Peter Fleischmann

Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola)

This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

Occurrence of Glycosidically Conjugated 1-Phenylethanol and Its Hydrolase β-Primeverosidase in Tea (Camellia sinensis) Flowers

A previous study found that 1-phenylethanol (1PE) was a major endogenous volatile compound in tea (Camellia sinensis) flowers and can be transformed to glycosically conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. In this study, four 1PE-Glys have been isolated from tea flowers. Three of them were determined as (R)-1PE β-d-glucopyranoside ((R)-1PE-Glu), (S)-1PE-Glu, and (S)-1PE β-primeveroside ((S)-1PE-Pri), respectively, on the basis of NMR, MS, LC-MS, and GC-MS evidence. The other one was identified as (R)-1PE-Pri on the basis of LC-MS and GC-MS data. Moreover, these 1PE-Glys were chemically synthesized as the authentic standards to further confirm their occurrences in tea flowers. 1PE-Glu had a higher molar concentration than 1PE-Pri in each floral stage and organ. The ratio of (R) to (S) differed between 1PE-Glu and 1PE-Pri. In addition, a 1PE-Gly hydrolase β-primeverosidase recombinant protein produced in Escherichia coli exhibited high hydrolysis activity toward (R)-1PE-Pri. However, β-primeverosidase transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in tea flowers. This study provides evidence of occurrences of 1PE-Glys in plants for the first time.

Isolation of all-trans lycopene by high-speed counter-current chromatography using a temperature-controlled solvent system.

The effect of solvent system, partition coefficient, retention of stationary phase, column, revolution speed, and flow rate of mobile phase are well known parameters to effect HSCCC (high-speed counter-current chromatography) separations. Temperature effects on chromatographic techniques like HPLC and GC are well studied, but the influence of temperature on CCC solvent systems is hardly investigated. This paper presents the influence of temperature on several key parameters (partition coefficient, settling time, volume ratios) in the hydrophobic HSCCC solvent system hexane:dichloromethane:acetonitrile (30:11:18, v/v/v) used for the isolation of lycopene from tomato paste at 10, 15, 20 and 25 degrees C.

Identification of (3S, 9R)- and (3S, 9S)-Megastigma-6,7-dien-3,5,9-triol 9-O-β- D -glucopyranosides as Damascenone Progenitors in the…

The progenitors of damascenone (1), the most intensive C13-norisoprenoid volatile aroma constituent of rose essential oil, were surveyed in the flowers of Rosa damascena Mill. Besides 9-O-β-D-glucopyranosyl-3-hydroxy-7,8-didehydro-β-ionol (4b), a stable progenitor already isolated from the residual water after steam distillation of flowers of R. damascena Mill., two labile progenitors were identified to be (3S, 9R)- and (3S, 9S)-megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranosides (2b) based on their synthesis and HPLC-MS analytical data. Compound 2b gave damascenone (1), 3-hydroxy-β-damascone (3) and 4b upon heating under acidic conditions.

Centrifugal precipitation chromatography, a powerful technique for the isolation of active enzymes from tea leaves (Camellia sinensis)

Centrifugal precipitation chromatography was developed approximately 10 years ago. In contrast to other counter-current chromatographic techniques, the centrifugal precipitation chromatography system is operated with two mutually miscible solutions separated by a cut-off membrane. Centrifugal precipitation chromatography was firstly introduced for the separation of proteins using an ammonium sulfate gradient. In this study we describe a novel approach using solvent-based protein precipitation for the isolation of active plant enzymes from tea leaves (Camellia sinensis) by centrifugal precipitation chromatography. We developed a gradient based on acetone and Tris-buffer, because the biological activity of carotenases in tea leaves cannot be preserved in the presence of ammonium sulfate. Parameters such as the critical solvent concentration, flow rate, buffer concentration, and sample load were determined and/or optimized. Subsequently, the newly developed separation protocol was successfully used for the isolation of active carotenoid cleavage enzymes from tea leaves. The isolated enzymes showed high enzymatic activities and purities and could be directly used for enzymatic assays and structure elucidation.

Enzymic pathways for formation of carotenoid cleavage products.

Degraded carotenoids (apocarotenoids, norisoprenoids) have been a subject of intensive research for several decades. From the perspective of human physiology and nutrition, the retinoids, acting as vitamins, signalling molecules, and visual pigments, attracted the greatest attention (Chapters 15 and 16). Plant scientists, however, detected a wealth of different apocarotenoids, presumably derived by the excentric cleavage of carotenoids in various species, the plant hormone abscisic acid (1, Scheme 6) being the best-investigated example. With the onset of fruit ripening, flower opening or senescence of green tissues, carotenoids are degraded oxidatively to smaller, volatile compounds. The natural biological functions of the reaction products are outlined in Chapter 15. As many of these apocarotenoids act as potent flavour compounds, food chemists and flavourists worldwide have investigated meticulously their structural and sensory properties. Many aspects of carotenoid metabolites and breakdown products as aroma compounds are presented in a comprehensive book [1].

Characterization of human papillomavirus 6b L1 virus-like particles isolated from silkworms using capillary zone electrophoresis.

Human papillomavirus 6b L1 virus-like particles (VLPs) were successfully expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system and rapidly purified using size exclusion chromatography after ultracentrifugation procedure and characterized by capillary zone electrophoresis (CZE). The average capillary electrophoresis migration time was 11 min with the relative standard deviation (RSD) of 0.3% of human papillomavirus 6b L1 VLPs. After this threefold fractionation, the CZE samples were still further investigated by dynamic light scattering and immuno blotting. The versatile technique, CZE not only proved to be a valuable tool for VLP characterization, but was also found to be reliable and precise. Thus CZE will also be an important option for the quality control of VLPs in pharmaceutical research level.

Purification and Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry of Aroma Compounds from Green Tea Products and Comparison to Bulk Analysis

A method for carbon isotope ratio (δ(13)C) analysis was developed for compound-specific isotope analysis of tea volatiles, and the values were compared with the δ(13)C value from bulk isotope analyses. The δ(13)C value of 2-phenylethanol liberated via enzymatic hydrolysis of the 2-phenylethyl β-primeveroside standard was examined first. Isotope fractionations for 2-phenylethyl β-primeveroside from preparative high-performance liquid chromatography (HPLC) were also analyzed. The enzymatic treatment and the preparative HPLC process did not cause carbon isotope fractionations, substantiating the strategies available for δ(13)C analysis of volatile compounds. On the basis of the gas chromatography-combustion-isotope ratio mass spectrometry data from 2-phenylethanol, it was possible to derive the conditions for enzyme treatment and preparative HPLC of the glycoconjugates of 2-phenylethanol, (Z)-3-hexenol, and benzyl alcohol isolated from green tea leaves. Larger variations in δ(13)C were found for individual volatile compounds compared with bulk analytical data from the leaves, indicating the potential to utilize this strategy in assigning the geographical origin of green tea.