13. "Purification and characterization of an extracellular α-L-arabinofuranosidase from Fusarium oxysporum".
P. Christakopoulos, P. Katapodis, D. G. Hatzinikolaou, D. Kekos and B. J. Macris.
Applied Biochemistry and Biotechnology, vol. 87, pages 127-133, (2000).   View at publisher's site   Request copy
Abstract: An α-L-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-L-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-L-arabinofuranosidase and an endo-(1→4)-β-D-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.

12. "Hemicellulolytic activity of Fusarium oxysporum grown on sugar beet pulp. Production of extracellular arabinanase".
T. Cheilas, T. Stoupis, P. Christakopoulos, P. Katapodis, D. Mamma, D. G. Hatzinikolaou, D. Kekos and B. J. Macris.
Process Biochemistry, vol. 35, pages 557-561, (2000).   View at publisher's site   Request copy
Abstract: Fusarium oxysporum F3 exhibited hemicellulolytic enzymic activity when grown on sugar beet pulp, a by-product of the sugar industry. The growth medium was specifically optimised for enhanced production of extracellular arabinanase. The optimum medium contained sugar beet pulp (4%, w/v) and corn steep liquor (6%, v/v) as carbon and nitrogen sources, respectively. Arabinanase activity as high as 0.25 U/ml of culture was obtained, which compared favourably to those reported for other microorganisms. Optimal arabinanase activity was observed at pH 6–7 and 50 °C. Investigation of the degradation of the main components of sugar beet pulp showed that arabinose containing polysaccharides and pectin were first degraded, followed by the glucose-containing polysaccharides.

11. "A novel lipolytic activity of Rhodotorula glutinis cells: Production, partial characterization and application in the synthesis of esters ".
D. G. Hatzinikolaou, E. Kourentzi, H. Stamatis, P. Christakopoulos, F. N. Kolisis, D. Kekos and B. J. Macris.
Journal of Bioscience and Bioengineering, vol. 88, pages 53-56, (1999).   View at publisher's site   Request copy
Abstract: Cell-bound lipase activity (10 pNPL units/g dry cell weight) was released when the yeast Rhodotorula glutinis was cultured in a 7-l stirred tank fermentor using palm-oil as the sole carbon source. The enzyme showed relative specificity towards medium chain organic acids since the apparent K_m values for pNPB (p-NitroPhenyl-Butyrate) and pNPL (p-NitroPhenyl-Laurate) were equal to 2.7 and 0.7 mM, respectively. In addition, 80% of this activity could be detected on the surface of the cells. The cell-bound nature of the enzyme increased its thermal stability showing half-life times of 200 and 60 min at 50 and 60°C, respectively, as well as good stability in organic solvents. Freeze-dried cell preparations were successfully used to catalyze the synthesis of fatty acid esters of butanol and heptanol in nearly anhydrous organic solvents. A conversion of 60–62% was obtained upon esterification of palmitic or oleic acid with butanol, within 96 h. The enzyme preparation was used in four consecutive batch reactions with only 10% loss of activity.

10. "Purification and Mode of Action of an Alkali-Resistant Endo-1,4-β-glucanase from Bacillus pumilus".
P. Christakopoulos, D. G. Hatzinikolaou, G. Fountoukidis, D. Kekos, M. Claeyssens and B. J. Macris.
Archives of Biochemistry and Biophysics, vol. 364, pages 61-66, (1999).   View at publisher's site   Request copy
Abstract: Alkaline endo-1,4-β-D-glucanase was secreted by Bacillus pumilus grown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pI values of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0–8.0 and 60 °C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30 °C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-β-D-glucoside, 4-nitrophenyl-β-D-cellobioside, and 4-nitrophenyl-β-D-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.

9. "Enhancement of pH-stability of a low molecular mass endoglucanase from Fusarium oxysporum by protein pegylation ".
P. Christakopoulos, E. Kourentzi, D. G. Hatzinikolaou, M. Claeyssens, D. Kekos and B. J. Macris.
Carbohydrate Research, vol. 314, pages 95-99, (1998).   View at publisher's site   Request copy
Abstract: The stability of the low molecular mass endoglucanase (23.2 kDa) from Fusarium oxysporum at alkaline pH was enhanced by chemical modification. Two distinct types of amino acid-specific modifiers were used. The first, either cyanuric chloride activated polyethylene glycol (CC–PEG) or polyethylene glycol succinimidyl succinate active ester (SS–PEG), react (more or less specifically) with protein amino groups. The second type, maleimide polyethylene glycol (Mal–PEG), is specific for cysteinyl residues. The enzyme lost almost all of its activity when modified with CC–PEG, whereas no inactivation was observed with SS–PEG and Mal–PEG. The modified endoglucanase showed remarkably enhanced alkaline pH stability. When acting upon cello-oligosaccharides and 4-methylumbelliferyl cello-oligosaccharides, the enzyme preferentially cleaved the internal glycosidic bonds. The modified enzymes mediated a decrease in the viscosity of carboxymethyl cellulose (CMC) associated with the release of only small amounts of reducing sugar. Thus, the modified enzyme retains the endo character of the native enzyme.

8. "An efficient and optimized purification procedure for the superoxide dismutase from Aspergillus niger. Partial characterization of the purified enzyme".
D. G. Hatzinikolaou, C. Tsoukia, D. Kekos and B. J. Macris.
Bioseparation, vol. 7, pages 39-46, (1997).   View at publisher's site   Request copy
Abstract: Cu,Zn-superoxide dismutase was isolated from Aspergillus niger mycelia, harvested at the mid-logarithmic growth phase. The purification scheme aimed at the optimization of the ethanol/chloroform extraction (Tsuchihashi extraction) through response surface methodology. Upon optimum extraction conditions, it was possible to obtain electrophoretically pure enzyme preparations, by the application of one step anion exchange chromatography. The enzyme yield of this simple purification procedure was above 75% while the specific activity of the final preparation was among the highest reported for eucariotic microorganisms. The purified enzyme exhibited similar physicochemical characteristics with other Aspergillus sp. superoxide dismutases revealing an apparent tetrameric structure with a subunit molecular weight of 19 kDa, and a pl of 5.95.

7. "A new glucose oxidase from Aspergillus niger: characterization and regulation studies of enzyme and gene".
D. G. Hatzinikolaou, O. C. Hansen, B. J. Macris, A. Tingey, D. Kekos, P. Goodenough and P. Stougaard.
Applied Microbiology and Biotechnology, vol. 46, pages 371-381, (1996).   View at publisher's site   Request copy
Abstract: A new glucose oxidase from Aspergillus niger was isolated and characterized. The enzyme showed different kinetic and stability characteristics when compared to a commercially available batch of A. niger glucose oxidase. The gene encoding the new glucose oxidase was isolated and DNA sequence analysis of the coding region showed 80% identity to the sequence of a glucose oxidase gene previously published. However, the similarity of the non-coding sequences up- and downstream of the open reading frame was much less, showing only 66% and 50% identity respectively. Despite the low degree of similarity between the promotor region of the new gene and the previously published one, the new glucose oxidase was likewise induced by calcium carbonate. In addition, we showed that this induction occurred on the transcriptional level. Observations concerning the effect of gluconolactone and the levels of glucose-6-phosphate isomerase upon calcium carbonate induction suggested that the enhancement of glucose oxidase biosynthesis by calcium carbonate was accompanied by a metabolic shift from glycolysis to the pentose phosphate pathway.

6. "Studies on localization and regulation of lipase production by Aspergillus niger".
J. B. Macris, E. Kourentzi and D. G. Hatzinikolaou.
Process Biochemistry, vol. 31, pages 807-812, (1996).   View at publisher's site   Request copy
Abstract: The lipase from strain BTL of Aspergillus niger was studied. The enzyme, which was mainly extracellular, was produced at elevated activity levels under optimum growth conditions. De novo biosynthesis of lipase occurred only in the presence of lipids and was completely repressed by glucose and glycerol. The reaction products, oleic acid and glycerol, showed differed inhibition patterns during triolein hydrolysis. The enzyme exhibited high specificity towards middle chain triglycerides and was possibly activated by double bonds in the fatty acid chain. It exhibited a marked stability against organic solvents.

5. "Production and partial characterization of extracellular lipase from Aspergillus niger".
D. G. Hatzinikolaou, J. B. Macris, P. Christakopoulos, D. Kekos, F. N. Kolisis and G. Fountoukidis.
Biotechnology Letters, vol. 18, pages 547-552, (1996).   View at publisher's site   Request copy
Abstract: The production and certain kinetic characteristics of extracellular lipase from Aspergillus niger were investigated. It was possible to substantially enhance the activity of excreted lipase by optimising the interaction between carbon and nitrogen sources applying a two-parameter complete experimental design and response surface analysis. The enzyme was partially purified and a number of kinetic characteristics such as optimum pH and temperature, thermal and pH stability and Km were determined and discussed. The elevated levels of lipase activity (40.5 U/ml) found in this work competed favourably with most of those reported for lipase hyperproducing fungi.