Activation of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine with poly(salicylic acid 5-aminodisulfide)

5-Aminosalicylic acid (5-ASA) inhibited by a mixed mechanism the peroxidase catalyzed oxidation of tetramethylbenzidine (TMB) in 0.015 M phosphate-citrate buffer (pH 6.4) supplemented with 5% DMSO and 5% DMF. Poly(salicylic acid 5-aminodisulfide) (poly(SAADS)) in 0.01 M phosphate buffer (pH 6.2-7.4) supplemented with 5% DMSO and 5% DMF effectively activated the peroxidase-catalyzed oxidation of TMB. The activation was quantitatively characterized by coefficients (M^–1) determined at different pH values: increased linearly with increase in pH up to the maximal value of 2.44·10⁵ M^–1 at pH 7.0. The activating effect of poly(SAADS) on the peroxidase-catalyzed oxidation of TMB is explained by the activator properties of polyelectrolyte, with its anionic form interacting with peroxidase sites responsible for the acid-base catalysis.     

Effects of Size of Carbohydrate Chain on Protease Digestion of Aspergillus niger Endo-β-1,4-glucanase

Three different carbohydrate-depleted enzymes were prepared from an endo-β-l,4-glucanase of Aspergillus niger IF031125 by treatment with endo-β-N-acetylglucosaminidase or α-mannosidase. They were purified by Concanavalin A-Sepharose affinity and DEAE ion-exchange column chromatographies. The molecular sizes of these enzymes had been decreased from 40 kDa containing 9.0% carbohydrate to 39, 38, and 37kDa with carbohydrate at 4.5, 1.3, and 0.8% (wt/wt), respectively. The native and these carbohydrate-depleted enzymes were compared in their enzymatic properties, and it was found that they were identical in their catalytic activities and both thermal and pH stabilities. However, the 37-kDa enzyme was more susceptible to proteolysis by Savinase, proteinase K, and Pronase E. On the other hand, the specific protease trypsin showed no such effect on activity of all enzymes. These results suggested that the core structure of the asparagine-linked sugar chain, which consisted of three monosaccharide residues, contributed to the high stability of the endo-β-l,4-glucanase against protease digestion.     

The Biotransformation of Propylene to Propylene Oxide by Methylococcus Capsulatus (Bath): 1. Optimization of Rates

The rates of propylene oxidation to propylene oxide by Methylococcus capsulatus (Bath) have been optimized in small shake flasks to establish the potential of this process for industrial application. It was shown that addition of the electron donors methanol, formaldehyde, formate or hydrogen stimulated the endogenous rate of propylene oxide formation 10 to 50 fold. Rates in excess of 500 nmol min^-1 mg dry weight cells^-1 have been obtained using methanol as the donor. These high rates could only be sustained for 3 to 4 min before loss of biocatalytic activity caused the rate of propylene oxide production to decline.     

Enhancement of ethene removal from waste gas by stimulating nitrification

The treatment of poorly water soluble waste gas compounds,such as ethene, is associated with low substrateconcentration levels in the liquid phase. This lowconcentration level might hamper the optimal development ofa microbial population. In this respect, the possible benefit ofintroducing nitrifying activity in the heterotrophic removal ofethene at moderate concentrations (< 1000 ppm) from awaste gas was investigated. Nitrifying activity is known to beassociated with (i) the production of soluble microbialproducts, which can act as (co-)substrates for heterotrophicmicro-organisms and (ii) the co-oxidation of ethene. Theused reactor configuration was a packed granular activatedcarbon biobed inoculated with the heterotrophic strain Mycobacterium E3. The nitrifying activity was introduced byregular submersion in a nitrifying medium prepared from (i)compost or (ii) activated sludge. In both cases a clearenhancement of the volumetric removal rate of ethene couldbe observed. When combined with a NH₃ dosage on adaily basis, a gradual increase of the volumetric removal rateof ethene could be observed. For a volumetric loading rateof 3 kg ethene-COD·m^-3·d^-1, the volumetric removal rate could thus be increased with a factor1.8, i.e. from 0.72 to a level of 1.26 kgethene-COD·m^-3·d^{- 1}.     

Enhanced dechlorination of chloroethenes by granular sludge under microaerophilic conditions

This study investigates an innovative dechlorination process using anaerobic granular sludge that was partially exposed to oxygen. The exposure supported a synchronously anaerobic and aerobic bioconversion process that combined reductive dechlorination with aerobic co-oxidation in a sludge granule. Experimental results showed that the highest dechlorination rates of tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride were 6.44, 2.98, 1.70 and 0.97 nmol/gVS day, at initial O₂ concentrations of 10, 100, 5 and 0%, respectively. Strictly anaerobic conditions favored the dechlorination of vinyl chloride while absolutely aerobic conditions were preferred for trichloroethene dechlorination. Microaerophilic conditions are suggested to ensure the overall biodegradation of the chlorinated ethenes present in groundwater as a mixture.     

Epoxypropane biosynthesis by Methylomonas sp. GYJ3: batch and continuous studies

Methylomonas sp. GYJ3 is a methanotrophic bacterium containing methane monooxygenase (MMO), which catalyses the epoxidation of propene to epoxypropane. In this study, the cell suspension of Methylomonas sp. GYJ3 has been used for epoxypropane biosynthesis from propene. When propene is epoxidized, the product epoxypropane is not further metabolized and accumulates extracellularly. Unfortunately, continuous production of epoxypropane is usually difficult due to exhaustion of reductant and the accumulation of toxic products. Hence, in order to address these problems, batch experiments were performed to explore the possibility of producing epoxypropane by a co-oxidation process. Methane was chosen as the most suitable electron-donating co-substrate since it did not result in molecular toxicity and provided abundant reductant for epoxidation. It was found that the maximum production of epoxypropane occurred in an atmosphere of 30% methane. Batch experiments also indicated that continuous removal of product was necessary to overcome the inhibition of epoxypropane. In continuous experiments, optimum mixed gaseous substrates were continuously circulated through the stirred tank bioreactor to remove product from the cell suspension. Initial epoxypropane productivity was 268 mol/day. The bioreactor has been allowed to operate continuously for 12 days without obvious loss of epoxypropane productivity, and more than 96% of initial MMO activity was retained.     

Peroxidase-Catalyzed Co-oxidation of 3,3",5,5"-Tetramethylbenzidine with 2-Amino-4-nitrophenol, 4,4"-Dihydroxydiphenylsulfone, and Their Polydisulfides in Aqueous and Micellar Media

The effects of different concentrations of 2-amino-4-nitrophenol (ANP) and of its polydisulfide (poly(ADSNP)) on peroxidase-catalyzed oxidation of 3,3"5,5"-tetramethylbenzidine (TMB) were studied at 20°C in reversed micelles of AOT (0.2 M) in heptane and in mixed reversed micelles of AOT (0.1 M)–Triton X-100 (0.1 M) in isooctane supplemented with 15% hexanol. The oxidation of TMB was activated nearly twofold in the presence of ANP and nearly fourfold in the presence of poly(ADSNP) in reversed micelles of AOT, whereas in the mixed micelles oxidation of the TMB–ANP pair was associated with inhibition of TMB conversion and poly(ADSNP) activated oxidation of TMB. The co-oxidation of TMB with 4,4"-dihydroxydiphenylsulfone (DDS) and with its polydisulfide (poly(DSDDS)) at different concentrations of phenol components was accompanied by activation of TMB conversion in 0.01 M phosphate buffer (pH 6.4) supplemented with 5% DMF and in reversed micelles of AOT in heptane. The effect of pH of the aqueous solution on the initial oxidation rate of the TMB–DDS and TMB–poly(DSDDS) pairs and also the effect of hydration degree of reversed micelles of AOT on conversion of the same pairs by peroxidase were studied. A scheme of peroxidase-dependent co-oxidation of aromatic amine–phenol pairs is proposed and discussed. A significant part of this scheme is a nonenzymatic exchange of phenoxyl radicals with amines and of aminyl radicals with phenols.     

Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines

Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117±12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9±0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106±11 nmol HCHO/min/mg SLO, and 3.2±0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides.