Influence of cultivation conditions on production of lignocellulolytic enzymes by Ceriporiopsis subvermispora

The aim of this work was to make a survey describing factors that influence the production of extracellular enzymes by white-rot fungus Ceriporiopsis subvermispora responsible for the degradation of lignocellulolytic materials. These factors were: carbon sources (glucose, cellulose, hemicellulose, lignin, maltose and starch), nitrogen sources (ammonium sulphate, potassium nitrate, urea, albumin and peptone), pH, temperature and addition of three different concentrations of Cu²⁺ and Mn²⁺. The cellulase and xylanase activities were similar in medium with different carbon sources and the highest cellulase and xylanase activities were measured in medium with urea and potassium nitrate as nitrogen sources, respectively. The highest laccase activity was observed in medium with lignin and peptone as carbon and nitrogen sources. In other experiments, time course of production of lignocellulolytic enzymes by white-rot fungus C. subvermispora in medium with lignin or glucose as carbon sources was observed.     

Multi-fed batch culture integrated with three-stage light irradiation and multiple additions of copper ions for the hyperproduction of ganoderic acid and Ganoderma polysaccharides by the medicinal mushroom Ganoderma lucidum

To further enhance the accumulation of the bioactive metabolite ganoderic acid (GA) by fermentation of the medicinal mushroom Ganoderma lucidum, a novel integrated strategy was developed by simultaneously adopting a strategy of multiple Cu²⁺ additions, three-stage light irradiation and multi-pulse feeding of carbon and nitrogen sources. Maximal GA content (i.e., 4.1 mg/100 mg DW) and production (i.e., 720.8 mg/L) were obtained using the novel integrated strategy. Not only the biomass but also the total GA production obtained in this work is the highest reported for a shaker flask culture of G. lucidum. This work is useful for the large-scale production of GA by G. lucidum fermentation.     

重金属Cd2+和Cu2+胁迫下泥蚶消化酶活性的变化

为了探讨重金属Cd²⁺和Cu²⁺胁迫对泥蚶消化酶活性的影响,运用酶学分析的方法,按《渔业水质标准》（GB 11607）规定的Cd²⁺、Cu²⁺最高限量值的1、2、5、10倍设置重金属离子Cd²⁺、Cu²⁺浓度及其组合,研究了在重金属Cd²⁺、Cu²⁺胁迫下,30d内泥蚶3种消化酶活性的变化规律。结果表明：与空白对照组相比,在重金属Cd²⁺、Cu²⁺或其组合的胁迫下,较低浓度组泥蚶的淀粉酶活性实验前期增强（即被诱导）,实验后期减弱（即被抑制）,较高浓度组泥蚶的淀粉酶活性从实验一开始就减弱,并保持在较低水平,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合Cu²⁺ > （Cd²⁺+Cu²⁺）组合 > Cd²⁺;泥蚶脂肪酶的活性实验前期增强,实验后期转为微减弱或减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺;泥蚶胃蛋白酶的活性实验前期增强,且活性呈现升高-降低-再升高-再降低的变化,实验后期分别表现微增强、微减弱和减弱,毒性比较,同一重金属高浓度 > 低浓度,不同重金属及其组合（Cd²⁺+Cu²⁺）组合 > Cu²⁺ > Cd²⁺。可见：环境中的Cd²⁺和Cu²⁺对泥蚶的消化酶活性起着明显的影响作用。     

Influence of Trace Elements Mixture on Bacterial Diversity and Fermentation Characteristics of Liquid Diet Fermented with Probiotics under Air-Tight Condition

Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ are often supplemented to the diet of suckling and early weaning piglets, but little information is available regarding the effects of different Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ mixtures on bacteria growth, diversity and fermentation characteristics of fermented liquid diet for piglets. Pyrosequencing was performed to investigate the effect of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ mixtures on the diversity, growth and fermentation characteristics of bacteria in the liquid diet fermented with Bacillus subtilis and Enterococcus faecalis under air-tight condition. Results showed that the mixtures of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ at different concentrations promoted Bacillus growth, increased bacterial diversity and lactic acid production and lowered pH to about 5. The importance of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ is different for Bacillus growth with the order Zn²⁺> Fe²⁺>Cu²⁺> I⁻ in a 21-d fermentation and Cu²⁺>I⁻>Fe²⁺>Zn²⁺ in a 42-d fermentation. Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ is recommended at a level of 150, 60, 150 and 0.6 mg/kg respectively for the production of fermented liquid diet with Bacillus subtilis. The findings improve our understanding of the influence of trace elements on liquid diet fermentation with probiotics and support the proper use of trace elements in the production of fermented liquid diet for piglets.     

Cu-ions mediated changes in growth,chlorophyll and other ion contents in a Cu-tolerantKoeleria splendens

The effects of Cu²⁺ on growth, chlorophyll and other ion contents ofKoeleria splendens originated from Cu-contaminated soil have been investigated in nutrient solution. The most evident Cu²⁺ effects concern the root growth, especially the root length. Since in plants grown under lower Cu²⁺ concentrations (4 and 8 μM) root elongation, biomass, chlorophyll, Mg²⁺, Fe²⁺, Ca²⁺ and K⁺ content were increased compared with the control, the development of an adaptive mechanism ofK. splendens to Cu²⁺ is suggested. High Cu²⁺ concentration (160 μM) caused a significant reduction in root length and biomass as well as a decreased rate of chlorophyll biosynthesis. The reduction of growth can be correlated with the toxic effect of Cu²⁺ on photosynthesis, root respiration and protein synthesis in roots. 160 μM Cu²⁺-treatment had a negative influence on the concentrations of Ca²⁺, Fe²⁺, Mg²⁺ and K⁺ and a positive influence on the Cu²⁺ concentration in the plant tissues. Loss of nutrients similar to the senescence response suggests that excess of Cu²⁺ leads to the progressive senescence of the plants. Our results demonstrate the existence of an adaptive mechanism ofK. splendens under low Cu²⁺ concentrations, while high Cu²⁺ quantities cause disturbances in plant function.     

Bioconversion of lignocellulose and simultaneous production of cellulase,ligninase and bioflocculants by Alcaligenes faecalis-X3

Lignocelluloses have been used as carbon sources for bioflocculant production. However, the low bioconversion efficiency of lignocellulose to bioflocculants is a major challenge. In this study, a lignocellulolytic strain of Alcaligenes faecalis-X3 was cultivated in ramie bio-degumming wastewater. Optimal production of ligninase, cellulase and bioflocculants (MBF-X3) was evaluated. The highest activity of MBF-X3 under the optimal conditions of pH 6.0 at 48 h of fermentation was 95.44%, with the maximum production of ligninase and cellulase (0.27 and 0.12 U/mL, respectively). The crude ligninase and cellulase had optimum activities at pH 5.0 and 40 °C and pH 6.0 and 50 °C, respectively. The cellulase activity was increased by Mn²⁺, Ca²⁺, Zn²⁺, and Mg²⁺ at 1 mM. The ligninase activity was significantly enhanced in the presence of Zn²⁺ at 10 mM. The flocculating activity of MBF-X3 was not changed by the addition of any metal cation. The results demonstrated that A. faecalis possesses an excellent enzyme system for the efficient bioconversion of lignocellulose into MBF-X3. Additionally, MBF-X3 has a high flocculating efficiency of Disperse Blue-2BLN (85.7%) at a dose of 1.0 g/L.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+

Citr⁺Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu²⁺. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu²⁺, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu²⁺. On the other hand, the Y_Glu value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu²⁺ enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu²⁺ stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP_i. In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum.The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H₂O → 1 butyrate^? + 2 HCO₃^? + 3 H⁺ + 2 H₂ (1) Glucose + 4 H₂O → 2 acetate^? + 2 HCO₃^? + 4 H⁺ + 4 H₂ (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases.The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations.The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP⁺.     

Effect of copper on germination and seedling growth ofMinuartia,Silene, Alyssum andThlaspi

The influence of increasing copper concentrations on seed germination, seedling survival and radicle length ofMinuartia hirsuta, Silene compacta, Alyssum montanum andThlaspi ochroleucum was studied. Seed germination was highly affected by the higher Cu²⁺ concentrations (80 and 160 μM), while lower Cu²⁺ concentrations seemed to be necessary for seed germination, even for the plants originated from non-Cu²⁺-rich soils (i.e. A. montanum). Nevertheless, plants originated from Cu²⁺-rich soils (M. hirsuta, S. compacta) showed a higher demand of Cu²⁺ for rapid seed germination. Cu²⁺ at higher concentrations severely reduced growth rate of radicle, especially inA. montanum andT. ochroleucum. These data clearly indicate the reduced suitability of the above mentioned plant species for reclamation on Cu²⁺ soils. Lower Cu²⁺-concentrations had no influence on seedling survival inM. hirsuta andS. compacta, but a progressive reduction of a number of survived seedlings with increasing Cu²⁺ concentration was found, that was more pronounced inA. montanum andT. ochroleucum.     

Effects of Combined Abiotic Stresses on Growth,Trace Element Accumulation,and Phytohormone Regulation in Two Halophytic Species

Trace element contamination of lands is a serious environmental problem that limits yield and threatens human health. To study the combined effect of high salinity and toxic levels of trace elements on halophytes, the performance of two marsh species, Atriplex halimus and Suaeda fruticosa, grown for 1 month with an irrigation solution supplemented with 200 mM NaCl and 400 μM Cd²⁺ or 400 μM Cu²⁺ was evaluated. The effect of the combined stress conditions on hormone signaling was also assessed. Biomass production and chlorophyll content decreased under Cd²⁺ stress in both species, whereas Cu²⁺ had a lower impact on plant performance. The different plant sensibilities to the two trace elements assayed indicate that each metal has a different effect on plants. Furthermore, the deleterious effect of metal toxicity was alleviated when NaCl was added to the irrigation solution, demonstrating that NaCl improves plant performance and tolerance of halophytic species to cope with trace element intoxication. Results show that both species accumulated important quantities of Cd²⁺ and Cu²⁺ in roots (Cd²⁺: 2,690–3,130 μg g^?1 DW and Cu²⁺: 2,070–2,770 μg g^?1 DW); this finding allows us to classify these species among the hyperaccumulator plants. Cd²⁺ and Cu²⁺ differently affected endogenous phytohormone contents in both species. Data suggest an essential involvement of roots on the regulation of tolerance to trace elements. Therefore, indole-3-acetic acid levels increased in roots of both species irrigated with high levels of Cd²⁺, which suggests that the auxin may stimulate root promotion and growth under these stress conditions. Other compounds, classically considered as “stress hormones” showed very different patterns of accumulation. Whereas, salicylic acid (SA) levels in roots and leaves increased in response to Cd²⁺, root contents of jasmonic acid (JA), and abscisic acid (ABA) decreased. In leaves, the rambling pattern of accumulation observed for JA and ABA suggested the lack of a specific role in regulation against trace element toxicity. Together, data suggest that SA could act as a specific signal that detects trace element toxicity, whereas JA and ABA promote general responses against abiotic stress.     

Capturing a Reactive State of Amyloid Aggregates: NMR-BASED CHARACTERIZATION OF COPPER-BOUND ALZHEIMER DISEASE AMYLOID β-FIBRILS IN A REDOX CYCLE*

The interaction of redox-active copper ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu⁺/Cu²⁺ with Aβ aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu⁺/Cu²⁺ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ∼60 μm hydrogen peroxide (H₂O₂) from a solution of 20 μm Cu²⁺ ions in complex with Aβ(1–40) in fibrils ([Cu²⁺]/[Aβ] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (∼1 mm). Furthermore, SSNMR ¹H T₁ measurements demonstrated that during ROS production the conversion of paramagnetic Cu²⁺ into diamagnetic Cu⁺ occurs while the reactive Cu⁺ ions remain bound to the amyloid fibrils. The results also suggest that O₂ is required for rapid recycling of Cu⁺ bound to Aβ back to Cu²⁺, which allows for continuous production of H₂O₂. Both ¹³C and ¹⁵N SSNMR results show that Cu⁺ coordinates to Aβ(1–40) fibrils primarily through the side chain N_δ of both His-13 and His-14, suggesting major rearrangements from the Cu²⁺ coordination via N_ϵ in the redox cycle. ¹³C SSNMR chemical shift analysis suggests that the overall Aβ conformations are largely unaffected by Cu⁺ binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu⁺ centers.     

Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper

Chick brain microsomal ATPase was strongly inhibited by Cu²⁺. (Na⁺ + K⁺)-ATPase was more susceptible to low levels of Cu²⁺ than Mg²⁺-ATPase. The inhibition of (Na⁺ + K⁺)-ATPase could be partially protected from Cu²⁺ in the presence of ATP in the preincubation period. When Cu²⁺ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu²⁺ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu²⁺ (1.0 μM) on (Na⁺ + K⁺)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K⁺ (5 mM), V with Na⁺ was markedly decreased in the presence of Cu²⁺ and K_m was about twice that of the control. However, at high K⁺ concentration (20 mM), the K_m for Na⁺ was not affected. At both low (25 mM) and high (100 mM) Na⁺, Cu²⁺ displayed non-competitive inhibition of the enzyme with respect to K⁺.On the basis of these data, we suggest that Cu²⁺ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu²⁺ interacts reversibly with the enzyme.     

Hsp27 suppresses the Cu2+-induced amyloidogenicity,redox activity,and cytotoxicity of α-synuclein by metal ion stripping

Aberrant copper homeostasis and oxidative stress have critical roles in several neurodegenerative diseases. Expression of heat-shock protein 27 (Hsp27) is elevated under oxidative stress as well as upon treatment with Cu²⁺, and elevated levels of Hsp27 are found in the brains of patients with Alzheimer and Parkinson diseases. We demonstrate, using steady-state and time-resolved fluorescence spectroscopy as well as isothermal titration calorimetry studies, that Hsp27 binds Cu²⁺ with high affinity (K_d ~10⁻¹¹ M). Treating IMR-32 human neuroblastoma cells with Cu²⁺ leads to upregulation of endogenous Hsp27. Further, overexpression of Hsp27 in IMR-32 human neuroblastoma cells confers cytoprotection against Cu²⁺-induced cell death. Hsp27 prevents the deleterious interaction of Cu²⁺ with α-synuclein, the protein involved in Parkinson disease and synucleinopathies. Hsp27 attenuates Cu²⁺- or Cu²⁺–α-synuclein-mediated generation of reactive oxygen species and confers cytoprotection on IMR-32 cells as well as on mouse primary neural precursor cells. Hsp27 prevents Cu²⁺–ascorbate or Cu²⁺–α-synuclein–ascorbate treatment-induced increase in mitochondrial superoxide level and mitochondrial disorganization in IMR-32 cells. Hsp27 dislodges the α-synuclein-bound Cu²⁺ and prevents the Cu²⁺-mediated amyloidogenesis of α-synuclein. Our findings that Hsp27 binds Cu²⁺ with high affinity leading to beneficial effects and that Hsp27 can dislodge Cu²⁺ from α-synuclein, preventing amyloid fibril formation, indicate potential therapeutic strategies for neurodegenerative diseases involving aberrant Cu²⁺ homeostasis.     

Effect of copper on the content and the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated rape seedlings (Brassica napus var.arvensis (Lam.) Thell.)

The influence of Cu²⁺ ions (in the form of CuCl₂) in the concentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell.). Ions Cu²⁺ acted on the seedlings either chronically from the beginning of the germination or acutely, during 3 to 72 h, on seven days old seedlings. The biosynthesis of both glucosinolates was followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into them in hypocotyl segments from seven days old intact etiolated seedlings. After the entry of small amounts of Cu²⁺ ions into the plants, stimulation of the glucosinolates formation occurs, as was found after three h action of Cu²⁺ ions. After the entry of a greater amount of Cu²⁺ ions into the plant, harmful effects appear, as was found after chronic two days action or after 24 and 48 hours acute action of Cu²⁺ ions. Later further stimulation of glucosinolate formation occurs, probably due to enhanced metabolism during reparation processes, as was manifested after chronic action of Cu²⁺ ions lasting four and eight days. The optimal effect of copper was found mainly in the concentration range 5×10^?4 M to 10^?5 M. Ions Cu²⁺ in higher concentration increased the uptake of sulphate ions by hypocotyl segments, and in lower concentrations increased the incorporation of³⁵S from³⁵SO₄ ^2? into the proteins.     

Cellular responses of developing Fucus serratus embryos exposed to elevated concentrations of Cu2+

Elevated concentrations of Cu²⁺ can have inhibitory effects on early development in plants and algae by targeting specific cellular processes. In the present study the effects of elevated Cu²⁺ on developmental processes in embryos of the brown algae Fucus serratus (Phaeophyceae) were investigated. Elevated Cu²⁺ was shown to inhibit fixation of the zygotic polar axis but not its formation. Actin localization was unaffected by elevated Cu²⁺ but polarized secretion, which occurs downstream, was inhibited. Significant differences in tolerance to Cu²⁺ were observed for polarization and rhizoid elongation of embryos derived from adults from Cu²⁺‐contaminated and uncontaminated locations. Moderate Cu²⁺ exposure inhibited the generation of cytosolic Ca²⁺ signals in response to hypo‐osmotic shocks. In contrast, cytosolic Ca²⁺ was elevated by treatments with high [Cu²⁺] and this coincided with production of reactive oxygen species. The results indicate that direct effects on signalling processes involved in polarization and growth may in part explain complex, concentration‐dependent effects of Cu²⁺ on early development.     

紫花苜蓿对铜胁迫生理响应的傅里叶变换红外光谱法研究

为阐明紫花苜蓿(Medicago sativa L.)对铜胁迫的耐性机理,采用准确度好、分辨率高和简便快捷的傅里叶变换红外光谱法(FTIR)研究在不同铜浓度(0、1、5、20、100 mol/L)处理时紫花苜蓿根、茎、叶化学组分的变化。结果表明:随着铜处理浓度的增加,紫花苜蓿根、茎、叶生物量变化不大。其根组织在2924 cm~(-1)处峰高处呈现出先下降后上升的趋势,反映了在低铜(5 mol/L)处理条件下紫花苜蓿分泌的有机酸不断螯合Cu,造成羧酸0-H的减少,但随着Cu含量的升高,其羧酸螯合力变弱,有机酸含量渐渐升高;根组织在1381 cm~(-1)处峰高先下降后上升,反映了含油脂化合物含量先下降后升高。可能与植物在细胞壁结构上增强抗逆性有一定关系,即低Cu处理下细胞壁可能通过提高阳离子交换能力(CEC),增强了耐Cu性;茎组织在2924,1643,1381,1064 cm~(-1)等处峰高无明显变化;叶组织所有峰值在低浓度(5 mol/L)Cu处理下变化不明显,高浓度(5 moL/L)Cu处理下所有峰值先升后降,随着这可能与可溶性糖及可溶性蛋白质等物质含量都呈现先升后降的趋势有关。这表明紫花苜蓿通过根部有机酸含量的变化和提高细胞壁阳离子交换能力,将吸收的Cu大部分积累在根部,阻止Cu向地上部分运输,有效地保护了植物地上部分组织。     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

Improvement of ganoderic acid and Ganoderma polysaccharide biosynthesis by Ganoderma lucidum fermentation under the inducement of Cu2+

The cell growth and total accumulation of bioactive metabolites were significantly improved by Cu²⁺ addition during the submerged fermentation of medicinal mushroom Ganoderma lucidum. A mathematical model, constructed by response surface methodology combination with full factorial design, was applied to study the synergic effect of Cu²⁺ addition concentration and addition time. The optimal Cu²⁺ inducement strategy for the cell growth were different from those for the biosynthesis of ganoderic acid (GA) and Ganoderma polysaccharide. A multiple additions strategy of Cu²⁺ by adding each 1 mM Cu²⁺ on day 2, 6, 8 and 2 mM Cu²⁺ on day 4 was developed to enhance total accumulation of GA and extracellular polysaccharides. The highest GA content reached 3.0 ± 0.1 mg per 100 mg DW, which was increased by 76.5% and 33.9% compared with the control without Cu²⁺ addition and the peak value predicted by the constructed mathematical model, respectively. While, relatively higher addition concentration of Cu²⁺ (i.e., 5 mM) on the culture of day 4 led to higher content and total production of intracellular polysaccharides. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010