Purification and characterization of polyphenol oxidase from corn tassel.

In this study, polyphenol oxidase (PPO) from corn tassel  was extracted and partially purified through  (NH4)2SO4 precipitation and gel filtration chromatography. Optimal temperatures for subsrates catechol and 4-methyl catechol were 40 °C and 30 °C, respectively. The optimal pH values were 8.0 for catechol and 6.0 for 4-methyl catechol. Catechol was the most suitible substrate (Km: 3.48 mM, Vmax: 1.0 Abs./ min.). The moleculer mass of PPO was determined as 158 kDa. In this work, sodium azide, ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) were found to inhibit the enzyme activity as 26.6 %,  22.2 % and 12.2 % ratio, respectively. Besides, the effects of carbohydrates such as sucrose, fructose, ribose and glucose on PPO activity were investigated. The enzyme was found to be activated 17 % by fructose and ribose, 16 % by glucose and 4 % by sucrose.

Cloning, purification and characterization of a thermostable ß-galactosidase from Bacillus licheniformis strain KG9.

A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.

The effects of organic pesticides on inner membrane permeability in Escherichia coli ML35.

We have tested whether some pesticides might cause inner membrane leakage in ML35 Escherichia coli cells, which express beta-galactosidase (lacZ; EC 3.2.1.23) constitutively but lack the permease (lacY) required for substrate entry. The activity of beta-galactosidase (indicative of substrate leakage through the inner membrane) was increased by various concentrations of pesticides, including the organometallic fungicides maneb and mancozeb, the insecticide Thiodan, and the herbicide Ally, as well as by antibiotics such as ampicillin, gramicidin D, and the calcium ionophore A23187. The enzyme activity was increased by up to approximately 30% when the E. coli ML35 strain was exposed to various concentrations (between 50 and 250 ppm) of both fungicides. Thiodan had only a slight effect on beta-galactosidase activity (increase of 12.8%), whereas, among the antibiotics, the calcium ionophore at 20 microg/ml caused a significant increase in enzyme activity by up to 61.8%. This effect is similar to that of sodium dodecyl sulfate, used as positive control ( approximately 70% increase). Accumulation of maneb and mancozeb by bacterial cells was also studied taking advantage of their metal content and using atomic absorption spectrophotometry. In parallel with the increase in enzyme activity, both fungicides accumulated in the cells as a function of their concentration. Time course experiments (3, 6, and 9 h) of fungicide accumulation and of bacterial growth at various pesticide concentrations were also carried out. Maneb seems to inhibit the bacterial growth better than mancozeb. In addition, maneb uptake increases with time up to 9 h at all tested concentrations, whereas the accumulation of mancozeb is similar at all the exposure times tested. This indicates a different uptake and/or metabolizing strategy by E. coli cells for the two fungicides.