l‐Rha‐ase. [ABSTRACT FROM AUTHOR]

Icariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain, Papiliotrema laurentii ZJU-L07, producing an intracellular α-L-rhamnosidase was isolated from the soil and mutagenized. The specific activity of α-L-rhamnosidase was 29.89 U·mg−1 through purification, and the molecular mass of the enzyme was 100 kDa, as assayed by SDS-PAGE. The characterization of the purified enzyme was determined. The optimal temperature and pH were 55 °C and 7.0, respectively. The enzyme was stable in the pH range 5.5–9.0 for 2 h over 80% and the temperature range 30–40 °C for 2 h more than 70%. The enzyme activity was inhibited by Ca2+, Fe2+, Cu2+, and Mg2+, especially Fe2+. The kinetic parameters of Km and Vmax were 1.38 mM and 24.64 μmol·mg−1·min−1 using pNPR as the substrate, respectively. When epimedin C was used as a nature substrate to determine the kinetic parameters of α-L-rhamnosidase, the values of Km and Vmax were 3.28 mM and 0.01 μmol·mg−1·min−1, respectively. The conditions of enzymatic hydrolysis were optimized through single factor experiments and response surface methodology. The icariin yield increased from 61% to over 83% after optimization. The enzymatic hydrolysis method could be used for the industrialized production of icariin. At the same time, this enzyme could also cleave the α-1,2 glycosidic linkage between glucoside and rhamnoside in naringin and neohesperidin, which could be applicable in other biotechnological processes. [ABSTRACT FROM AUTHOR]

Icariside I, the glycosylation product of icaritin, is a novel effective anti-cancer agent with immunological anti-tumor activity. However, very limited natural icariside I content hinders its direct extraction from plants. Therefore, we employed a computer-aided protein design strategy to improve the catalytic efficiency and substrate specificity of the α-L-rhamnosidase from Thermotoga petrophila DSM 13995, to provide a highly-efficient preparation method. Several beneficial mutants were obtained by expanding the active cavity. The catalytic efficiencies of all mutants were improved 16–200-fold compared with the wild-type TpeRha. The double-point mutant DH was the best mutant and showed the highest catalytic efficiency (kcat/KM: 193.52 s−1 M−1) against icariin, which was a 209.76-fold increase compared with the wild-type TpeRha. Besides, the single-point mutant H570A showed higher substrate specificity than that of the wild-type TpeRha in hydrolysis of different substrates. This study provides enzyme design strategies and principles for the hydrolysis of rhamnosyl natural products.

Recently enzyme preparations of microbial origin become increasingly important in different industries. Preparations of α-L-rhamnosidase are used in the pharmaceutical industry as well as in scientific work as a tool for analytical research. We have obtained purified α-L-rhamnosidase preparations from Penicillium tardum, Eupenicillium erubescens and Cryptococcus albidus microorganism strains which are effective enzyme producers. The aim of the study was to estimate the ability of germanium coordination compounds to enhance enzyme catalytic activity. The effects of 11 heterometal mixed ligand tartrate (malate-)germanate compounds at 0.01 and 0.1% concentration on the activity of α-L-rhamnosidase preparations from Penicillium tardum IMV F-100074, Eupenicillium erubescens and Cryptococcus albidus 1001 were studied at 0.5 and 24 h exposition. The inhibitory effect of [Ni(bipy)3]4[{Ge2(OH)2(Tart)2}3Cl2]·15H2 on P. tardum α-L-rhamnosidase was revealed. All studied compounds except [CuCl(phen)2][Ge(OH)(HMal)2] were shown to increase activity of P. tardum α-L-rhamnosidase at a longer term of exposition. Activity of E. erubescens α-L-rhamnosidase was shown to be stimulated by d-metal cation-free compounds. C. albidus α-L-rhamnosidase occurred to be insensitive to all compounds studied.

The α‐L‐Rhamnosidase (Rha) is a useful glycoside hydrolase for selectively hydrolyzing the terminal L‐rhamnose residues in flavonoids, being vital to food and pharmaceutical industries. However, Rha suffers from low recyclability and poor stability in harsh environments. Herein, we explored five typical metal‐organic frameworks (MOFs) as porous carriers to immobilize Rha, and the activities of the resultant Rha@MOF composites were compared with the free enzyme. The locations of the enzyme in MOFs were proved by a series of characterization techniques. It was found that Rha@Ce‐BTC (with enzyme immobilization efficiency of 23 % and enzyme loading content of 8.8 %) showed the highest enzymatic activity. The immobilized Rha@Ce‐BTC showed 80 % residual activity after five consecutive cycles, suggesting a limited leaching effect. Also, Rha@Ce‐BTC manifested markedly enhanced enzyme‐substrate affinity and catalytic efficiency compared to free Rha, as supported by Michaelis‐Menten kinetic studies. Accordingly, the Ce‐BTC would be an appealing carrier for enzyme immobilization, and the as‐designed enzyme/Ce‐BTC composites are promising candidates for industrial use with remarkably high activity, recyclability, and storage stability. [ABSTRACT FROM AUTHOR]

In this study, we found the addition of sorbitol could improve the thermostability of α‐l‐rhamnosidase from Aspergillus niger. When α‐l‐rhamnosidase with sorbitol was heat‐treated at 60°C, 65°C, and 70°C, the half‐life t1/2 increased by 28‐, 18‐, and 9‐fold, respectively. Inactivation thermodynamic analysis showed that both Ea and ΔG≠ of α‐l‐rhamnosidase increased. Through the response surface methodology (RSM) analysis, the higher hesperidin conversion (63.26%) by α‐l‐rhamnosidase was attained with 0.7 M sorbitol at 60°C and pH 4.5 for 10 min. Furthermore, hesperidin could be completely hydrolyzed after 10 hr of reaction. Overall, the results indicated that the addition of sorbitol improved the thermostability of α‐l‐rhamnosidase and increased the enzymatic conversion of hesperidin to hesperetin‐7‐O‐glucoside (HMG). It also provided a simple and efficient way to increase enzymatic conversion of other valuable flavonoid monomers due to the broad substrate specificities of α‐l‐rhamnosidase from A. niger. Practical applications: Hesperetin‐7‐O‐glucoside (HMG), a derhamnosylation product of hesperidin, is considered as a synthetic precursor for novel and efficient sweeteners and is important in food, functional food, and nutraceutical industries. Compared to chemical hydrolysis methods, the enzymatic conversion of hesperidin is milder and has the advantages of high specificity. Adding sorbitol can improve the thermostability of α‐l‐rhamnosidase and increase the enzyme efficacy against hesperidin. This study gave more evidence that adding sorbitol could improve the thermostability of enzymes and provide a better choice for improving biotransformation potency of enzymes. [ABSTRACT FROM AUTHOR]

In recent years, cold-adapted enzymes are increasingly used in industrial processes such as the food, textile and beverage industries. Moreover, cold-active enzymes are usually thermolabile and can be inactivated with little heat. This is especially important in reactions where it is necessary to inactivate an enzyme after it has completed its function, while maintaining conditions that allow other enzymes involved in the reaction to function. Among these enzymes, glycosidases play an important role, which are used in medical technological processes, the food industry, biotechnology for the purification and processing of raw materials, as well as in many other areas of human activity. Therefore, the aim of this work was to study the ability of the psychrotolerant bacterium Pseudomonas mandelii U1 to produce glycosidases, in particular a-L-rhamnosidases, and also to investigate their physicochemical properties and substrate specificity. Methods. Glycosidase activities were determined by Romero and Davis methods, protein - by Lowry method. Results. The study of enzymatic activities in the dynamics of growth indicates that already on the third day of cultivation in the supernatant of the culture liquid of P. mandelii U1 α-L-rhamnosidase activity (0.09 U/mg protein) was noted. On the fifth day of cultivation, in addition to α-L-rhamnosidase (0.09 U/mg protein), β-D-glucosidase (0.09 U/mg protein) and α-D-glucosidase (0.09 U/mg protein) activities were identified. On the seventh and ninth days of cultivation, the spectrum of glycosidase activities was wider, except for α-L-rhamnosidase (0.2 and 0.16 U/mg protein, respectively), β-D-glucosidase (0.02 and 0.05 U/mg protein, respectively) and α-D-glucosidase (0.04 and 0.08 U/mg of protein, respectively), α-D-mannosidase (0.025 and 0.025 U/mg protein, respectively), α-D-fucosidase (0.025 and 0.05 U/mg protein, respectively), N-acetyl-β-D-glucosaminidase (0.025 and 0.025 U/mg protein, respectively) and N-acetyl-β-D-galactosaminidase (0.025 and 0.025 U/mg protein, respectively). Since among the studied glycosidase activities, α-L-rhamnosidase was the highest, subsequent studies were aimed at investigating its properties. It was shown that P. mandelii U1 α-L-rhamnosidase has the pH optimum of action at 5.0, and the temperature optimum - at 4 °C. Conclusions. The temperature optimum of P. mandelii U1 α-L-rhamnosidase preparation isolated from moss in Antarctica, Galindez Island, is 4 °C, the optimum pH is 5.0, the enzyme is able to hydrolyze as synthetic substrates p-nitrophenyl-α-L-rhamnopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl-α-D-mannopyranoside, and natural substrates - naringin, neohesperidin and rutin, which suggests the possibility of its use in the future in food technologies, in particular in food processing and waste degradation at low temperatures. [ABSTRACT FROM AUTHOR]

Catalytic efficiency and thermostability are the two most important characteristics of enzymes. However, it is always tough to improve both catalytic efficiency and thermostability of enzymes simultaneously. In the present study, a computational strategy with double‐screening steps was proposed to simultaneously improve both catalysis efficiency and thermostability of enzymes; and a fungal α‐l‐rhamnosidase was used to validate the strategy. As the result, by molecular docking and sequence alignment analysis within the binding pocket, seven mutant candidates were predicted with better catalytic efficiency. By energy variety analysis, A355N, S356Y, and D525N among the seven mutant candidates were predicted with better thermostability. The expression and characterization results showed the mutant D525N had significant improvements in both enzyme activity and thermostability. Molecular dynamics simulations indicated that the mutations located within the 5 Å range of the catalytic domain, which could improve root mean squared deviation, electrostatic, Van der Waal interaction, and polar salvation values, and formed water bridge between the substrate and the enzyme. The study indicated that the computational strategy based on the binding energy, conservation degree and mutation energy analyses was effective to develop enzymes with better catalysis and thermostability, providing practical approach for developing industrial enzymes. [ABSTRACT FROM AUTHOR]

Calceorioside B, a multifunctional phenylethanol glycosides (PhGs) derivative, exhibits a variety of notable properties, such as antithrombotic, anti-tumorigenic, anti-neocoronavirus, anti-inflammatory, and neuroprotective effects. However, the large-scale production of calceorioside B is routinely restricted by its existence as an intermediary compound derived from plants, and still unachieved through excellent and activity chemical synthesis. Here, a total of 51 fungal endophytes were isolated from four PhGs-producing plants, and endophyte Simplicillium sinense EFF1 from Echinacea purpurea was identified with the ability to de-rhamnosing isoacteoside to generate calceorioside B. According to the RNA-transcription of EFF1 under the various substrates, a key gene CL1206.Contig2 that undertakes the hydrolysis function was screened out and charactered by heterologous expression. The sequence alignment, phylogenetic tree construction and substrate specificity analysis revealed that CL1206 was a novel α-L-rhamnosidase that belongs to the glycosyl hydrolase family 78 (GH78). The optimum catalytic conditions for CL1206 were at pH 6.5 and 55 °C. Finally, the enzyme-catalyzed approach to produce calceorioside B from 50 % crude isoacteoside extract was explored and optimized, with the maximum conversion rate reaching 69.42 % and the average producing rate reaching 0.37 g-1.L-1.h−1, which offered a great biocatalyst for potential industrial calceorioside B production. This is the first case for microorganism and rhamnosidase to show the hydrolysis ability to caffeic acid-modified PhGs. [ABSTRACT FROM AUTHOR]

[Display omitted] • DthRha could efficiently hydrolyze the α-1-rhamnosidic bond of epimedium flavonoids. • Icaritin preparation by two thermostable enzymes with high productivity. • Major parts of total flavonoids extract of epimedium were transformed into icaritin. The thermostable GH78 family α-L-rhamnosidase DthRha from Dictyoglomus thermophilum DSM3960 was found to have the ability to remove the α-L-rhamnose moiety that was directly linked to the aglycone by an α-1-rhamnosidic bond at the C-3 position of epimedium flavonoids. It exhibited high specific activity on p- nitrophenyl-α-L-rhamnopyranoside (pNPR) (281.06 U/mg), followed by epimedin C (30.42 U/mg), icariin (9.74 U/mg), baohuoside I (1.02 U/mg) and 2'-O-rhamonosylicariside II (3.74 U/mg). In addition, the thermostable glucosidase Dth3 from Dictyoglomus thermophilum DSM3960, which exhibits β-xylosidase activity and β-glucosidase activity, was purified. The main enzymatic properties of the two thermostable glycosidases were compared, and these glycosidases were successfully utilized in the preparation of pharmacologically active icaritin from the total flavonoid extract of epimedium. Finally, 5 g/L of the total flavonoid extract of epimedium was completely transformed into 207.95 mg/L icaritin at pH 5.5 and 80 °C for a total of 4 h. This is the first report on the efficient biotransformation of the total flavonoid extract of epimedium into icaritin by two thermostable enzymes with high productivity. The cost of icaritin preparation will be substantially reduced by converting all the major ingredients in the total flavonoid extract of epimedium. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosidase [EC 3.2.1.40], enzyme of the hydrolase family has a wide range of applications: in the food industry, for example, in winemaking to improve the quality and aroma of wines, in the production of citrus juices and drinks to remove bitter components (naringin) that improves the quality and nutritional value of these products; in research as an analytical tool for studying the structure of complex carbohydrate-substituted biopolymers. For the successful use of α-L-rhamnosidases in various biotechnological processes, an important aspect is the development of ways to increase their activity. The main factors affecting the growth and metabolism of microorganisms, including the synthesis of enzymes, are the physicochemical conditions of cultivation, the composition of the nutrient medium, the introduction of substances that raise the yield of the enzyme, which is manifested in an increase in its activity. At present, one of the priority directions of modern research is the study of the effect of various effector compounds that are capable to modify the studied enzymatic activity. In this work, which is a continuation of previous studies, a number of mixed-ligand and mixed-ligand-different-metal coordination germanium compounds of with xylaric acid (H5Xylar), 1,10-phenanthroline (Phen), 2,2-bipyridine (bipy) and ions of 3d-metals (Fe2+, Ni2+, Cu2+, Zn2+) were selected as effectors. Study of the effect of these complexes on the activity of Eupenicillium erubescens, Cryptococcus аlbidus and Penicillium tardum α-L-rhamnosidases were the aim of this work. Materials and methods. The objects of research were α-Lrhamnosidases from Eupenicillium erubescens 248, Cryptococcus albidus 1001, and Penicillium tardum IMV F-100074. The α-L-rhamnosidase activity was determined by the Davis method using naringin as a substrate. We used 12 coordination compounds of germanium as modifiers of enzyme activity, the composition and structure of which were established using a combination of physical and chemical research methods: elemental analysis, thermogravimetry, IR spectroscopy and X-ray structural analysis. Structures of seven compounds are deposited in the Cambridge Crystallographic Database. When studying the effect of various compounds on the activity of enzymes, concentrations of 0.1 and 0.01 % were used, exposure times were 0.5 and 24 hours. The test compounds were dissolved in 0.1 % dimethyl sulfoxide. UV-spectra of absorption of native and chemical modified preparations of the enzymes were studied by spectrophotometer-fluorimeter DeNovix DS-11 in the range of 220-340 nm, concentration of the enzyme preparation 1.0 mg of protein/mL. Results. Analysis of the totality of the obtained data (exposure time 24 h, concentration 0.1 %) regarding the effect of the studied compounds on the activity of E. erubescens, C. albidus and P. tardum α-L-rhamnosidases showed that the influence of the studied modifiers for the activity of α-L-rhamnosidases varies depending on the producer strain. Our data allow us to present the following series of modifiers in accordance with an increase in their effect on the activity of enzymes of different producers: E. еrubescens: 12 < 11 < 5 < 3 < 4=10 < 1 < 3 < 8 < 2 < 6 < 7; C. albidus: 10 < 11 < 12 < 9 < 3 < 1=5 < 8=4 < 2 < 6 < 7; P. tardum: 12=2 < 3 < 4 < 11 < 5 < 8 < 1 < 9 < 6 < 10 < 7. Conclusions. The results obtained allow us to conclude that compound (7)(-tris(bipyridine) nickel(II) μ-dihydroxyxylaratogermanate(IV)) is the most effective activator of α-L-rhamnosidases of all three micromycete strains, compound (6)(tris(phenanthroline)nickel(II) μ-dihydroxyxylaratogerman ate(IV)) - on α-L-rhamnosidase from E. erubescens and C. albidus, while compound (10)-(copper(II) μ-dihydroxyxylaratogermanate(IV)-cuprate(II)) - only of P. tardum α-L-rhamnosidase. [ABSTRACT FROM AUTHOR]

The directed search for extremophilic producers in order to obtain hydrolytic enzymes with increased thermal stability has an unconditional practical potential for use in the food and feed industry to improve the quality of the final product. The aim of the work was to study the ability of collection strains of thermophilic fungi to show α-L-rhamnosidase, α-galactosidase, cellulase, β-mannanase, keratinase and caseinolytic activity. Methods. Micromycetes were grown under submerged conditions in test tubes at 42 °C for 8-14 days. Enzymatic activities were studied in the culture liquid supernatant. p-Nitrophenyl-α-D-galactopyranoside, naringin, guar gum galactomannan and Na-carboxymethylcellulose were used as substrates to determine α-galactosidase, α-L-rhamnosidase, β-mannanase and cellulase activities, respectively. Casein and crushed defatted feathers were served as substrates for the determination of proteolytic activity. Results. The enzymatic activity of 50 strains of micromycetes belonging to 17 species was investigated. The studied group showed high activity: 94 % of the strains had at least one, 34 % - two, 26 % - from three to five enzyme activities. The most active keratinase producers were Thielavia terrestris 1920 and 62, Rhizomucor tauricus 1909, Chrysosporium thermophilum 2050, Thermoascus thermophilus 92 and Thermoascus aurantiаcus 2052 (10-26 U/mL). The highest α-L-rhamnosidase activity was observed in T. terrestris 62 (0.35 U/mL), and carboxymethylcellulase activity -in Thermomyces lanuginosus 2046. Six strains showed α-galactosidase (0.05-0.2 U/mL) and four strains - β-mannanase (5-130 U/mL) activity. Conclusions. As a result new strains producing proteolytic and glycolytic enzymes were isolated among thermophilic micromycetes. Soil thermophilic micromycetes can be used as producers of proteolytic and glycolytic enzymes. Of particular interest are the cultures of Acremonium thermophilum 1963, Corynascus thermophilum 2050, C. sepedonium 1899 and 65068, T. thermophilus 1946, which are capable of producing complexes of proteases and glycosidases in the culture liquid. This indicates that these strains are promising for use as destructors in various technologies processing of complex raw materials. [ABSTRACT FROM AUTHOR]

The aim of the work was to investigate the possibility of practical use substrate specificity of α-Lrhamnosidase Cryptococcus albidus. p-Nitrophenyl derivatives of monosaccharides were used to determine the activity and specificity of the enzyme. The ability to hydrolyze of natural substrates was evaluated by Davis and high-performance liquid chromatography methods. It was shown that the enzyme exhibits narrow specificity towards the glycon of synthetic substrates and hydrolyzes only p-nitrophenyl-α-L-rhamnopyranoside (Km 4.5 mM) and p-nitrophenyl-α-D-glucopyranoside (Km 10.0 mM). C. albidus α-L-rhamnosidase the most active degrades naringin (Km 0.77 mM), releasing prunin and naringenin. Km for neohesperidin was 3.3 mM. The efficacy of the naringin hydrolysis in grapefruit and pomelo juice was 98 and 94% in 60 min (40 оC, 2 U/ml). As the result of treatment by α -L-rhamnosidase of green tea and orange juice, there was a decrease in the content of rutin, narirutin and hesperidin, indicating that the α -1,2- and α -1,6-linked rhamnose can be cleaved from natural flavonoids. Thus, the study shows the efficiency of treating citrus juices and green tea with C. albidus α -L-rhamnosidase for the purpose of improving their taste qualities and obtaining bioavailable flavonoids glucosides.

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The efficiency of the process of obtaining biotechnologically active substances, in particular enzymes, by microbiological synthesis largely depends on the successful optimization of the conditions of producer cultivation. The aim was to determine the influence of the main components of nutrient medium and culture conditions on the level of extracellular α-L-rhamnosidase activity of Penicillium restrictum. Methods. The micromycetes culture was grown in a submerged cultivation condition at 25 °C for 7 days. α-L-Rhamnosidase activity in the supernatant culture liquid of P. restrictum was determined using n-nitrophenyl- α-L-rhamnopyranoside. Xylose, arabinose, glucose, galactose, rhamnose, mannose, lactose, maltose, sucrose, and mannitol were used as carbon sources. Sodium nitrate, sodium nitrite, ammonium sulfate, ammonium nitrate, yeast autolysate, yeast extract, peptone, urea, and soy flour were used as nitrogen sources. To study the effect of cultivation conditions, the strain was grown on medium optimized for carbon and nitrogen sources, changing the initial pH of the medium (3.0-8.0), temperature (25-42 °C) and volume of the medium (50-250 ml). Results. It was found that the highest rates of P. restrictum α-Lrhamnosidase activity were observed on the 7th day of cultivation in case of rhamnose using a as carbon source at a concentration of 5 g/l and ammonium sulfate as nitrogen source - 0.8 g/l. The most effective for α-L-rhamnosidase synthesis by P. restrictum was the use a medium with an initial pH value of 6.0, temperature of 25 °C and 100 ml of nutrient medium in flasks. Conclusions. It was shown the possibility of P. restrictum to synthesize a new highly active α-Lrhamnosidase in submerged cultivation conditions. The combination of rhamnose and ammonium sulfate was found to provide α-rhamnosidase activity (1.2 U/ml). The established physical and chemical conditions of P. restrictum cultivation can be used for production of α-L-rhamnosidase for biotechnological purposes. [ABSTRACT FROM AUTHOR]

α-L-Rhamnosidase has attracted lots of attention due to its industrial potential applications. The applicability of α-L-rhamnosidase, however, was limited by their low ligand affinity on the industrial scale. In order to improve the affinity of α-L-rhamnosidase for industrial use, we investigated the variation of its affinity by amino acid replacement. Particularly, the enzyme affinity of a α-L-rhamnosidase from Aspergillus niger JMU-TS528 (rRha1) was measured with the semi-conservative amino acid (homology between 30% -80%) replaced. As a result, the enzyme affinity of the two mutants, R404S and N578D, were increased by 1.45-fold and 2.3-fold, respectively, showing that these two mutants could be the promising candidates for industrial use. To test if these mutations bring negative effect on the enzyme properties, we also determined the other enzymatic properties of these mutants and showed no negative effect. To understand the improvement of enzyme affinity, the conformational flexibility of (α/α) 6 -barrel catalytic domain were examined by molecular dynamics (MD) simulation, and demonstrated that the conformations of these mutants are more flexible, which could influence the affinity of substrates to the enzyme and hence the enzyme activity. This work not only enhanced the enzyme affinity of a α-L-rhamnosidase, making rRha1 a promising candidate for industrial processes, but also provided an effective technical strategy for improving affinity of other enzymes. [ABSTRACT FROM AUTHOR]

Studies of microscopic fungi of extreme ecosystems are of interest both in terms of researching the mechanisms of adaptation processes and in connection with the high biotechnological potential of micromycetes. im. To investigate a-Lrhamnosidase, mannan-degrading and proteolytic activity of micromycetes cultures isolated from the Chrnobyl Exclusion Zone. Methods. Naringin was used as a substrate for determining a-L-rhamnosidase activity, p-nitrophenyl-a-D-galactopyrano-side - for a-galactosidase activity, and galactomannan guar - for ß-mananase activity. Screening of proteolytic activities was performed using 10 % gelatin medium and 1 % casein agar. Results. It was shown that 42 % of the tested strains showed a-L-rhamnosidase (0.01-1.1 U/ml), 75 % - a-galactosidase (0.05-3.0 U/ml), 50 % - ß-mannanase (0.5-45 U/ml) and 74 % - proteolytic activity. Three glycosidase activities were observed in Eupenicillium pinetorum, Trichoderma viride, Eurotium herbariorum, Aspergillus flavipes, Aspergillus ochraceus, Aspergillus flavus, Penicillium adametzii, Penicillium decumbens, Penicillium restrictum, Penicillium variabile, Penicillium verrucosum. Conclusions. High degradation activity of A. flavipes, P. decumbens, P. clavigerum, P. restrictum, P. roseopurpureum and P. sacculum strains against substrates containing terminal rhamnose and galactose residues, ability to hydrolyze galactomannan, casein and gelatin were showed. It has been shown that technogenic pollution areas can be a source of new producers of biotechnologically important enzymes. [ABSTRACT FROM AUTHOR]

[Display omitted] l -Swainsonine (l -SWN) is a known inhibitor of α- l -rhamnosidase (IC 50 0.13 µM), but its isomers have not been thoroughly investigated. In the present study, we focused on the 8- epi - l -SWN (6) and its analogues 9a–c and synthesized them from chiral lactam 1 as a common intermediate. 8- epi - l -SWN (6) showed moderate inhibition (IC 50 87 µM), but this was about 670-fold lower than l -SWN. The introduction of a (6 S)-C-methyl group into 6 significantly reduced its α- l -rhamnosidase inhibitory activity (9b : IC 50 329 μM). On the other hand, among the analogues 9a–c , (6 R)-C-benzyl 8- epi - l -SWN 9c was approximately 8.7 times better than (6 R)-C-methyl-8- epi - l -SWN 9a. These results suggest that the α- l -rhamnosidase inhibitory activity may be further enhanced by introducing a long or bulky substituent at the C6 position of l -SWN. [ABSTRACT FROM AUTHOR]

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The K m of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while k cat / K m value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin. • A novel α-L-rhamnosidase from Spirochaeta thermophila was characterized. • St-Rha showed excellent thermostability and methanol tolerance. • The highest production yield of prunin was obtained using methanol cosolvent systems. • Mechanism of methanol tolerance for α-L-rhamnosidase was studied for the first time. [ABSTRACT FROM AUTHOR]

Isoquercitrin is a flavonoid with important applications in the pharmaceutical and nutraceutical industries. However, a low yield and high production cost hinders the industrial preparation of isoquercitrin. In the present study, isoquercitrin was prepared by biotransformation of rutin using α-L-rhamnosidase from Aspergillus niger JMU-TS528 combined with high-speed countercurrent chromatography (HSCCC) purification. As a result, the optimum transformation pH, temperature, and time were pH 4.0, 60 °C, and 60 min, respectively. The Km and Vmax were 0.36 mM and 0.460 mmol/min, respectively. For isoquercitrin production, the optimal rutin concentration and transformation time were approximately 1000 mg/L and 60 min. The rutin transformation rate reached 96.44%. The isoquercitrin was purified to a purity of 97.83% using one-step purification with HSCCC. The isoquercitrin was identified using UPLC-Q-TOF-MS. The comprehensive results indicated that the biotransformation procedure using the α-L-rhamnosidase from A. niger JMU-TS528 combined with HSCCC was a simple and effective process to prepare isoquercitrin, which might facilitate the production of isoquercitrin for industrial use. [ABSTRACT FROM AUTHOR]