Effects of Oral Supplementation with Paprika Xanthophylls on Human Skin Moisture.

Carotenoids, classified into carotenes and xanthophylls, are natural lipophilic pigments that are widely distributed in plants. Red paprika is unique in its high levels of various xanthophylls. Dietary paprika xanthophylls have been shown to reduce UV-induced photo damage by the strong antioxidant activity in the skin. However, the precise effects of paprika xanthophylls on skin condition are still unknown. Here we show that skin moisture is enhanced by the intake of red paprika supplements including seven xanthophylls. We conducted a 4-week randomized, single-blind, parallel-group controlled trial to clarify the effects of dietary paprika xanthophylls on facial skin. The results showed that the moisture was significantly higher in the paprika intake group than in the control (21.0±8.9 vs 13.4±6.0 (A.U.)). There was no significant difference between the paprika and control groups in other parameters such as viscoelasticity, the number of wrinkles, and the amount of water evaporation. On the other hand, the number of brown stains in the paprika group increased significantly, to 190±26 from 173±30 (p < 0.05). In vitro experiments, quantitative real-time PCR showed that paprika extract led to increases in the gene expression of Aquaporin 3 (AQP3) and hyaluronic acid synthase 3 (HAS3) in cultured keratinocytes. Western blotting showed that the paprika extract enhanced AQP3 expression. Taken together, taking supplements containing paprika xanthophylls may provide beneficial effects of moisture on facial skin. The study provides new insights into understanding the role of paprika xanthophylls in the skin.

Capsanthin Production in Escherichia coli by Overexpression of Capsanthin/Capsorubin Synthase from Capsicum annuum.

Capsanthin, a characteristic red carotenoid found in the fruits of red pepper (Capsicum annuum), is widely consumed as a food and a functional coloring additive. An enzyme catalyzing capsanthin synthesis was identified as capsanthin/capsorubin synthase (CCS) in the 1990s, but no microbial production of capsanthin has been reported. We report here the first successful attempt to biosynthesize capsanthin in Escherichia coli by carotenoid-pathway engineering. Our initial attempt to coexpress eight enzyme genes required for capsanthin biosynthesis did not detect the desired product. The dual activity of CCS as a lycopene ß-cyclase as well as a capsanthin/capsorubin synthase likely complicated the task. We demonstrated that a particularly high expression level of the CCS gene and the minimization of byproducts by regulating the seven upstream carotenogenic genes were crucial for capsanthin formation in E. coli. Our results provide a platform for further study of CCS activity and capsanthin production in microorganisms.

Pathway engineering for high-yield production of lutein in Escherichia coli.

Lutein is an industrially important carotenoid pigment, which is essential for photoprotection and photosynthesis in plants. Lutein is crucial for maintaining human health due to its protective ability from ocular diseases. However, its pathway engineering research has scarcely been performed for microbial production using heterologous hosts, such as Escherichia coli, since the engineering of multiple genes is required. These genes, which include tricky key carotenoid biosynthesis genes typically derived from plants, encode two sorts of cyclases (lycopene ε- and ß-cyclase) and cytochrome P450 CYP97C. In this study, upstream genes effective for the increase in carotenoid amounts, such as isopentenyl diphosphate isomerase (IDI) gene, were integrated into the E. coli JM101 (DE3) genome. The most efficient set of the key genes (MpLCYe, MpLCYb and MpCYP97C) was selected from among the corresponding genes derived from various plant (or bacterial) species using E. coli that had accumulated carotenoid substrates. Furthermore, to optimize the production of lutein in E. coli, we introduced several sorts of plasmids that contained some of the multiple genes into the genome-inserted strain and compared lutein productivity. Finally, we achieved 11 mg/l as lutein yield using a mini jar. Here, the high-yield production of lutein was successfully performed using E. coli through approaches of pathway engineering. The findings obtained here should be a base reference for substantial lutein production with microorganisms in the future.

Function of second glucan binding site including tyrosines 54 and 101 in Thermus aquaticus amylomaltase.

Amylomaltase from Thermus aquaticus catalyzes three types of transglycosylation reaction, as well as a weak hydrolytic reaction of alpha-1,4 glucan. From our previous study [Fujii et al., Appl. Environ. Microbiol., 71, 5823-5827 (2005)], tyrosine 54 (Y54) was identified as an amino acid controlling the reaction specificity of this enzyme. Since Y54 is not located around the active site but in the proposed second glucan binding site that is 14 A away from catalytic residues, the functions of Y54 and the second glucan binding site are of great interest. In this study, we introduced mutations into another tyrosine (Y101) in the second glucan binding site. The obtained mutated enzymes were subjected to all four types of enzyme assay and the effects of mutations on the reaction specificities of these enzymes were comprehensively investigated. These studies indicated that the amino acid substitution at Y54 or Y101 for removing their aromatic side chain increases cyclization activity (intra-molecular transglycosylation reaction) but decreases disproportionation, coupling and hydrolytic activities (inter-molecular reactions). The superimposition of the reported structures of the enzyme with and without substrate analog revealed the occurrence of a conformational change in which a donor binding site becomes open. From lines of evidence, we conclude that the binding of glucan substrate to the second glucan binding site through an interaction with the aromatic side chains of Y54 and Y101 is a trigger for the enzyme to take a completely active conformation for all four types of activity, but prevents the cyclization reaction to occur since the flexibility of the glucan is restricted by such binding.

Nanostructure and physical properties of cellulose nanofiber-carbon nanotube composite films.

We studied the nanostructure and physical properties of cellulose nanofiber-multi-walled carbon nanotube (CNF-MWNT) composite films prepared via MWNT aqueous dispersion using 4-O-methyl-α-d-glucuronoxylan as a MWNT dispersion aid. The composite film had high electrical conductivity (1.05S/cm), good mechanical properties (Young's modulus: 10.1GPa, tensile strength: 173.4MPa) and a low coefficient of thermal expansion (7ppm/K). FE-SEM imaging showed that the carbon nanotubes dispersed homogeneously and made reinforcing networks in the matrix of cellulose nanofibers. Improvement in the physical properties of cellulose nanofiber film by adding MWNTs is due to this composite structure.

Use of random and saturation mutageneses to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses.

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of alpha-1,4 glucans to produce cyclic alpha-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.