20. "Modeling of the simultaneous hydrolysis–ultrafiltration of whey permeate by a thermostable β-galactosidase from Aspergillus niger".
D. G. Hatzinikolaou, E. Katsifas, D. Mamma, A. D. Karagouni, P. Christakopoulos and D. Kekos.
Biochemical Engineering Journal, vol. 24, pages 161-172, (2005).  View at publisher's site  Request copy
Abstract: A wild type strain of Aspergillus niger, denoted as BTL, produced elevated levels of β-galactosidase when grown in a low cost medium that contained wheat bran as the sole carbon and energy source. The enzyme was collected, concentrated and partially purified from the culture supernatant. Its kinetic and stability properties were thoroughly examined towards its potential use for the hydrolysis of acid whey permeate lactose. The β-galactosidase of A. niger BTL showed increased pH and thermal stability, with activation energy for the first order deactivation constant equal to 180 kJ/mol at pH 3.5. Lactose hydrolysis by the enzyme was described by Michaelis–Menten kinetics with competitive inhibition only from galactose. An integrated process, concerning the simultaneous hydrolysis–ultrafiltration of whey lactose that incorporated the specific kinetic properties of the β-galactosidase was developed and modeled. The model proved very successful in predicting the behavior of a continuous laboratory hydrolysis–ultrafiltration set up, specifically designed for that purpose. The validated model was finally used in a number of computer simulations in order to investigate the effect of the various process parameters on the overall system performance.

19. "Biodegradation of Phenol by Acclimatized Pseudomonas putida Cells Using Glucose as an Added Growth Substrate".
D. Mamma, E. Kalogeris, N. Papadopoulos, D. G. Hatzinikolaou, P. Christrakopoulos and D. Kekos.
Journal of Environmental Science and Health, Part A Toxic/Hazardous Substances and Environmental, vol. 39, pages 2093 – 2104, (2004).  View at publisher's site  Request copy
Abstract: Biodegradation of phenol, a pollutant derived from many industrial processes, was achieved through acclimatized Pseudomonas putida cells. The strategy to overcome the inhibitory effect of phenol on microbial growth involved the addition of glucose, a conventional carbon source. A factorial experimental design was employed in order to optimize the initial phenol and glucose concentrations. The optimum conditions found were applied in 2-lt bioreactors. The development of acclimatized cells and the use of glucose as an added growth substrate resulted in a significant phenol degradation rate of 60.7 mg^.L^-1h^-1 with a complete removal of 1200 mg^.L^-1 phenol.

18. "Biochemical and catalytic properties of two intracellular β-glucosidases from the fungus Penicillium decumbens active on flavonoid glucosides".
D. Mamma, D. G. Hatzinikolaou and P. Christakopoulos.
Journal of Molecular Catalysis B: Enzymatic, vol. 27, pages 183-190, (2004).  View at publisher's site  Request copy
Abstract: In the presence of rutin as sole carbon source,Penicillium decumbens produces two intracellular β-glucosidases named GI and GII, with molecular masses of 56,000 and 460,000 Da, respectively. The two proteins have been purified to homogeneity. GI and GII composed of two and four equal sub-units, respectively and displayed optimal activity at pH 7.0 and temperature 65–75 °C. Both β-glucosidases were competitively inhibited by glucose and glucono-δ-lactone. GI and GII exhibited broad substrate specificity, since they hydrolyzed a range of (1,3)-, (1,4)- and (1,6)-β-glucosides as well as aryl β-glucosides. Determination of kcat/Km revealed that GII hydrolyzed 3–8 times more efficiently the above-mentioned substrates. The ability of GI and GII to deglycosylate various flavonoid glycosides was also investigated. Both enzymes were active against flavonoids glycosylated at the 7 position but GII hydrolyzed them 5 times more efficiently than GI. Of the flavanols tested, both enzymes were incapable of hydrolyzing quercetrin and kaempferol-3-glucoside. The main difference between GI and GII as far as the hydrolysis of flavanols is concerned, was the ability of GII to hydrolyze the quercetin-3-glucoside.

17. "Biochemical Characterization of the Multi-enzyme System Produced by Penicillium decumbens Grown on Rutin".
D. Mamma, E. Kalogeris, D. G. Hatzinikolaou, A. Lekanidou, D. Kekos, B. J. Macris and P. Christakopoulos.
Food Biotechnology, vol. 18, pages 1-18, (2004).  View at publisher's site  Request copy
Abstract: Penicillium decumbens produced a set of enzymes, including a monoxygenase and two glycosidases, which degrade rutin, a nontoxic flavonoid glycoside, to water-soluble products. The monoxygenase (quercetinase) cleaves the heterocyclic ring in quercetin, the aglycone part of rutin. The glycosidases (α-L-rhamnosidase and β-glucosidase) hydrolyze the bonds between quercetin and rutinose, and between glucose and rhamnose, the constituent monosaccharides of rutinose. Simultaneous production of the three enzymes was optimized following the examination of a number of culture conditions. Maximum enzyme activities were observed when the fungus was grown at 30°C with an initial pH of 7.0, using 8.0 g/L rutin and 9.0 g/L di-ammonium hydrogen phosphate as carbon and nitrogen sources, respectively. The enzymes were purified to electrophoretic homogeneity by a series of consecutive chromatographic steps including anion and cation exchange as well as gel filtration. The purified quercetinase revealed an apparent tetrameric structure, with a reduced molecular mass of 45 kDa. α-L-Rhamnosidase showed an apparent molecular mass of 58 kDa and the purified β-glucosidase was a tetramer exhibiting a reduced molecular mass of 120 kDa.

16. "Study of the mechanisms of cigarette smoke gas phase cytotoxicity".
C. Piperi, A. E. Pouli, N. A. Katerelos, D. G. Hatzinikolaou, A. J. Stavridou and M. C. Psallidopoulos.
Anticancer Research, vol. 23, pages 2185-2190, (2003).  View at publisher's site  Request copy
Abstract: BACKGROUND: The cytotoxicity of cigarette smoke (CS) in humans is well-documented, but the mechanism behind CS toxicity and carcinogenicity remains unknown. We are interested in the toxicological effects of CS gas phase and the biological mechanisms of its action. MATERIALS AND METHODS: Gas phase CS cytotoxicity was measured by Wst-1 and LDH assays, in cultured cells. The mechanism of cell death was investigated by flow cytometric analysis using Annexin V and PI staining. Gas phase CS-induced oxidative damage was evaluated by estimating cellular glutathione (GSH) levels. Protein modifications (nitration of tyrosines) induced by gas phase CS and activation of key signalling proteins (Mitogen-activated protein kinase, MAPK) were detected by immunoblotting. RESULTS: The cytotoxicity of gas phase CS was found to be dose-dependent. The mechanism of cell death was found to be both apoptotic and necrotic depending on the concentrations used. Exposure to gas phase CS resulted in depletion of cellular GSH levels, increased nitrotyrosine immunoreactivity and phosphorylation of p44/42 MAPK proteins. CONCLUSION: These results suggest that the CS gas phase alone contributes significantly to the deleterious effects of CS in cellular systems.

15. "The cytotoxic effect of volatile organic compounds of the gas phase of cigarette smoke on lung epithelial cells".
A. E. Pouli, D. G. Hatzinikolaou, C. Piperi, A. Stavridou, M. C. Psallidopoulos and J. C. Stavrides.
Free Radical Biology & Medicine, vol. 34, pages 345-355, (2003).  View at publisher's site  Request copy
Abstract: Health effects of cigarette smoke (CS) in humans are well known from both clinical and epidemiological studies. However, the mechanism behind CS toxicity and carcinogenicity remains mainly unknown. Recent studies have pointed to the major importance of the gas phase of CS in generating its cytotoxic effects. In the current study, an exposure system capable of introducing the gas phase of mainstream cigarette smoke deprived of its volatile organic constituents (VOCs) was used to study the role of the nonorganic components of the gas phase on the cytotoxicity of smoke to monolayer cultures of mouse lung epithelial cells. Cell viability was measured by Wst-1 and the lactate dehydrogenase (LDH) assays. In cells treated with increasing doses of mainstream cigarette smoke gas phase (one to nine puffs), a dose-dependent increase in cytotoxicity was observed (one puff, 95% viability; nine puffs, 40% viability). Cell viability of cultures exposed to gas phase with only the nonorganic components was found to be equivalent to control, unexposed cultures, indicating that removal of VOCs resulted in almost eliminating the cytotoxic ability of the gas phase of CS. Furthermore, the removal of VOCs seems to reduce the effects of protein tyrosine nitration mediated through the gas phase constituents. The results obtained suggest the important and decisive role of VOCs in inducing cytotoxic effects.

14. "Comparative growth studies of the extreme thermophile Sulfolobus acidocaldarius in submerged and solidified substrate cultures".
D. G. Hatzinikolaou, E. Kalogeris, P. Christakopoulos, D. Kekos and B. J. Macris.
World Journal of Applied Microbiology and Biotechnology, vol. 17, pages 229-234, (2001).  View at publisher's site  Request copy
Abstract: An attempt was made, for the first time, to exploit cultures on solidified substrates (SSC) as an alternative to submerged cultures (SmC) for growing extremophilic microorganisms. The extreme thermophilic archaebacterium Sulfolobus acidocaldarius was grown on a number of carbon sources and, in all experiments, biomass yields and growth rates were always higher in SSC than in the corresponding SmC. Inoculum age significantly affected growth characteristics on both types of fermentation. Heavy growth of the microorganism in SSC was observed on low-cost carbon sources such as starch. Wheat bran significantly enhanced growth characteristics when used to supplement starch media. The results of this work show that cultures on solid surfaces could be a promising alternative method for growing extreme thermophiles.