Determination of para-synephrine and meta-synephrine positional isomers in bitter orange-containing dietary supplements by LC/UV and LC/MS/MS

Dietary supplements that contain bitter orange (Citrus aurantium) fruit as an integrated component have rapidly replaced ephedra-containing dietary supplements for use as weight loss products. However, the safety of bitter orange-containing supplements has been questioned because synephrine, an adrenergic alkaloid and a key component of bitter orange fruit, has potential adverse health effects. Conflicting reports have stated that synephrine exists as the para (p) and/or meta (m) positional isomers in some bitter orange-containing supplements and this is problematic because the p- and m-isomers have distinctly different pharmacological and metabolic activities. Two liquid chromatographic (LC) methods have been developed for the baseline separation and quantitation of p- and m-synephrine in bitter orange-containing supplements. An isocratic LC method that utilizes ultraviolet (UV) absorbance detection and a gradient LC method that utilizes tandem mass spectrometry (MS/MS) detection were optimized for separation of the isomers within a run time of 25 min. Terbutaline was utilized as an internal standard compound in both LC methods. The LC/UV and LC/MS/MS methods demonstrated limits of quantitation (LOQs) for synephrine of ≈30 ng (on-column) and ≈0.02 ng (on-column), respectively, and each method exhibited analytical linearity over three orders of magnitude. Both LC methods were used to evaluate the synephrine levels in a limited selection of commercially available bitter orange-containing supplements. Significantly, m-synephrine was not detected in any of the tested dietary supplements.     

Isolation and structural determination of a novel flavonol triglycoside and 7 compounds from the leaves of oriental raisin tree (Hovenia dulcis) and their antioxidative activity

The hot water and ethanol extracts of oriental raisin tree (Hovenia dulcis Thunb) leaves showed DPPH radical scavenging activities. Antioxidants were purified and isolated from hot water and ethanol extracts by various column chromatographic procedures with the guided assay of DPPH radical scavenging. The structure of a novel flavonol triglycoside was determined to kaempferol 3-O-α-l-rhamnopyranoside-7-O-[α-d-glucopyranosyl(1→3)-α-l-rhamnopyranoside] (4). In addition, 7 known compounds were identified as caffeine (1), kaempferol 3,7-O-α-l-dirhamnopyranoside (2), kaempferol 3-O-α-l-rhamnopyranosyl( 1→6)-O-β-d-glucopyranosyl(1→2)-O-β-d-glucopyranoside (3), E-3-carboxy-2-petenedioate 5-methyl ester (5), quercetin 3-O-α-l-rhamnopyranoside (6), kaempferol 3-O-α-l-rhamnopyranoside (7), and quercetin 3-O-β-d-glucopyranoside (8). Compound 1–3 and 5–8 were newly identified in this plant. Quercetin glycosides (5, 7) showed higher DPPH radical scavenging activity than other compounds.     

Determination of <Emphasis Type="SmallCaps">d</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-Amino Acids in Collagen from Pig and Cod Skins by UPLC Using Pre-column Fluorescent Derivatization

A simple and sensitive analytical method for the quantification of d,l-amino acids by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection is described. The reaction of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] with d,l-hydroxyproline (Hyp), glycine (Gly), d,l-aspartic acid (Asp), and d,l-proline (Pro) effectively proceeds at 55 °C for 20 min in the presence of 3% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). The mixture was simultaneously separated within 20 min on an ACQUITY UPLCTM BEH C18 (1.7 μm, 100 mm?×?2.1 mm i.d.) by gradient elutions using water–acetonitrile containing 0.2% formic acid as the mobile phase. Peak resolution was in the range of 1.62 (d,l-Asp), 2.99 (d,l-Pro), and 6.74 (d,l-Hyp). A good linearity was achieved from the calibration curves (r²?>?0.9984) in the range of 1.0~100 pmol; the limit of detection (S/N?=?3) was 42.0–250 fmol, the inter-day and intra-day assay precisions were all less than 6.23%, and the mean recoveries (%) of the d,l-amino acids spiked in the collagen from pig and dried cod skins were 87.58–107.41%. The derivatives of the free d,l-amino acids in the collagen were successfully identified by the proposed procedure. To the best of our knowledge, for the first time, these three kinds of d-amino acids, which were d-Asp, d-Pro, and d-Hyp, were detected in the collagen samples.     

Enzymatische Bestimmung von S?uren in Wein I. Mitteilung ?pfels?ure, Milchs?ure, Citronens?ure

Zusammenfassung Nach Ausschaltung von Störungsmöglichkeiten durch Farbstoffe und Gerbstoffe kann die enzymatische Bestimmung vonl-Malat,l-Lactat und Citrat in Wein einwandfrei durchgeführt werden. Äpfelsäure kommt in Wein nur in derl-Form vor, Lactat dagegen sowohl in derd- als auch in derl-Form. Die beiden Antipoden stehen in keinem bestimmten Verhältnis zueinander, jedoch überwiegt bei hohen Milchsäuregehalten der Anteil derl-Form.

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Enzymatic determination of acids in wine I. Malic-, lactic- and citric-acid

Summary The enzymatic determination ofl-malate with MDH,l-lactate with LDH and citrate with citrate — lyase was carried out in more than 100 samples of white and red wine, after elimination of interfering substances, such as tans and colouring matters. The contents ofl-malate and citrate agreed very well with the results, found by a fractionation method on silica gel. Comparing the content of total lactate, found by the latter method, with the results of enzymatic determination ofl-lactate, it was obvious, thatd- andl-compounds occur in a different ratio, but at high lactic acid concentrations, however, the level of thel-form is higher.

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Auszug aus der Dissertation vonSiegfried Looser: Beitrag zur enzymatischen Analyse von organischen Säuren und Kohlenhydraten in Lebensmitteln. Technische Hochschule München 1968.     

Structure elucidation and immunological activity of a novel pectic polysaccharide from the stems of Avicennia marina

The structure of HAM-3-IIb-II, an acidic pectic polysaccharide in Avicennia marina, was characterized in detail and the immunological activity was investigated in this study. The results showed that HAM-3-IIb-II contained Rha, Ara, Gal, Glc, and GalA in a molar ratio of 1:2.8:2.4:6.1 and that HAM-3-IIb-II is a type I rhamnogalacturonan with branches of arabinose chains, galactosyl chains, and arabinogalactosyl chains. The backbone of HAM-3-IIb-II is mainly composed of disaccharide repeat units, →4)-α-d-GalA-(1 → 2)-α-l-Rhap-(1→. These repeats are predominantly linked to the O-4 of 1,2,4-linked α-l-Rhap in the branches of arabinose chains, galactosyl chains, and arabinogalactosyl chains. HAM-3-IIb-II had a significant effect on lipopolysaccharide-induced B lymphocyte proliferation, but it had little effect on concanavalin A-induced T lymphocyte proliferation compared with the control. This study provides a foundation for the exploration and utilization of A. marina in the future.     

Extraction and pharmacological properties of bioactive compounds from longan (Dimocarpus longan Lour.) fruit — A review

Mature longan (Dimocarpus longan Lour.) fruit has a succulent, edible and white aril, which has gained popularity as an exotic fruit in temperate regions. It is prized on the international market resulting in an increased production with significant contributions to local economic development. Longan fruit contains significant amounts of bioactive compounds such as corilagin, ellagic acid and its conjugates, 4-O-methylgallic acid, flavone glycosides, glycosides of quercetin and kaempferol, ethyl gallate 1-??-O-galloyl-d-glucopyranose, grevifolin and 4-O-??-l-rhamnopyranosyl-ellagic acid. The fruit has been used in the traditional Chinese medicinal formulation, serving as an agent in relief of neural pain and swelling. The application of ultrasonic-assisted extraction or high pressure-assisted extraction greatly increases the yield from longan pericarp or seeds. In recent years, some pharmacological activities such as anti-tyrosinase, anti-glycated and anticancer activities, and memory-enhancing effects of longan aril, pericarp or seed extract have been found, implicating a significant contribution to human health. Regarding the increasing cultivation area and increasing quantity of longan fruit in the world, further utilization of this fruit is expected in an effort to use more efficiently the inherent bioactive compounds. The paper reviews the recent advances in the extraction and pharmacological activities of bioactive compounds from longan fruit. Some novel pharmacological potential of longan fruit is also discussed in this paper.     

Luminescent enzymatic flow sensor for d- and l-lactate assay in beer

 A bioluminescent flow sensor, previously developed for the determination of both d- and l-lactate in clinical samples, was utilized to carry out the same assay in beer. The sensor monitored the reduced form of nicotinamide adenine dinucleotide, produced by nylon-immobilized d- and l-lactate dehydrogenase, by means of bacterial bioluminescent enzymes immobilized on a separate nylon coil. The preparation of beer samples was very simple as only a modification of pH and a dilution were necessary. The recoveries ranged from 91% to 104%, and the relative standard deviations at the 1 mmol 1^–1 level were 4.6% and 6.7% for l- and d-lactate respectively. The response was linear in the range 0.1–10 mmol 1^–1 for both d- and l-lactate. The total amount of lactate determined by bioluminescent biosensor (x) and by HPLC (y) showed a very good correlation (y=0.654 x+88.1, n=29, r=0.918). The flow injection system developed allowed the determination of not only the total but also the individual contents of d- and l-lactate in beer, and the timely discovery of the unwanted presence of lactic acid bacteria. Received: 14 January 1999 / Revised version: 29 April 1999     

Prebiotic effects of structurally identified galacto-oligosaccharides produced by β-galactosidase from Aspergillus oryzae

Novel galacto-oligosaccharides were produced by β-galactosidase from Aspergillus oryzae using lactose, and their structural characteristics and prebiotic effects were examined. Highly purified oligosaccharide fraction (HP) was prepared from a crude one (low purified, LP) by gel-filtration on Biogel P-2 column, which was further purified into S1 and S2 fractions by prep-HPLC. S1 and S2 were comprised of galactose (Gal) and glucose (Glc) in the ratio of 2 to 1. ESI-MS-MS and methylation analysis indicated that S1 and S2 were trisaccharides with structures of β-d-Galp-(1,6)-β-d-Galp-(1,4)-β-d-Glcp and β-d-Galp-(1,3)-β-d-Galp-(1,4)-β-d-Glcp, respectively. Herein, LP and HP were used as the carbon sources for determining the prebiotic activity score of probiotics including Lactobacillus and Bifidobacterium species. LP and HP at 1% and 2% (w/v) were observed at all positive scores on several probiotics, especially, B. infantis ATCC 15697 at the 2% level of HP (p<0.05). Consequently, structurally identified trisaccharides of HP can significantly enhance the growth of B. infantis.     

High-performance liquid chromatographic analysis of biogenic amines in pharmaceutical products containing Citrus aurantium

A reverse-phase (RP)-HPLC method is reported for determining L-tyrosine, p-octopamine, synephrine, tyramine and hordenine as chemical markers of the species Citrus aurantium in raw material, dry extracts and phytotherapeutic herbal formulations. Using RP-HPLC with diode array detection (DAD) and gradient elution, the amines were determined in 12 different products from different Brazilian states labelled as containing C. aurantium. The presence of the amines was confirmed by mass spectrometry using electrospray ionisation (ESI-MS/MS). This RP-HPLC method allowed the separation of the amines from complex mixtures containing caffeine, ephedrine, salicin and other raw materials (e.g. Garcinia camboja, Phaseolus vulgaris, Caralluma fimbriata, Cassia nomane, Ephedra sp. and Cordia ecalyculata). The method proved useful and selective for inspecting herbal medicines containing p-synephrine and structural analogues. The herbal products analysed had a p-synephrine content ranging from 0.005 to 4.0% (w/w).     

Chiral amino acid analysis of vinegars using gas chromatography – selected ion monitoring mass spectrometry

 Twenty-five vinegars were examined quantitatively for their content of free amino acid (AA) enantiomers using chiral gas chromatography/mass spectrometry. Vinegars manufactured from grape must contained l-proline (l-Pro) as the major AA. Balsamic vinegars (aceto balsamico di Modena), with one exception, contained the highest amounts of l-AAs (861–2000 mg l^–1) as well as d-AAs (46–361 mg l^–1). The amounts of d-Pro and d-alanine (d-Ala) increased in the course of maturation. Sherry vinegars had a AA pattern similar to that of balsamic vinegars but with much lower amounts: concentrations of l-AAs were 244 mg l^–1 and 456 mg l^–1 and of d-AAs were 18 mg l^–1 and 19 mg l^–1. The l-AA content of cider vinegars was very low (34 mg l^–1 and 44 mg l^–1) and only traces of d-AAs (<2 mg l^–1) were found. In spirit vinegars few d-AAs and low amounts of most l-AAs were detected, with the exception of l-glutamic acid (l-Glu) (210–847 mg l^–1), probably added as a flavour enhancer. The AA content of spirit vinegars blended with wine vinegar was influenced by the portion of wine vinegar added. Rice vinegars had concentrations of l-AAs from as low as 36 mg l^–1 to as high as 6860 mg l^–1, and the concentrations of d-AAs ranged from 6 mg l^–1 to 531 mg l^–1. All vinegars declared as "produced by microbial fermentation" contained d-Ala, d-aspartic acid, and d-Glu as typical bacterial markers. From the data it is concluded that chiral AA analysis can be used to distinguish among fermented and synthetic vinegars and to identify raw materials used for their production. In particular, the amount of d-Pro can be used as proof of the maturation process of balsamic vinegar. Received: 11 December 1997 / Revised version: 12 May 1998     

Chiral amino acid analysis of vinegars using gas chromatography – selected ion monitoring mass spectrometry

 Twenty-five vinegars were examined quantitatively for their content of free amino acid (AA) enantiomers using chiral gas chromatography/mass spectrometry. Vinegars manufactured from grape must contained l-proline (l-Pro) as the major AA. Balsamic vinegars (aceto balsamico di Modena), with one exception, contained the highest amounts of l-AAs (861–2000 mg l^–1) as well as d-AAs (46–361 mg l^–1). The amounts of d-Pro and d-alanine (d-Ala) increased in the course of maturation. Sherry vinegars had a AA pattern similar to that of balsamic vinegars but with much lower amounts: concentrations of l-AAs were 244 mg l^–1 and 456 mg l^–1 and of d-AAs were 18 mg l^–1 and 19 mg l^–1. The l-AA content of cider vinegars was very low (34 mg l^–1 and 44 mg l^–1) and only traces of d-AAs (<2 mg l^–1) were found. In spirit vinegars few d-AAs and low amounts of most l-AAs were detected, with the exception of l-glutamic acid (l-Glu) (210–847 mg l^–1), probably added as a flavour enhancer. The AA content of spirit vinegars blended with wine vinegar was influenced by the portion of wine vinegar added. Rice vinegars had concentrations of l-AAs from as low as 36 mg l^–1 to as high as 6860 mg l^–1, and the concentrations of d-AAs ranged from 6 mg l^–1 to 531 mg l^–1. All vinegars declared as "produced by microbial fermentation" contained d-Ala, d-aspartic acid, and d-Glu as typical bacterial markers. From the data it is concluded that chiral AA analysis can be used to distinguish among fermented and synthetic vinegars and to identify raw materials used for their production. In particular, the amount of d-Pro can be used as proof of the maturation process of balsamic vinegar. Received: 11 December 1997 / Revised version: 12 May 1998     

Estrogen-like activity of Adenophora triphylla var. japonica water extract in MCF-7 cells

This study investigated the estrogen-like activity of Adenophora triphylla var. japonica, which has been shown to have pharmacological activities. Water extracts from A. triphylla (ATWE) could bind to estrogen receptors and displaced binding of E2 to ERα and ERβ. ATWE stimulated the proliferation of estrogen receptor-positive MCF-7 cells in a dose dependent manner (p<0.05). ATWE induced proliferation was blocked by the addition of the estrogen antagonist ICI 182,780 (p<0.05). Moreover, ATWE treatment caused a significant increase in mRNA expression of estrogen-responsive genes (pS2, PR, and cathepsin D; p<0.05). These results indicate that A. triphylla has estrogen-like activity and could be used to improve estrogen deficiency-related menopausal symptoms or diseases in postmenopausal women.     

Synthesis of oligosaccharides with lactose and N-acetylglucosamine as substrates by using β-d-galactosidase from Bacillus circulans

In the present study, β-d-galactosidase from Bacillus circulans was proved to be a suitable biocatalyst for the production of N-acetyl-oligosaccharides with lactose and N-acetylglucosamine (GlcNAc) as biocatalyst. During the hydrolysis of lactose, apart from no ultraviolet absorption oligosaccharides such as β-d-Galp-(1 → 6)-d-Glcp (6′-allolactose) and β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-Glcp (4′-galactosyl-lactose), the formation of four N-acetyl-oligosaccharides was followed by high-performance liquid chromatography with a diode-array detector. The four N-acetyl-oligosaccharides were isolated from the reaction mixture and identified to be as β-d-Galp-(1 → 4)-d-GlcpNAc (LacNAc, I), β-d-Galp-(1 → 6)-d-GlcpNAc (allo-LacNAc, II), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (III), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (IV) by authentic standards and the spike technique or high-resolution mass spectrometry with an electrospray ionization source and nuclear magnetic resonance spectroscopy. Furthermore, the effects of synthetic conditions including reaction temperature, concentration of substrate, molar ratio of donor/acceptor and enzyme concentration on the formation of N-acetyl-oligosaccharides were examined. We found that the optimal synthetic conditions were different for production of oligosaccharides with β-(1 → 4) linkages and β-(1 → 6) linkage. The optimal reaction conditions for I, III and IV were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 1.0 U/mL of enzyme. Under such conditions, the N-acetyl-oligosaccharides formed were composed of 28.75% of I, 2.29% of II, 9.47% of III and 5.67% of IV. On the other hand, suitable reaction conditions found for II were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 2.0 U/mL of enzyme.     

Bacterial reporter strains for d-xylose content analysis in arabinoxylans

A pair of ice nucleation and fluorescence reporter strains induced specifically by d-xylose were developed and optimized, to monitor d-xylose content in hydrolyzed arabinoxylans samples. Reporter strains were developed by fusing the Escherichia coli xylose isomerase promoter P _xylA to the promoterless native inaZ gene of Pseudomonas syringae and synthetic gfp gene of Aequorea victoria and transferring final constructs to E. coli DH5α. Because of relatively low initial response toward the target analyte, signal improvement by promoter region length adjustment has been implemented. In both cases, this approach proved to be successful, although the manner in which reporter strains have responded to it was dissimilar. The specificity and quantitative response of obtained reporter strains have been confirmed, and the response ranges for NIxylA100 and gfpxylA300 have been established as 9 × 10^?6–9 × 10^?1 g l^?1 and 9 × 10^?3–9 g l^?1, respectively. The performance of developed reporter strains has been assessed in comparison with high-performance anion-exchange chromatography and demonstrated 15–18 % difference between data obtained with reporter strains and chromatography analysis, which for microbial sensors is an acceptable dissimilarity. The developed microbial reporter strains present an alternative for other analytical methods used for monosaccharide quantification and enable quantification of d-xylose in arabinoxylans samples.     

Chemical composition,antioxidant and antibacterial activities of the essential oils isolated from Tunisian Thymus capitatus Hoff. et Link.

The chemical composition, antioxidant and antibacterial activities of essential oils isolated by hydrodistillation from the aerial parts of Tunisian Thymus capitatus Hoff. et Link. during the different phases of the plant development, and from different locations, were evaluated. The chemical composition was analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The main components of the essential oils were carvacrol (62–83%), p-cymene (5–17%), γ-terpinene (2–14%) and β-caryophyllene (1–4%). The antioxidant activity of the oils (100–1000 mg l⁻¹) was assessed by measurement of metal chelating activity, the reductive potential, the free radical scavenging (DPPH) and by the TBARS assay. The antioxidant activity was compared with that of synthetic antioxidants: butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Both the essential oils and BHA and BHT showed no metal chelating activity. Although with the other methodologies, there was a general increase in the antioxidant activity, with increasing oil concentration, maxima being obtained in the range of 500 and 1000 mg l⁻¹ for flowering and post-flowering phase oils. Major differences were obtained according to the methodology of antioxidant capacity evaluation. Antibacterial ability of Th. capitatus essential oils was tested by disc agar diffusion against Bacillus cereus, Salmonella sp., Listeria innocua, four different strains of Staphylococus aureus (C15, ATCC25923, CFSA-2) and a multi-resistant form of S. aureus (MRSA-2). Antibacterial properties were compared to synthetic antibiotics. Higher antibacterial activity was observed with the flowering and the post-flowering phase essential oils.     

Effect of l-lysine on the physicochemical properties of pork sausage

Effects of l-lysine on physicochemical properties of pork sausage were investigated. WHC values significantly increased using 0.8% l-lysine, but significantly decreased at 0.4% l-lysine (p<0.05), compared with controls. l-Lysine increased pH values and a* values, but significantly decreased CL, cohesiveness, L* values, and b* values (p<0.05), compared with controls. Addition of 0.4% l-lysine significantly (p<0.05) increased hardness, springiness, and chewiness values, compared with controls. SEM analysis showed that addition of 0.8% l-lysine induced formation of a smoother, more compact, and more uniform gel matrix, compared with controls. DSC analysis indicated that addition of 0.8% l-lysine significantly (p<0.05) increased thermal transition temperatures and enthalphy values, compared with controls, showing that interactions between l-lysine and myofibrillar proteins affected the properties of pork sausages.     

Liquid chromatographic determination of caffeine and adrenergic stimulants in food supplements sold in Brazilian e-commerce for weight loss and physical fitness

Methyl-xanthines and adrenergic stimulants, such as caffeine and synephrine, are commonly added to food supplements due to their stimulating and thermogenic effects. In addition, the abusive consumption of food supplements with ergogenic and aesthetic purposes has been observed worldwide. This work describes the study of caffeine, p-synephrine, hordenine, octopamine, tyramine, ephedrine and salicin as stimulants in dietary supplements marketed in Brazil for weight loss and physical fitness claims. A total of 94 different products were acquired from 30 Brazilian websites. Thus, the sampling of marketed supplements was performed in virtual commerce (e-commerce) with claims of weight loss, appetite reduction, fat burning and metabolism acceleration. The developed analytical method involved the separation of the stimulants by HPLC with diode array detection (HPLC-DAD) by using a gradient elution of flow rate (0.7–2.5 ml min^?1) and mobile phase composition (0.1% H₃PO₄/methanol). The validated method was applied to the study of 46 dietary supplements. Caffeine, p-synephrine and ephedrine were found to be present as stimulants in 52% of the studied samples marketed as encapsulated or bulk forms. Caffeine was found to be present in concentrations that represent doses from 25.0 to 1476.7 mg day^–1. Synephrine was found in concentrations that represent doses from 59.1 to 127.0 mg day^–1. Ephedrine was found to be associated with caffeine in one formulation at a concentration representing a 26.1 mg day^–1 dosage.     

Effect of sulphur dioxide on the activity of trypsin

The effects of sulphur(IV) oxospecies [S(IV)] on the rate of hydrolysis of N-benzoyl-d,l-arginine-p-nitroanilide by bovine trypsin are described. The enzyme is partly inhibited in a biphasic process: a rapid reduction of activity in a process capable of being saturated by S(IV) is followed by a change, on the timescale of hours, whose rate is independent of the concentration of S(IV).     

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.     

A novel α-l-rhamnosidase with potential applications in citrus juice industry and in winemaking

The production of monoglycosylated flavonoids by α-l-rhamnosidases (EC 3.2.1.40) is an interesting development in biocatalysis. Applications of rhamnosidases in industry include removal of bitterness caused by naringin from citrus juices. In the present work, a psychrotolerant bacterial strain with α-l-rhamnosidase activity was isolated. The α-l-rhamnosidase was found to be able to degrade naringin and was purified and characterized. The α-l-rhamnosidase from Brevundimonas sp. Ci19 was able to release both rhamnose and prunin from naringin. The enzyme was partially purified with a performance of 2.7-fold purification. The α-l-rhamnosidase showed an optimum pH between 6.00 and 7.00 with substantial residual activity at pH 5.00 (85.3 %). The optimum temperature was between 20 and 37 °C. The enzyme showed activation in the presence of Ca²⁺ and Cd²⁺ ions and at a high ethanol concentration level (10 % v/v). Activity was found for β-d-glucosidase (EC 3.2.1.21) in the partially purified extract, but it was inactive in the acid pH region. This result indicates the potential for inactivation of β-d-glucosidase along with the high level of α-l-rhamnosidase activity necessary for the production of flavonoid glycosides. The α-l-rhamnosidase from Brevundimonas sp. Ci19 showed interesting properties for potential use not only in the citrus juice industry but also in winemaking.