Inhibitory kinetics of citric acid on beta-N-acetyl-d-glucosaminidase from prawn (Litopenaeus vannamei).

NAGase (EC.3.2.1.52) from crustaceans has the important roles in immunity, molting and digestion of chitinous foods. In this paper, the effects of citric acid on the activity of NAGase from Litopenaeus vannamei for the hydrolysis of pNP-NAG have been studied. The results showed that appropriate concentrations of citric acid could lead to reversible inhibition on NAGase and IC(50) was estimated to be 5.00 +/- 0.35 mM. Using the plots of Lineweaver-Burk, the inhibition of NAGase by citric acid belongs to competitive type, the inhibitory equilibrium constant for citric acid binding with free NAGase, K(I), is 3.26 +/- 0.25 mM. The inhibitory kinetics of citric acid on NAGase in the appropriate concentrations of citric acid has been studied using the kinetic method of substrate reaction. The time course of NAGase for the hydrolysis of pNP-NAG in the presence of different concentrations of citric acid showed that at each citric acid concentration, the rate decreased with increasing time until a straight line was approached. The results show that the inhibition of NAGase by citric acid is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction on citric acid with NAGase.

[Comparison of soil respiration in natural Castanopsis carlesii forest and plantation forest].

By using the Li-8100 open soil carbon flux system, the dynamic change of soil respiration rate in natural Castanopsis carlesii and plantation of Castanopsis carlesii forests in Geshikao Nature Reserve in Fujian Province of China were measured from January 2011 to December 2011, with the relationship between the dynamic changes and the relation affecting factors analyzed. The monthly variation of soil respiration in the two types of forests were both single-peaked,with the peaks appeared in early June [7.03 micromol x (m2 x s) (-1)] andlate July [5.12 micromol x (m2 x s)(-1)], respectively. The average annual soil respiration rates of the two forests were 3.74 micromol x (m2 x s)(-1) and 3.05 micromol x (m2 x s)(-1), respectively, showing significant difference. Soil temperature was the main factor affecting soil respiration, explaining 80.1% and 81.0% of the monthly variation of soil respiration. There was a significant positive correlation between the soil respiration rate and soil moisture content in natural Castanopsis carlesii forest, but lower correlation in plantation of Castanopsis carlesii forest. The soil respiration had extremely significant correlation with the litterfall mass of the current month and the month before. The Q10 values of soil respiration in natural Castanopsis carlesii and plantation of Castanopsis carlesii forests were 1.86 and 2.01, and the annual CO2 fluxes were 14.34 t x (hm2 x a)(-1) and 11.18 t x (hm2 x a)(-1), respectively. The soil respiration declined by 22.03% after natural forest was changed to plantation forest.

[Concentration characteristics and ecological risk of persistent organic pollutants in the surface sediments of Tianjin coastal area].

Surface sediments were sampled from various surface water bodies in Tianjin coastal area, and four types of persistent organic pollutants including polycyclic aromatic hydrocarbons (PAHs), organic pesticides (OCPs), polychlorinated biphenyl (PCBs) and polybrominated diphenyl ethers (PBDEs) were measured. The purposes were to investigate the concentration characteristics of the four types of pollutants and to assess their ecological risks. The results showed that all the 16 priority PAHs were detected from the 10 sediments sampled from the Tianjin coastal area. The total concentrations of the 16 PAHs ranged from 274.06 microg x kg(-1) to 2656.65 microg x kg(-1), with the average being 1 198.51 microg x kg(-1). Combustion of fossil fuel such as coal and gasoline was the major source of PAHs in the surface sediments, while input of petroleum products might occur in some locations. In the Dagu discharging river, the total concentration of 22 OCPs was 3 103.36 microg x kg(-1), the total concentration of 35 PCBs and the total concentration of 14 PBDEs were 87.31 microg x kg(-1) and 13.88 microg x kg(-1), respectively. The concentrations of OCPs, PCBs and PBDEs in other sampling locations were low. The total organic carbon in the surface sediments had good correlation with PAHs but not with OCPs, PCBs and PBDEs, and this might be due to the fact that PAHs mainly came from area pollution while the other compounds mainly came from point pollution. In the sediments, PAHs (particularly low molecular weight PAHs) had high ecological risk; in multiple locations, the concentrations of naphthalene and/or acenaphthene exceeded their probable effect concentrations, indicating that they were most likely causing adverse effects to benthonic organisms. In the Dagu discharging river, the concentrations of heptachlor epoxide and lindane (gamma-BHC) exceeded their probable effect concentrations and were most likely causing adverse effects to benthonic organisms. The OCPs in other sampling locations had low ecological risk. Overall, PCBs and PBDEs had low ecological risk.

[Microbial degradation of 2, 2', 4, 4'-tetrabrominated diphenyl ether under anaerobic condition].

Polybrominated diphenyl ethers are emerging contaminants in the environment, among which 2, 2' 4, 4'-tetrabrominated diphenyl ether (BDE-47) is one of the most frequently detected and the most toxic to organisms. Using BDE-47 as the object, this study investigated the degradation of BDE-47 by six enriched cultures containing Dehalococcoides species. The purpose is to understand the microbial degradation of BDE-47 under anaerobic condition and its kinetics. Serum bottles of 100 mL were used to construct the microcosms and enrich the anaerobic microorganisms. For each culture, two treatments were performed. One used BDE-47 as the only energy source with the designed concentration of 200 microg x L(-1), and the other used both BDE-47 and trichloroethene (TCE) as the energy source with the designed concentrations being 200 microg x L(-1) and 13 mg x L(-1), respectively. After three months of experiment, two enriched cultures (6M6B and T2) could obviously degrade BDE-47, producing BDE-17 and BDE-4 (and small amount of DE). The presence of TCE inhibited the degradation of BDE-47 by 6M6B and T2 cultures to a certain degree. The PCR-DGGE assay was performed to compare the microbial community structure of various enrichments and it was found that Acetobacterium sp. had good correlation with the degradation of BDE-47. Under three different initial BDE-47 concentrations (50 microg x L(-1), 250 microg x L(-1) and 500 microg x L(-1)), the degradation rates of BDE-47 were 0.003 3, 0.001 4 and 0.001 0 d(-1), respectively. This study demonstrated that BDE-47 could be reductively degraded by bacteria under anaerobic condition, producing BDE-17 and BDE-4. Acetobacterium sp. might play an important role in the degradation of BDE-47. High concentration of BDE-47 might inhibit the activity of the BDE-47 degrading bacteria to a certain degree.

[Enhanced bioremediation of coking plant soils contaminated with polycyclic aromatic hydrocarbons].

Soil samples contaminated with polycyclic aromatic hydrocarbons (PAHs) were collected from Beijing Coking Plant. The purposes were to isolate PAHs degrading bacteria from the soils, determine their appropriate living condition, enrich them and apply them in the enhanced bioremediation of the contaminated soils. Using each of the 16 USEPA priority PAHs as the sole carbon source, PAHs degrading bacteria were isolated using the method of plate streaking and identified by genetic analysis. In total seven species of PAHs degrading bacteria were obtained. When mixed, these bacteria could degrade the 16 (2-6 cyclic) PAHs studied at appropriate concentrations. In the liquid medium, when the total concentration of the 16 PAHs (sigma PAH16) was 17 microg/mL, single bacteria could grow well and degrade the PAHs. However, when sigma PAH16 was 166 microg/mL, the growth and activity of either single PAHs degrading bacteria or a mixture of the seven PAHs degrading bacteria were inhibited. Aiming at the contaminated soils from Beijing coking plant, five treatments were performed, i.e., control (C), addition of nutrient (N), addition of nutrient and PAHs degrading bacteria (N + B), addition of nutrient and surfactant (N +S), addition of nutrient and PAHs degrading bacteria and surfactant (N + B + S). After five weeks of experiment, compared to the C treatment, the mean removal rate of the 16 PAHs in the N + B treatment was increased 32%, and the mean removal rate of the 16 PAHs in the N + B + S treatment was increased 46% (the mean removal rate of the 10 4-6 cyclic PAHs was increased 52%). The addition of PAHs degrading bacteria and surfactant could significantly enhance the degradation of PAHs in the soils. This study provides evidence for the enhanced bioremediation of PAHs contaminated soil for Beijing coking plant and other coking plants.

Inhibitory effects of fatty acids on the activity of mushroom tyrosinase.

The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.

Inactivation kinetics of polyphenol oxidase from pupae of blowfly (Sarcophaga bullata) in the dimethyl sulfoxide solution.

The effects of dimethyl sulfoxide (DMSO) on the activity of polyphenol oxidase (PPO, EC 1.14.18.1) from blowfly pupae for the oxidation of L-3,4-dihydroxyphenylalanine were studied. The results showed that low concentrations of DMSO could lead to reversible inactivation to the enzyme. The IC(50) value, the inactivator concentration leading to 50% activity lost, was estimated to be 2.35 M. Inactivation of the enzyme by DMSO was classified as mixed type. The kinetics of inactivation of PPO from blowfly pupae in the low concentrations of DMSO solution was studied using the kinetic method of the substrate reaction. The rate constants of inactivation were determined. The results show that k(+0) was much larger than k'(+0), indicating that the free enzyme molecule was more fragile than the enzyme-substrate complex in the DMSO solution. It was suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.

Inactivation kinetics of beta-N-acetyl-D-glucosaminidase from green crab (Scylla serrata) in dioxane solution.

Beta-N-acetyl-D-glucosaminidase (NAGase, EC.3.2.1.52), which catalyzes the cleavage of N-acetylglucosamine polymers, is a composition of chitinase and cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into N-acetylglucosamine (NAG). In this investigation, A NAGase from green crab (Scylla serrata) was purified and the effects of dioxane on the enzyme activity for the hydrolysis of p-Nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were studied. The results show that appropriate concentrations of dioxane can lead to reversible inactivation of the enzyme and the inactivation is classified as mixed type. The value of IC50, the dioxane (inactivator) concentration leading to 50% activity lost, is estimated to be 0.68%. The kinetics of inactivation of NAGase in the appropriate concentrations of dioxane solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results showed that k+0 is much larger than k'+0, indicating the free enzyme molecule is more fragile than the enzyme-substrate complex in the dioxane solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by dioxane.