Isolate-specific effects of ultraviolet radiation on photosynthesis,growth and mycosporine-like amino acids in the microbial mat-forming cyanobacterium <Emphasis Type="Italic">Microcoleus </Emphasis><Emphasis Type="Italic">chthonoplastes</Emphasis>

Microcoleus chthonoplastes constitutes one of the dominant microorganisms in intertidal microbial mat communities. In the laboratory, the effects of repeated daily exposure to ultraviolet radiation (16:8 light:dark cycle) was investigated in unicyanobacterial cultures isolated from three different localities (Baltic Sea = WW6; North Sea = STO and Brittany = BRE). Photosynthesis and growth were measured in time series (12–15 days) while UV-absorbing mycosporine-like amino acids (MAAs) and cellular integrity were determined after 12 and 3 days exposure to three radiation treatments [PAR (22 μmol photon m⁻² s⁻¹) = P; PAR + UV-A (8 W m⁻²) = PA; PAR + UV-A + UV-B (0.4 W m⁻²) = PAB]. Isolate-specific responses to UVR were observed. The proximate response to radiation stress after 1-day treatment showed that isolate WW6 was the most sensitive to UVR. However, repeated exposure to radiation stress indicated that photosynthetic efficiency (F _v/F _m) of WW6 acclimated to UVR. Conversely, although photosynthesis in STO exhibited lower reduction in F _v/F _m during the first day, the values declined over time. The BRE isolate was the most tolerant to radiation stress with the lowest reduction in F _v/F _m sustained over time. While photosynthetic efficiencies of different isolates were able to acclimate to UVR, growth did not. The discrepancy seems to be due to the higher cell density used for photosynthesis compared to the growth measurement. Apparently, the cell density used for photosynthesis was not high enough to offer self-shading protection because cellular damage was also observed in those filaments under UVR. Most likely, the UVR acclimation of photosynthesis reflects predominantly the performance of the surviving cells within the filaments. Different strategies were observed in MAAs synthesis. Total MAAs content in WW6 was not significantly different between all the radiation treatments. In contrast, the additional fluence of UV-A and UV-B significantly increased MAAs synthesis and accumulation in STO while only UV-B fluence significantly increased MAAs content in BRE. Regardless of the dynamic photosynthetic recovery process and potential UV-protective functions of MAAs, cellular investigation showed that UV-B significantly contributed to an increased cell mortality in single filaments. In their natural mat habitat, M. chthonoplastes benefits from closely associated cyanobacteria which are highly UVR-tolerant due to the production of the extracellular UV-sunscreen scytonemin.     

UV effects on photosynthesis and DNA in propagules of three Antarctic seaweeds (<Emphasis Type="Italic">Adenocystis utricularis</Emphasis>, <Emphasis Type="Italic">Monostroma hariotii</Emphasis> and <Emphasis Type="Italic">Porphyra endiviifolium</Emphasis>)

Ozone depletion is highest during spring and summer in Antarctica, coinciding with the seasonal reproduction of most macroalgae. Propagules are the life-stage of an alga most susceptible to environmental perturbations therefore, reproductive cells of three intertidal macroalgal species Adenocystis utricularis (Bory) Skottsberg, Monostroma hariotii Gain, and Porphyra endiviifolium (A and E Gepp) Chamberlain were exposed to photosynthetically active radiation (PAR), PAR + UV-A and PAR + UV-A + UV-B radiation in the laboratory. During 1, 2, 4, and 8 h of exposure and after 48 h of recovery, photosynthetic efficiency, and DNA damage were determined. Saturation irradiance of freshly released propagules varied between 33 and 83 μmol photons m⁻² s⁻¹ with lowest values in P. endiviifolium and highest values in M. hariotii. Exposure to 22 μmol photons m⁻² s⁻¹ PAR significantly reduced photosynthetic efficiency in P. endiviifolium and M. hariotii, but not in A. utricularis. UV radiation (UVR) further decreased the photosynthetic efficiency in all species but all propagules recovered completely after 48 h. DNA damage was minimal or not existing. Repeated exposure of A. utricularis spores to 4 h of UVR daily did not show any acclimation of photosynthesis to UVR but fully recovered after 20 h. UVR effects on photosynthesis are shown to be species-specific. Among the tested species, A. utricularis propagules were the most light adapted. Propagules obviously possess good repair and protective mechanisms. Our study indicates that the applied UV dose has no long-lasting negative effects on the propagules, a precondition for the ecological success of macroalgal species in the intertidal.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

Effects of solar ultraviolet radiation on photochemical efficiency of <Emphasis Type="Italic">Chaetoceros curvisetus</Emphasis> (Bacillariophyceae)

To assess the short- and long-term impacts of Ultraviolet radiation (UVR, 280–400 nm) on the red tide alga Chaetoceros curvisetus, we exposed cells to three different solar radiation treatments–PAB:280–700 nm, PA:320–700 nm, and P:400–700 nm under 20°C incubated temperature. Short-term exposures were investigated: the photochemical efficiency (Φ_PSII) versus irradiance curves under six levels of solar radiation by covering the incubators with a variable number of neutral density screens (the irradiance thus varied from 100 to 3%) lasting 1 h, and long-term exposures were designed to assess how the cells acclimate to solar radiation (the growth, UV_abc and ratio of repair to damage rates of D1 protein were detected). A significant decrease in the photochemical efficiency (Φ_PSII) at high irradiance (100% of incident solar radiation, 261.6 Wm⁻²) was observed in short-term exposure (1 h). UVR-induced photoinhibition was reduced to 7% in 3% solar radiation (4.08 Wm⁻²), compared with 66% in 100% solar radiation (261.6 Wm⁻²). In long-term experiments (11 days) using batch cultures, cell densities during the first 6 days were relatively constant for treatment P, and decreased slightly under PA and PAB treaments, reflecting a change in the irradiance experienced in the laboratory to that of incident solar irradiance. Thereafter, cell density increased and UV-induced photoinhibition decreased with the following days, indicating acclimation to solar UV. At the end of experiment, cells were found to exhibit both higher ratios of repair to UV-related damage and increased concentrations of UV-absorbing compounds, whose maximum absorption was found to be at 329 nm. Our data indicate that C. curvisetus is sensitive to ultraviolet radiation, but was able to acclimate relatively rapidly (ca. 6 days) by synthesizing UV-absorbing compounds and by increasing the rates of repair processes of D1 protein in PSII.     

UV-absorbing compounds in <Emphasis Type="Italic">Porphyra haitanensis</Emphasis> (Rhodophyta) with special reference to effects of desiccation

The intertidal red alga Porphyra haitanensis Chang et Zheng is episodically desiccated and exposed to high levels of solar radiation at low tide during emersion. However, little has been documented on the relationship between the stresses during desiccation and related chemical compounds. We found that P. haitanensis thalli, when desiccated under indoor (artificial radiation) or outdoor (solar radiation) conditions, with or without UV radiation (UVR: 280–400 nm), contained significantly higher concentrations of UV-absorbing compounds (peak at 336 nm) than those maintained submerged (without desiccation). Solar UVR had no effect on the content of UV-absorbing compounds. Even though the concentration of these compounds decreased with time in all treatments, a slower decrease was observed in the desiccated samples. The samples with higher levels of UV-absorbing compounds showed higher photochemical efficiency of photosystem II (PS II) during the exposure or subsequent recovering process than samples with low concentration of UV-absorbing compounds, reflecting their protective role. The concentration of these compounds varied in different parts of the thallus, with the middle and marginal parts containing 60–80% more UV-absorbing compounds than the basal parts in both female and male plants. In addition, the marginal parts of male thalli contained more UV-absorbing compounds than the corresponding parts of female thalli. Our data suggest that desiccation plays a key role in this alga to maintain high concentration of UV-absorbing compounds, and that this might provide a beneficial advantage to compete in the intertidal zone where the organism is normally exposed to high levels of UVR.     

A new species of <Emphasis Type="Italic">Cortinarius</Emphasis> sect. <Emphasis Type="Italic">Orellani</Emphasis> from Japan

Cortinarius breviradicatus sp. nov., found in deciduous forests, is described and illustrated from Niigata, Japan. It is characterized by its medium-sized to large dark brown basidiocarp, acutely conical pileus, and rooting stipe, and by subglobose to broadly ellipsoid basidiospores. In addition, the extracting solution from its basidiocarps exhibits a strong fluorescence around 400–430 nm in ultraviolet radiation (250 nm), which was observed in a species of Cortinarius sect. Orellani. The new species belongs to the section Orellani. The differences between the new taxon and similar species are briefly discussed.     

Photoresponses of late instar <Emphasis Type="Italic">Chaoborus punctipennis</Emphasis> larvae to UVR

With a few clear exceptions (e.g., Daphnia) it is uncertain if most aquatic invertebrates can detect and respond to ultraviolet radiation (UVR). It is known that many aquatic invertebrates are vulnerable to UVR and that anthropogenically-induced increases in surface UVR have occurred in recent decades. We examined the photoresponses of late larval instars of Chaoborus punctipennis to different combinations of UVA (320–400 nm), UVB (300–320 nm) and visible light (400–700 nm) to determine whether the larvae can detect and/or avoid UVR. To accomplish this, we exposed late instar C. punctipennis larvae to a directional light source of UVR only (peak wavelength at 360 nm), visible light only or visible plus various wavebands of UVR. We examined negative phototaxis for 10 min at a quantum flux of 2.62 x 10¹³ quanta s^–1 cm^–2 (S.D. = 3.63 x 10¹² quanta s^–1 cm^–2). In the dark, larvae stayed close to the surface of the experimental vessels. Under all treatments containing visible light the larvae exhibited negative phototaxis and occupied the bottom of the vessels. Under UVR only, the larvae occupied the middle of the water column. Our results suggest that late instar C. punctipennis larvae are unable to detect and avoid UVB and short UVA wavelengths but they can detect long UVA wavelengths.     

The short- and long-term response of <Emphasis Type="Italic">Scrippsiella trochoidea</Emphasis> (Pyrrophyta) to solar ultraviolet radiation

To assess the short- and long-term impacts of UV radiation (UVR, 280–400 nm) on the microalga Scrippsiella trochoidea, we exposed cells to three different radiation treatments (PAB: 280–700 nm, PA: 320–700 nm, and P: 400–700 nm). A significant decrease in the photochemical efficiency (Φ_PSII) at high irradiance (100% of incident solar radiation, 216.0 W m⁻²) was observed. Photoinhibition was reduced from 62.7 to 10.9% when the cells were placed in 12% solar radiation (26.1 W m⁻²). In long-term experiments (11 days) using batch cultures, cell densities during the first 5 days were decreased under treaments P, PA, and PAB, reflecting a change in the irradiance experienced in the laboratory to that of incident solar irradiance. Thereafter, specific growth rates increased and UV-induced photoinhibition decreased, indicating acclimation to solar UV. Cells were found to exhibit both higher ratios of repair to UV-related damage, shorter period for recovery and increased concentrations of UV-absorbing compounds (UV_abc), whose maximum absorption was found to be at 336 nm. Our data indicate that S. trochoidea is sensitive to ultraviolet radiation, but was able to acclimate relatively rapidly (ca. 6 days) by synthesizing UV_abc and by increasing the rates of repair processes of D1 protein in PSII.     

Evaluation of the toxicity of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis> extract

In this study, the methanol extract of Arthrospira (Spirulina) platensis was examined for acute and subchronic toxicities. The extract did not produce any sign of toxicity within 7 days after feeding it at a single high dose of 6 g kg⁻¹ body weight to female and male Swiss mice. For the subchronic toxicity test, the extract at doses of 6, 12, and 24 mg kg⁻¹ body weight was orally administered to six male and six female Wistar rats daily for 12 weeks. Throughout the study period, we did not observe any abnormalities on behavior, food and water intakes, and health status among the treated animals. The hematology and clinical chemistry parameters of treated groups did not significantly differ from those of the controls in both sexes. Postmortem examination of the test groups also showed no abnormalities in both gross and histological findings. These results thus suggest that the methanol extract of A. platensis did not cause acute or subchronic toxicity in our experimental animals.     

Optimization and mechanism of diacetyl accumulation by <Emphasis Type="Italic">Enterobacter</Emphasis><Emphasis Type="Italic">aerogenes</Emphasis> mutant UV-3

A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of α-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0–7.5, and temperature between 36 and 39°C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l⁻¹. Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%. Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH₄)₂SO₄, 0.1% citric acid, 0.2% MnSO₄ and 0.2% MgSO_4, and this was determined by one-factor-at-a-time approach. Data from the five level central composite designs demonstrated that initial pH of 7.0, temperature of 37°C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl production. The maximum yield of diacetyl could reach 1.35 g l⁻¹ in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of α-acetolactate and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3.     

UV-A Mediated Modulation of Photosynthetic Efficiency,Xanthophyll Cycle and Fatty Acid Production of <Emphasis Type="Italic">Nannochloropsis</Emphasis>

Nannochloropsis, a green microalga, is a source for commercially valuable compounds as extensively described and, in particular, is recognised as a good potential source of eicosapentaenoic acid (20:5ϖ3), an important polyunsaturated fatty acid for human consumption for prevention of several diseases. Climate change might include variation in the ultraviolet (UV) levels as one of the consequences derived from the anthropogenic activity. This paper shows the response of Nannochloropsis cultures exposed for 7 days to UV-A (320–400 nm) added to photosynthetically active radiation (PAR; 400–700 nm). Growth rates and photosynthetic activity were assessed to determine the impact of UV-A increased levels on the cell growth and basic metabolism activity. Xanthophyll pigments (zeaxanthin and violaxanthin), carotenoids (canthaxanthin and β-carotene) and polyunsaturated fatty acids (myristic, palmitic, palmitoleic, arachidonic and eicosapentaenoic acids) were measured for assessing the antioxidant response of the microalgae to added UV-A radiation to PAR. The results show that the modulated use of UV-A radiations can lead to increased growth rates, which are sustained in time by an increased light transduction activity. The expected antioxidant response to the incident UV-A radiation consisted of increases in zeaxanthin and β-carotene contents—synthesis of antioxidant carotenoids—and increases in the saturated fatty acids to polyunsaturated fatty acids ratio. The results suggest that modulated UV-A radiation can be used as a tool to stimulate value molecules accumulation in microalgae through an enhanced both light transduction process and antioxidant response, while sustaining cell growth.     

Effects of realistically simulated,elevated UV irradiation on photosynthesis and pigment composition of the alpine snow alga <Emphasis Type="Italic">Chlamydomonas nivalis</Emphasis> and the arctic soil alga <Emphasis Type="Italic">Tetracystis</Emphasis> sp. (Chlorophyceae)

An indoor sun simulator was used to provide elevated UV-B radiation (280–315 nm) in combination with realistic ratios to PAR (400–700 nm) and UV-A radiation (315–400 nm) in order to test the physiological response of a soil- and snow microalga during a three-day stress scenario, which may occasionally occur in their respective arctic and alpine habitats.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

An investigation of <Emphasis Type="Italic">Clostridium</Emphasis> species present in nutraceutical preparations of <Emphasis Type="Italic">Arthrospira platensis</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) for human consumption

The presence of the anaerobic spore former Clostridium in Arthrospira platensis destined for human consumption is generally not assessed during quality assurance procedures. As this nutraceutical is administered as complementary medicine to the immunocompromised, this study aimed to investigate the presence of these potential pathogens. Anaerobic counts performed on tablets from a single manufacturer indicated an excess of 10⁵ CFU/endospores g⁻¹ tablet for three different A. platensis batches. Tests for coliforms for use as “indicators” of pathogens in the tablets were negative. Using classic culture techniques, five species of Clostridium were isolated. Subsequent use of PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting of tablets showed a divergent microbial population, with a predominance of anaerobic endospore formers, including Clostridium. Sequencing of a 1.5 kb 16S rDNA clone library and phylogenetic analyses of prominent operational taxonomic units confirmed the presence of an additional five Clostridium spp. and other genera in the tablets. A composite molecular ladder, using 16S rRNA DGGE amplicons of 17 representative bacterial species was constructed to assist in identifying anaerobes present in tablets sourced from three different A. platensis manufacturers. Results indicated that commercial A. platensis preparations were contaminated with potentially hazardous clostridia and other anaerobic species. Results suggest that certain commercial A. platensis preparations require stringent microbial quality assurance measures to ensure safe use as a nutraceutical for the immunocompromised and the general public.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.