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Munish Puri - One of the best experts on this subject based on the ideXlab platform.
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Updates on Naringinase: structural and biotechnological aspects
Applied Microbiology and Biotechnology, 2012Co-Authors: Munish PuriAbstract:Naringinases has attracted a great deal of attention in recent years due to its hydrolytic activities which include the production of rhamnose, and prunin and debittering of citrus fruit juices. While this enzyme is widely distributed in fungi, its production from bacterial sources is less commonly known. Fungal Naringinase are very important as they are used industrially in large amounts and have been extensively studied during the past decade. In this article, production of bacterial Naringinase and potential biotechnological applications are discussed. Bacterial rhamnosidases are exotype enzymes that hydrolyse terminal non-reducing α- l -rhamnosyl groups from α- l -rhamnose containing polysaccharides and glycosides. Structurally, they are classified into family 78 of glycoside hydrolases and characterized by the presence of Asp567 and Glu841 in their active site. Optimization of fermentation conditions and enzyme engineering will allow the development of improved rhamnosidases for advancing suggested industrial applications.
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citrus peel influences the production of an extracellular Naringinase by staphylococcus xylosus mak2 in a stirred tank reactor
Applied Microbiology and Biotechnology, 2011Co-Authors: Munish Puri, Aneet Kaur, Colin J Barrow, Ram Sarup SinghAbstract:Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce Naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca+2 caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in Naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l−1) in the fermentation medium further increased Naringinase production (8.9 IU ml−1) in the bioreactor. Thus, availability of Naringinase renders it attractive for potential biotechnological applications in citrus processing industry.
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Citrus peel influences the production of an extracellular Naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor
'Springer Science and Business Media LLC', 2011Co-Authors: Munish Puri, Kaur Aneet, Barrow, Colin J., Singh, Ram SarupAbstract:Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce Naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca+2 caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in Naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 gl-1) in the fermentation medium further increased Naringinase production (8.9 IUml-1) in the bioreactor. Thus, availability of Naringinase renders it attractive for potential biotechnological applications in citrus processing industry.Published online 5 October 201
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response surface optimization of medium components for Naringinase production from staphylococcus xylosus mak2
Applied Biochemistry and Biotechnology, 2010Co-Authors: Munish Puri, Aneet Kaur, Ram Sarup Singh, Anubhav SinghAbstract:Response surface methodology was used to optimize the fermentation medium for enhancing Naringinase production by Staphylococcus xylosus. The first step of this process involved the individual adjustment and optimization of various medium components at shake flask level. Sources of carbon (sucrose) and nitrogen (sodium nitrate), as well as an inducer (naringin) and pH levels were all found to be the important factors significantly affecting Naringinase production. In the second step, a 22 full factorial central composite design was applied to determine the optimal levels of each of the significant variables. A second-order polynomial was derived by multiple regression analysis on the experimental data. Using this methodology, the optimum values for the critical components were obtained as follows: sucrose, 10.0%; sodium nitrate, 10.0%; pH 5.6; biomass concentration, 1.58%; and naringin, 0.50% (w/v), respectively. Under optimal conditions, the experimental Naringinase production was 8.45 U/mL. The determination coefficients (R 2) were 0.9908 and 0.9950 for Naringinase activity and biomass production, respectively, indicating an adequate degree of reliability in the model.
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covalent immobilization of Naringinase for the transformation of a flavonoid
Journal of Chemical Technology & Biotechnology, 2005Co-Authors: Munish Puri, Harsimran Kaur, John F KennedyAbstract:Abstract:Naringinase(EC3.2.1.40)from Penicillium spwasimmobilizedbycovalentbindingtowoodchipsto improve its catalytic activity. The immobilization of Naringinase on glutaraldehyde-coated woodchips(600mg woodchips, 10U Naringinase, 45 ◦ C, pH 4.0 and 12h) through 1% glutaraldehyde cross-linkingwas optimized. The pH–activity curve of the immobilized enzyme shifted toward a lower pH comparedwith that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of theenzyme. The immobilized Naringinase was stable during storage at 4 ◦ C. No loss of activity was observedwhen the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency ofimmobilization was 120%, while soluble Naringinase afforded 82% efficacy for the hydrolysis of standardnaringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76%debittering efficiency. 2005 Society of Chemical Industry Keywords: woodchips; covalent binding; debittering; Naringinase; kinnow mandarin juice
Huinong Cai - One of the best experts on this subject based on the ideXlab platform.
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Optimizing culture medium for debittering constitutive enzyme Naringinase production by Aspergillus oryzae JMU316
2020Co-Authors: Dongxiao Chen, Tiangui Niu, Huinong CaiAbstract:The objective of this study was to investigate nutrient requirements for extracellular constitutive Naringinase production by Aspergillus oryzae JMU316. The one-factor-at-a-time method was used to determine the impact of different carbon and nitrogen sources on Naringinase production. Naringin exhibited the highest Naringinase activity compared to all other carbon sources and pomelo pericarp powder produced comparable Naringinase activity. Pomelo pericarp powder was selected as carbon source because it is a waste of fruit process, which means that it is a cheap resource and has additional environmental benefits. Peptone proved to be the most suitable nitrogen source for Naringinase production. Subsequently, the orthogonal matrix method was used to further optimize the concentration of pomelo pericarp powder, peptone, and minerals. The optimal concentration of the components were 15 g pomelo pericarp powder, 12 g peptone, 0.2 g CaCl 2 , 0.4 g NaCl, 2 g MgSO 4 ·7H 2 O and 1 g K 2 HPO 4 in 1 L distilled water for producing 408.28 IU/mL Naringinase activity. The effects of medium components on Naringinase were in the order of pomelo pericarp powder, K 2 HPO 4 , NaCl, peptone, CaCl 2 , MgSO 4 ·7H 2 O. This two-step optimization strategy used in this study can be widely applied to other microbial fermentation processes
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development and evaluation of an hplc method for accurate determinations of enzyme activities of Naringinase complex
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Anfeng Xiao, Feng Chen, Ya Qi Wang, Huinong CaiAbstract:An HPLC method that can separate naringin, prunin, and naringenin was used to help accurately measure the activities of Naringinase and its subunits (α-l-rhamnosidase and β-d-glucosidase). The activities of the Naringinase and β-d-glucosidase were determined through an indirect calculation of the naringenin concentration to avoid interference from its poor solubility. The measured enzymatic activities of the Naringinase complex, α-l-rhamnosidase, and β-d-glucosidase were the as same as their theoretical activities when the substrates’ (i.e., naringin or prunin) concentrations were 200 μg/mL, and the enzyme concentrations were within the range of 0.06–0.43, 0.067–0.53, and 0.15–1.13 U/mL, respectively. The β-d-glucosidase had a much higher Vmax than either Naringinase or α-l-rhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step of the enzyme reaction. The reliability of the method was finally validated through the repeatability test, indicating its feasibility for the determinat...
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Identification and characterization of Aspergillus aculeatus JMUdb058 for Naringinase production
Wei sheng wu xue bao = Acta microbiologica Sinica, 2013Co-Authors: Ni Hui, Anfeng Xiao, Huinong Cai, Feng Chen, Wenjin SunAbstract:OBJECTIVE A new Naringinase-producing strain, JMUdb058 was identified and characterized. METHODS The strain was identified by morphological observation and 28S rDNA homogeneous analysis. Naringinase was identified by monitoring the hydrolysis of naringin to prunin and naringenin using a reversed phase High Performance Liquid Chromatography (HPLC). The regulation of Naringinase expression was studied by measuring Naringinase activity of 11 different carbon sources and 7 nitrogen sources in shaking cultivation. The Naringinase-producing capacity was investigated in both solid-state fermentation and submerged fermentation. RESULTS The macro-morphology and micro-morphology of JMUdb058 corresponded to the characteristics of Aspergillus section Nigri Gams, and the 28S rDNA sequences showed homogeneity at 100% to Aspergillus aculeatus. Crude enzymes prepared by both submerged fermentation and solid-state fermentation could hydrolyze naringin to prunin and naringenin. In addition, the enzyme could remove naringin from citrus juice effectively. Carbon resources, including hesperidin, naringin, rutin and rhamnose, and organic nitrogen resources, i. e., tryptone, soybean meal, yeast extract and corn syrup were shown to express the Naringinase. The strain had an outstanding ability to yield Naringinase in the solid-state fermentation, which showed an alpha-L-rhamnosidase activity of 5903 U/gds by HPLC, and the Naringinase of 1939U/gds by HPLC and 72232 U/gds by Davis method. CONCLUSION It is the first time to report a stain of Aspergillus aculeatus can produce Naringinase, carbon source containing rhamnose groups are able to induce the enzyme expression. The stain JMUdb058 is a new microorganism source for high yield of Naringinase, in particularly by the solid-state fermentation.
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Purification and characterization of a Naringinase from Aspergillus aculeatus JMUdb058.
Journal of agricultural and food chemistry, 2013Co-Authors: Yuelong Chen, Feng Chen, Huinong CaiAbstract:A Naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This Naringinase had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric analysis revealed that the three larger subunits were β-D-glucosidases and that the smallest subunit was an α-L-rhamnosidase. The Naringinase and its α-L-rhamnosidase and β-D-glucosidase subunits all had optimal activities at approximately pH 4 and 50 °C, and they were stable between pH 3 and 6 and below 50 °C. This Naringinase was able to hydrolyze naringin, aesculin, and some other glycosides. The enzyme complex had a K(m) value of 0.11 mM and a k(cat)/K(m) ratio of 14,034 s(-1) mM(-1) for total Naringinase. Its α-L-rhamnosidase and β-D-glucosidase subunits had K(m) values of 0.23 and 0.53 mM, respectively, and k(cat)/K(m) ratios of 14,146 and 7733 s(-1) mM(-1), respectively. These results provide in-depth insight into the structure of the Naringinase complex and the hydrolyses of naringin and other glycosides.
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Purification and Characterization of a Naringinase from Aspergillus aculeatus JMUdb058
2013Co-Authors: Yuelong Chen, Feng Chen, Huinong CaiAbstract:A Naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This Naringinase had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric analysis revealed that the three larger subunits were β-d-glucosidases and that the smallest subunit was an α-l-rhamnosidase. The Naringinase and its α-l-rhamnosidase and β-d-glucosidase subunits all had optimal activities at approximately pH 4 and 50 °C, and they were stable between pH 3 and 6 and below 50 °C. This Naringinase was able to hydrolyze naringin, aesculin, and some other glycosides. The enzyme complex had a Km value of 0.11 mM and a kcat/Km ratio of 14 034 s–1 mM–1 for total Naringinase. Its α-l-rhamnosidase and β-d-glucosidase subunits had Km values of 0.23 and 0.53 mM, respectively, and kcat/Km ratios of 14 146 and 7733 s–1 mM–1, respectively. These results provide in-depth insight into the structure of the Naringinase complex and the hydrolyses of naringin and other glycosides
George W J Fleet - One of the best experts on this subject based on the ideXlab platform.
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Looking glass inhibitors : synthesis of a potent Naringinase inhibitor L-DIM [1,4 -dideoxy -1,4 -imino -L -mannitol), the enantiomer of DIM [1,4 -dideoxy -1,4 -imino -D -mannitol) a potent α-D-mannosidase inhibitor
Tetrahedron-asymmetry, 2007Co-Authors: Anders E. Håkansson, Luisa Guglielmini, Emma L. Evinson, Jeroen Van Ameijde, Graeme Horne, Robert J Nash, Atsushi Kato, George W J FleetAbstract:Abstract The synthesis of l -DIM [1,4-dideoxy-1,4-imino- l -mannitol] and of 1,4-imino- d - glycero - l - talo -heptitol from d - glycero - d - gulo -heptono-1,4-lactone depends on the use of pentan-3-one to form two five ring ketals as described by Burke, rather than the formation of one five ring and one six ring ketal formed with acetone. l -DIM, the enantiomer of the potent α- d -mannosidase inhibitor DIM [1,4-dideoxy-1,4-imino- d -mannitol] is a good inhibitor of Naringinase (an α- l -rhamnosidase) with a K i of 3.63 μM. 1,4-Imino- d - glycero - l - talo -heptitol is a moderate inhibitor of Naringinase.
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l swainsonine and other pyrrolidine inhibitors of Naringinase through an enzymic looking glass from d mannosidase to l rhamnosidase
Tetrahedron Letters, 1996Co-Authors: Benjamin G Davis, Robert J Nash, Andrew A Bell, Alison A Watson, Rhodri C Griffiths, George M Jones, Colin Smith, George W J FleetAbstract:Abstract The synthesis and inhibitory properties towards Naringinase (L-rhamnosidase) of L-(+)-swainsonine and of a number of more highly oxygenated analogues, and of some monocyclic equivalents, are reported. L-(+)-swainsonine and 1,4,6-trideoxy-1,4-imino-L-mannitol are powerful and specific inhibitors of Naringinase.
Feng Chen - One of the best experts on this subject based on the ideXlab platform.
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comparative analyses of aromas of fresh Naringinase treated and resin absorbed juices of pummelo by gc ms and sensory evaluation
Flavour and Fragrance Journal, 2015Co-Authors: Peng Hong, Anfeng Xiao, Feng Chen, Hao Sun, Yan Hong ChenAbstract:Resin adsorption and Naringinase treatment represent two major methods for debittering naringin in citrus juices, but their effects on the aroma profiles of citrus juices have not been intensively investigated yet. In this study, the volatiles of the fresh, Naringinase-treated and resin-absorbed juices of pummelo were investigated by gas chromatography coupled with a mass spectrometer (GC-MS) and sensory evaluation. The fresh juice was analyzed to have 24 volatiles, among which (E)-3-hexen-1-ol and 1-hexanol had the highest concentrations. After the Naringinase treatment, the juice showed 32 volatiles, among which (Z)- and (E)-linalool oxides were the most dominant in concentration; while the resin adsorption captured most of the volatiles. Principal component analysis (PCA) showed that Naringinase treatment led to an increase in some compounds, particularly aldehydes, whereas resin adsorption decreased the amounts of a lot of volatile compounds. The sensory evaluation demonstrated the fresh and Naringinase-treated juices had similar aroma profiles, whereas the resin-adsorbed juice had a much weaker aroma intensity than its counterparts. These results indicated that the Naringinase treatment is more desirable than the resin adsorption for debittering citrus juice because the Naringinase treatment could maintain the aroma profile close to the original, fresh juice. Copyright © 2015 John Wiley & Sons, Ltd.
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development and evaluation of an hplc method for accurate determinations of enzyme activities of Naringinase complex
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Anfeng Xiao, Feng Chen, Ya Qi Wang, Huinong CaiAbstract:An HPLC method that can separate naringin, prunin, and naringenin was used to help accurately measure the activities of Naringinase and its subunits (α-l-rhamnosidase and β-d-glucosidase). The activities of the Naringinase and β-d-glucosidase were determined through an indirect calculation of the naringenin concentration to avoid interference from its poor solubility. The measured enzymatic activities of the Naringinase complex, α-l-rhamnosidase, and β-d-glucosidase were the as same as their theoretical activities when the substrates’ (i.e., naringin or prunin) concentrations were 200 μg/mL, and the enzyme concentrations were within the range of 0.06–0.43, 0.067–0.53, and 0.15–1.13 U/mL, respectively. The β-d-glucosidase had a much higher Vmax than either Naringinase or α-l-rhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step of the enzyme reaction. The reliability of the method was finally validated through the repeatability test, indicating its feasibility for the determinat...
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Identification and characterization of Aspergillus aculeatus JMUdb058 for Naringinase production
Wei sheng wu xue bao = Acta microbiologica Sinica, 2013Co-Authors: Ni Hui, Anfeng Xiao, Huinong Cai, Feng Chen, Wenjin SunAbstract:OBJECTIVE A new Naringinase-producing strain, JMUdb058 was identified and characterized. METHODS The strain was identified by morphological observation and 28S rDNA homogeneous analysis. Naringinase was identified by monitoring the hydrolysis of naringin to prunin and naringenin using a reversed phase High Performance Liquid Chromatography (HPLC). The regulation of Naringinase expression was studied by measuring Naringinase activity of 11 different carbon sources and 7 nitrogen sources in shaking cultivation. The Naringinase-producing capacity was investigated in both solid-state fermentation and submerged fermentation. RESULTS The macro-morphology and micro-morphology of JMUdb058 corresponded to the characteristics of Aspergillus section Nigri Gams, and the 28S rDNA sequences showed homogeneity at 100% to Aspergillus aculeatus. Crude enzymes prepared by both submerged fermentation and solid-state fermentation could hydrolyze naringin to prunin and naringenin. In addition, the enzyme could remove naringin from citrus juice effectively. Carbon resources, including hesperidin, naringin, rutin and rhamnose, and organic nitrogen resources, i. e., tryptone, soybean meal, yeast extract and corn syrup were shown to express the Naringinase. The strain had an outstanding ability to yield Naringinase in the solid-state fermentation, which showed an alpha-L-rhamnosidase activity of 5903 U/gds by HPLC, and the Naringinase of 1939U/gds by HPLC and 72232 U/gds by Davis method. CONCLUSION It is the first time to report a stain of Aspergillus aculeatus can produce Naringinase, carbon source containing rhamnose groups are able to induce the enzyme expression. The stain JMUdb058 is a new microorganism source for high yield of Naringinase, in particularly by the solid-state fermentation.
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Purification and characterization of a Naringinase from Aspergillus aculeatus JMUdb058.
Journal of agricultural and food chemistry, 2013Co-Authors: Yuelong Chen, Feng Chen, Huinong CaiAbstract:A Naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This Naringinase had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric analysis revealed that the three larger subunits were β-D-glucosidases and that the smallest subunit was an α-L-rhamnosidase. The Naringinase and its α-L-rhamnosidase and β-D-glucosidase subunits all had optimal activities at approximately pH 4 and 50 °C, and they were stable between pH 3 and 6 and below 50 °C. This Naringinase was able to hydrolyze naringin, aesculin, and some other glycosides. The enzyme complex had a K(m) value of 0.11 mM and a k(cat)/K(m) ratio of 14,034 s(-1) mM(-1) for total Naringinase. Its α-L-rhamnosidase and β-D-glucosidase subunits had K(m) values of 0.23 and 0.53 mM, respectively, and k(cat)/K(m) ratios of 14,146 and 7733 s(-1) mM(-1), respectively. These results provide in-depth insight into the structure of the Naringinase complex and the hydrolyses of naringin and other glycosides.
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Purification and Characterization of a Naringinase from Aspergillus aculeatus JMUdb058
2013Co-Authors: Yuelong Chen, Feng Chen, Huinong CaiAbstract:A Naringinase from Aspergillus aculeatus JMUdb058 was purified, identified, and characterized. This Naringinase had a molecular mass (MW) of 348 kDa and contained four subunits with MWs of 100, 95, 84, and 69 kDa. Mass spectrometric analysis revealed that the three larger subunits were β-d-glucosidases and that the smallest subunit was an α-l-rhamnosidase. The Naringinase and its α-l-rhamnosidase and β-d-glucosidase subunits all had optimal activities at approximately pH 4 and 50 °C, and they were stable between pH 3 and 6 and below 50 °C. This Naringinase was able to hydrolyze naringin, aesculin, and some other glycosides. The enzyme complex had a Km value of 0.11 mM and a kcat/Km ratio of 14 034 s–1 mM–1 for total Naringinase. Its α-l-rhamnosidase and β-d-glucosidase subunits had Km values of 0.23 and 0.53 mM, respectively, and kcat/Km ratios of 14 146 and 7733 s–1 mM–1, respectively. These results provide in-depth insight into the structure of the Naringinase complex and the hydrolyses of naringin and other glycosides
Maria H L Ribeiro - One of the best experts on this subject based on the ideXlab platform.
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high affinity water soluble system for efficient Naringinase immobilization in polyvinyl alcohol dimethyl sulfoxide lens shaped particles
Journal of Molecular Recognition, 2012Co-Authors: Mario A P Nunes, Pedro Fernandes, Maria H L RibeiroAbstract:Polyvinyl alcohol (PVA) is a water-soluble, biocompatible and biodegradable synthetic polymer whose application in the immobilization of biological agents for use in biocatalysis has shown promising results. This study aimed to investigate and optimize the immobilization of Naringinase from Penicillium decumbens in PVA networks, targeting for the hydrolysis of naringin. Variables such as the most suitable cross-linker, catalyst, inorganic salt, co-solvents and solidification process were identified as key issues for PVA-based methods to form lens-shaped particles, while retaining high enzyme activity and stability. Major improvements were established for better and more reproducible immobilization conditions, namely, by designing a new immobilization apparatus to produce uniform lens-shaped particles. The common problems of PVA-based entrapment were significantly mitigated, through the use of selected cross-linker, glutaraldehyde (GA), and co-solvent, dimethyl sulfoxide (DMSO), which decreased the toxicity of the immobilization process and allowed the control of membrane porosity, respectively. The relevance of DMSO and GA and their interaction and effect on the swelling ratio, encapsulation efficiency and residual activity of PVA biocatalysts were established. The immobilization of Naringinase in PVA under a DMSO concentration of 60%, cross-linked with 1% GA, and particle lens size of 3.5–4.0 mm, width of 100–300 µm and average particle volume of 12.5 ± 0.92 µL, allowed an encapsulation efficiency of 98.6% and an average residual activity of 87% ± 3.6%. The kinetic characterization of the immobilized Naringinase showed no changes in pH profile, whereas hydrolytic activity increased up to 60 °C. Immobilization in PVA/DMSO/GA lens-shaped particles enhanced the storage stability of Naringinase. Moreover, these Naringinase bio-immobilizates retained a conversion rate higher than 78% after 23 runs. Copyright © 2012 John Wiley & Sons, Ltd.
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enzymatic synthesis of the flavone glucosides prunin and isoquercetin and the aglycones naringenin and quercetin with selective α l rhamnosidase and β d glucosidase activities of Naringinase
Enzyme Research, 2011Co-Authors: Helder Vilareal, Antonio J Alfaia, Rosario M Bronze, Antonio R T Calado, Maria H L RibeiroAbstract:The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished through enzymatic catalysis. Naringinase is an enzyme complex, expressing both α-L-rhamnosidase and β-D-glucosidase activities, with application in glycosides hydrolysis. To produce monoglycosylated flavonoids with Naringinase, the expression of β-D-glucosidase activity is not desirable leading to the need of expensive methods for α-L-rhamnosidase purification. Therefore, the main purpose of this study was the inactivation of β-D-glucosidase activity expressed by Naringinase keeping α-L-rhamnosidase with a high retention activity. Response surface methodology (RSM) was used to evaluate the effects of temperature and pH on β-D-glucosidase inactivation. A selective inactivation of β-D-glucosidase activity of Naringinase was achieved at 81.5°C and pH 3.9, keeping a very high residual activity of α-L-rhamnosidase (78%). This was a crucial achievement towards an easy and cheap production method of very expensive flavonoids, like prunin and isoquercetin starting from naringin and rutin, respectively.
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α rhamnosidase and β glucosidase expressed by Naringinase immobilized on new ionic liquid sol gel matrices activity and stability studies
Journal of Biotechnology, 2011Co-Authors: Helder Vilareal, Antonio J Alfaia, Emilia M Rosa, Joao N Rosa, Pedro M P Gois, Anta Nio R T Calado, Maria H L RibeiroAbstract:Abstract Novel ionic liquid (IL) sol–gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α - l -rhamnosidase and β - d -glucosidase activities expressed by Naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol–gel matrices and used on Naringinase immobilization. The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α -rhamnosidase expressed by Naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM] > [BMIM] > [EMIM] > [C 2 OHMIM] > [BIM] and [OMIM] ≈ [E 2 -MPy] ≫ [E 3 -MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α -rhamnosidase efficiencies: [BMIM]BF 4 ≫ [C 2 OHMIM]BF 4 ≫ [BIM]BF 4 . Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E 3 -MPy] and [E 2 -MPy] allowed an impressive difference in the α -rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α -rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF 6 − , BF 4 − and Tf 2 N − ) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL. The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and Naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf 2 N] and TMOS/Glycerol@[C 2 OHMIM][PF 6 ] and on TMOS/Glycerol. An improved stability and efficiency of α - l -rhamnosidase and β -glucosidase expressed by encapsulated Naringinase on TMOS/Glycerol@[OMIM][Tf 2 N] and TMOS/Glycerol@[C 2 OHMIM][PF 6 ] were achieved. In addition to these advantageous, with ILs as sol–gel templates, environmental friendly processes can be implemented.
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Enzymatic Synthesis of the Flavone Glucosides, Prunin and Isoquercetin, and the Aglycones, Naringenin and Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase
Hindawi Limited, 2011Co-Authors: Hélder Vila-real, Antonio J Alfaia, Rosario M Bronze, Antonio R T Calado, Maria H L RibeiroAbstract:The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished through enzymatic catalysis. Naringinase is an enzyme complex, expressing both α-L-rhamnosidase and β-D-glucosidase activities, with application in glycosides hydrolysis. To produce monoglycosylated flavonoids with Naringinase, the expression of β-D-glucosidase activity is not desirable leading to the need of expensive methods for α-L-rhamnosidase purification. Therefore, the main purpose of this study was the inactivation of β-D-glucosidase activity expressed by Naringinase keeping α-L-rhamnosidase with a high retention activity. Response surface methodology (RSM) was used to evaluate the effects of temperature and pH on β-D-glucosidase inactivation. A selective inactivation of β-D-glucosidase activity of Naringinase was achieved at 81.5∘C and pH 3.9, keeping a very high residual activity of α-L-rhamnosidase (78%). This was a crucial achievement towards an easy and cheap production method of very expensive flavonoids, like prunin and isoquercetin starting from naringin and rutin, respectively
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Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase
2011Co-Authors: The Aglycones, Antonio J Alfaia, Rosario M Bronze, Antonio R T Calado, Hélder Vila-real, Maria H L RibeiroAbstract:Copyright © 2011 Hélder Vila-Real et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The production of flavonoid glycosides by removing rhamnose from rutinosides can be accomplished through enzymatic catalysis. Naringinase is an enzyme complex, expressing both α-L-rhamnosidase and β-D-glucosidase activities, with application in glycosides hydrolysis. To produce monoglycosylated flavonoids with Naringinase, the expression of β-D-glucosidase activity is not desirable leading to the need of expensive methods for α-L-rhamnosidase purification. Therefore, the main purpose of this study was the inactivation of β-D-glucosidase activity expressed by Naringinase keeping α-L-rhamnosidase with a high retention activity. Response surface methodology (RSM) was used to evaluate the effects of temperature and pH on β-D-glucosidase inactivation. A selective inactivation of β-D-glucosidase activity of Naringinase was achieved at 81.5 ◦ C and pH 3.9, keeping a very high residual activity of α-L-rhamnosidase (78%). This was a crucial achievement towards an easy and cheap production method of very expensive flavonoids, like prunin and isoquercetin starting from naringin and rutin, respectively. 1
