Optimization of intracellular product release from Neisseria denitrificans using microfluidizer.

Disruption of Neisseria denitrificans cells by microfluidizer was optimized using a factorial experiments design. The pH, pretreatment time, cell concentration, NaCl, ethylenediamine tetraacetic acid (EDTA) and Triton X-100 concentrations showed significant impact on disruption process and the process was optimized using central composite design and response surface methodology (RSM). Investigation revealed optimum conditions: 90 min pretreatment at pH 9.0 containing 110 g L(-1) cells (dry cell weight), 50 mM NaCl, 10 mM EDTA, and 0.2% Triton X-100. At optimized conditions, the disruption rate increased twofold, up to 5.62 ± 0.27 × 10(-3) MPa(-a); meanwhile, yield of intracellular content was increased by 26%, with 1 g of cells resulting in 113.2 ± 8.2 mg proteins, 12.1 ± 0.7 mg nucleic acids, 21.0 ± 1.2 mg polysaccharides, 0.99 ± 0.08 kU glucose-6-phosphate dehydrogenase (G6PD), and 10,100 ± 110 kU restriction endonuclease NdeI endonuclease. Particle size distribution analysis revealed nearly twofold larger cell lysate particles with diameter of 120 nm. For optimal release of intracellular content, 9200 J/g of energy was needed (95% confidence), yielding 6900 J/g energy savings. Model equations generated from RSM on cell disruption of N. denitrificans were found adequate to determine significant factors and its interaction. The results showed that optimized combination of known pretreatment and disruption methods could considerably improve cell disruption efficiency.

Intensification of biocatalytical processes by synergistic substrate conversion. Fungal peroxidase catalyzed N-hydroxy derivative oxidation in presence of 10-propyl sulfonic acid phenoxazine.

Many industrial pollutants, xenobiotics, and industry-important compounds are known to be oxidized by peroxidases. It has been shown that highly efficient peroxidase substrates are able to enhance the oxidation of low reactive substrate by acting as mediators. To explore this effect, the oxidation of two N-hydroxy derivatives, i.e., N-hydroxy-N-phenyl-acetamide (HPA) and N-hydroxy-N-phenyl-carbamic acid methyl ester (HPCM) catalyzed by recombinant Coprinus cinereus (rCiP) peroxidase has been studied in presence of efficient substrate 3-(4a,10a-dihydro- phenoxazin-10-yl)-propane-1-sulfonic acid (PPSA) at pH 8.5. The bimolecular constant of PPSA cation radical reaction with HPA was estimated to be (2.5 +/- 0.2).10(7) M(-1) s(-1) and for HPCM was even higher. The kinetic measurements show that rCiP-catalyzed oxidation of HPA and HPCM can increase up to 33,000 times and 5,500 times in the presence of equivalent concentration of high reactive substrate PPSA. The mathematical model of synergistic rCiP-catalyzed HPA-PPSA and HPCM-PPSA oxidation was proposed. Experimentally obtained rate constants were in good agreement with those calculated from the model confirming the synergistic scheme of the substrate oxidation. In order to explain the different reactivity of substrates, the docking of substrates in the active site of the enzyme was calculated. Molecular dynamic calculations show that the enzyme-substrate complexes are structurally stable. The high reactive PPSA exhibited higher affinity to enzyme active site than HPA and HPCM. Furthermore, the orientation of HPA and HPCM was not favorable for proton transfer to the distal histidine, and different substrate reactivity was explained by these diversities.