Decrease In Specific Activity Research Articles

Implication of an unfavorable residue (Thr346) in intrinsic flexibility of firefly luciferase

In order to better understand the functional role of an unusual residue (Thr346) of firefly luciferase mutagenesis at this residue was performed. Firefly luciferase, catalyzes the bioluminescence reaction and is an excellent tool as a reporter in nano-system biology studies. Nonetheless, the enzyme rapidly loses its activity at temperatures above 30°C and this leads to reduced sensitivity and precision in analytical applications. Residue Thr346 in a connecting loop (341–348) of firefly luciferase is located in a disallowed region of Ramachandran plot. In this study, we have substituted this residue (T346) with anomalous dihedral angles with Val, Gly and Pro to clarify the role of this residue in structure and function of the enzyme using site-directed mutagenesis. Substitution of this unfavorable residue (T346) with atypical dihedral angles (ψ, φ) with other residues brought about an increase of thermostability and decrease of specific activity. Structural and functional properties of the mutants were analyzed using different spectroscopic methods. It seems that this residue is a critically conserved residue to support the functional flexibility for a fast kinetic bioluminescence reaction at the expense of lower stability.

Modulation of Lysozyme Activity by Lead Administered by Different Routes. In Vitro Study and Analysis in Blood, Kidney, and Lung

Modifications in the enzyme activity of lysozyme, a protein implied in the defence barrier of the organism, can be a good biomarker of alterations in the immune system as a result of exposure to toxic metal, such as lead. The aim of this work was to evaluate the effect of a 200 ppm dose of lead on lysozyme activity in blood, kidney, and lung, and also on tissue structure. Previously, the effect of lead acetate on lysozyme activity in vitro was determined; the in vitro results indicated that lead produced a decrease in enzyme activity. The activity loss was 16 % at 200 ppm of lead. Lead acetate was administered to Wistar rats by oral and intraperitoneal injections. An increase in lysozyme activity was observed in blood when lead was administered by introperitoneal route and in kidney by the oral route. The possible immunostimulation in kidney was discarded because of the structural alterations observed in the tissue. In lung, the decrease in specific lysozyme activity, for both routes of lead exposure, seems to indicate immunosupression, which was in accordance with the structural alterations observed in this tissue.

Teaching biochemistry and molecular biology using dihydrofolate reductase as an expression system

The enzyme dihydrofolate reductase (DHFR) converts dihydrofolic acid to tetrahydrofolic acid, thereby regulating the synthesis of purine and certain amino acids. Since DHFR plays such an important role in cell proliferation and growth, it was the first enzyme targeted for chemotherapy. The objective of this project is to teach molecular and biochemical techniques by having students induce gene expression, purify and enzymatically assay for DHFR activity. During this semester‐long project, students induce expression in recombinant bacteria and purify the DHFR protein using IMAC (immobilized metal affinity chromatography) resin. Students check purity via SDS‐PAGE electrophoresis and identify the fraction (insoluble vs. soluble) containing DHFR. Additionally, they design PCR primers for site‐directed mutagenesis, verify PCR success via restriction enzyme digestion and agarose gel electrophoresis, transform into expression competent cells, and confirm the amino acid mutation by DNA sequencing. Students then examine specific activity of the wildtype DHFR protein and compare it with the mutant DHFR protein. They also determine protein concentrations using the Bradford assay and perform Western blot analysis to detect glutathione S‐transferase (GST) and histidine (His) tags. All student groups successfully purified DHFR and detected both GST and His tags by Western blot analysis. Site‐directed mutagenesis showed a decrease in specific activity ranging from 32–122% compared to the wild‐type protein. This project provides students with the opportunity to gain hands‐on experience with a multitude of laboratory techniques as well as the opportunity to design and implement cutting edge research approaches.

Synthesis of silica particles and their application as supports for alcohol dehydrogenases and cofactor immobilizations: Conformational changes that lead to switch in enzyme stereoselectivity

FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60–70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-(p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (Vmax and Km) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.

Estimation of radioactive contamination of soils from the “Balapan” and the “Experimental field” technical areas of the Semipalatinsk nuclear test site

In spite of the long history of the research, radioactive contamination of the Semipalatinsk nuclear test site (SNTS) in the Republic of Kazakhstan has not been adequately characterized. Our cartographic investigation has demonstrated highly variable radioactive contamination of the SNTS. The Cs-137, Sr-90, Eu-152, Eu-154, Co-60, and Am-241 activity concentrations in soil samples from the “Balapan” site were 42.6–17646, 96–18250, 1.05–11222, 0.6–4865, 0.23–4893, and 1.2–1037 Bq kg−1, correspondingly. Cs-137 and Sr-90 activity concentrations in soil samples from the “Experimental field” site were varied from 87 up to 400 and from 94 up to 1000 Bq kg−1, respectively. Activity concentrations of Co-60, Eu-152, and Eu-154 were lower than the minimum detectable activity of the method used. Concentrations of naturally occurring radionuclides (K-40, Ra-226, U-238, and Th-232) in the majority of soil samples from the “Balapan” and the “Experimental field” sites did not exceed typical for surrounding of the SNTS areas levels. Estimation of risks associated with radioactive contamination based on the IAEA clearance levels for a number of key radionuclides in solid materials shows that soils sampled from the "Balapan" and the “Experimental field” sites might be considered as radioactive wastes. Decrease in specific activity of soil from the sites studied up to safety levels due to Co-60, Cs-137, Sr-90, Eu-152, Eu-154 radioactive decay and Am-241 accumulation-decay will occur not earlier than 100 years. In contrast, soils from the “Experimental field” and the “Balapan” sites (except 0.5–2.5 km distance from the “Chagan” explosion point) cannot be regarded as the radioactive wastes according safety norms valid in Russia and Kazakhstan.

GISTS: Gastrointestinal Stromal Tumors

1664 Objectives A high specific activity (SA) of 111In-labeled tracers will allow administration of low amounts of tracer to prevent receptor saturation and/or side-effects. To increase the SA, we studied the effect of the buffer used during the labeling procedure: NaAc, NH4Ac, HEPES and MES-buffer. The effect of ageing of the 111InCl3 stock and cadmium contamination, the decay product of 111In, was also examined in these buffers. Methods Escalating amounts of 111InCl3 were added to 1 µg of the DTPA- and DOTA-compound (Exendin-3, Octreotide and anti-CAIX antibody, cG250). Five volumes of MES, HEPES, NH4Ac or NaAc (0.1 M, pH 5.5) were added. After 20 min at 20 °C (DTPA-conjugated peptides), at 95 °C (DOTA-Exendin-3 and DOTA-Octreotide) or 45 °C (DOTA-cG250), the labeling efficiency was determined by ITLC. The effect of ageing of the 111InCl3 stock was examined on the labeling efficiency of DTPA-Exendin-3 The effect of escalating concentrations of Cd2+, the decay product of 111In, on labelling efficiency of DTPA-Exendin-3 was determined. Results Specific activities obtained for DTPA-Octreotide and DOTA-cG250 were 5 times higher in MES and HEPES buffer. Radiolabeling of DTPA-Exendin-3, DOTA-Exendin-3 and DTPA-cG250 in MES and HEPES buffer resulted in two times higher specific activities than in NaAc and NH4Ac. Labeling of DTPA-Exendin-3 decreased with 66% and 73% for NaAc and NH4Ac, respectively at day 12 after the production date of 111InCl3, while for MES and HEPES the maximal decrease in specific activity was 10% and 4% at day 12, respectively. The presence of 0.1 nmol Cd2+ in the labeling mixture of DTPA-Exendin-3 in NaAc and NH4Ac markedly reduced labeling efficiency, whereas Cd2+ concentrations up to 100 nmol did not affect the labeling efficiency in MES and HEPES buffer. Conclusions In conclusion, we showed improved labeling of DTPA- and DOTA-conjugated compounds with 111In in HEPES and MES buffer. The enhanced labeling efficiency could be due to the reduced competitive chelation of cadmium

Anammox in a UASB reactor treating saline wastewater

The feasibility of an anammox (anaerobic ammonium oxidation) UASB (upflow anaerobic sludge blanket) bioreactor to treat ammonium-rich brines was investigated in batch and continuous-flow experiments. The evidence from batch tests indicated that the anammox activity was significantly inhibited under highly saline conditions while the inhibition was reversible. Saline shock loading of 30 g NaCl l −1 caused a 67.5% decrease in specific anammox activity (SAA) compared to reference biomass (not exposed to salt). However, the acclimatized biomass displayed a SAA value just 45.1% lower than that of the reference biomass. When transferred from brine to freshwater, the salt-exposed biomass resumed its activity by 43.1%. Subsequent to appropriate acclimatization, careful manipulation nitrite concentration, nitrogen loading rate (NLR), and other operational parameters for 77 days, the results of continuous-flow experiment revealed that under the salinity of 30 g NaCl l −1 the reactor performed well and the substrate removal capacity (nitrogen removal rate, NRR, of 448 ± 15 mgl −1 d −1) was comparable to the condition of freshwater (NRR of 464 ± 13 mgl −1 d −1). However, the anammox UASB bioreactor was prone to malfunction under transient operating conditions.

Impact of site-specific N-glycosylation on cellular secretion, activity and specific activity of the plasma phospholipid transfer protein

The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed mutagenesis and expressed in Chinese hamster ovary Flp-In cells. The apparent size of each of the six PLTP mutants was slightly less than that of wild type by Western blot, indicating that all six sites are glycosylated or utilized. The size of the carbohydrate at each N-glycosylation site ranged from 3.14 to 4.2 kDa. The effect of site-specific N-glycosylation removal on PLTP secretion varied from a modest enhancement (15% and 60%), or essentially no effect, to a reduction in secretion (8%, 14% and 32%). Removal of N-glycosylation at any one of the six glycosylation sites resulted in a significant 35-78% decrease in PLTP activity, and a significant 29-80% decrease in PLTP specific activity compared to wild type. These data indicate that although no single N-linked carbohydrate chain is a requirement for secretion or activity, the removal of the carbohydrate chains had a quantitative impact on cellular secretion of PLTP and its phospholipid transfer activity.

Tools for Resistance Monitoring in Oriental Fruit Moth (Lepidoptera: Tortricidae) and First Assessment in Brazilian Populations

In southern Brazilian apple (Malus spp.) orchards, predominantly organophosphates are used to control the oriental fruit moth, Cydia molesta (Busck) (Lepidoptera: Tortricidae), but control failures often occur. Therefore the susceptibility of three C. molesta Brazilian populations was investigated to five insecticides of different groups and modes of action, in comparison with a susceptible laboratory strain mass reared in southern France for >10 yr. At the same time, comparative biochemical and genetic analysis were performed, assessing the activities of the detoxification enzymatic systems and sequencing a gene of insecticide molecular target to find out markers associated with resistance. The three Brazilian populations were significantly resistant to chlorpyrifos ethyl compared with the reference strain. One of the field populations that had been frequently exposed to deltamethrin treatments showed significant decreasing susceptibility to this compound, whereas none of the three populations had loss of susceptibility to tebufenozide and thiacloprid compared with the reference strain. All three populations had slight but significant increases of glutathione transferase and carboxylesterases activities and significant decrease of specific acetylcholinesterase activities compared with the reference. Only the most resistant population to chlorpyriphos exhibited a significantly higher mixed function oxidase activity than the reference. The acetylcholinesterase of females was significantly less inhibited by carbaryl in the Brazilian populations than in the reference strain (1.7-2.5-fold), and this difference was not expressed in the male moth. However, no mutation in the MACE locus was detected. These biological and molecular characterizations of adaptive response to insecticides in C. molesta provide tools for early detection of insecticide resistance in field populations of this pest.

P03-296 - Oxidative stress markers in patients treated with typical and atypical antipsychotics

IntroductionStudies performed in schizophrenia patients have generally suggested the presence of a compromised antioxidant system, but this is not always consistent with specific observed parameters, which on the whole, show evidences of dysregulation. There are also controversies regarding the oxidative stress status in patients treated with typical vs. atypical antipsychotics.AimIn this context, the aim of the present work was to evaluate the specific activity of some peripheral antioxidant defences like superoxide dismutase (SOD) and glutathione peroxidase (GPX) and the level of a lipid peroxidation maker (malondialdehyde-MDA), in schizophrenic patients treated with typical (haloperidol) or atypical (olanzapine, quetiapine and risperidone) antipsychotics, compared with age-matched healthy subjects.MethodsThe subjects of this study (n = 45), consisted of 35 patients who met DSM-IV criteria for schizophrenia and 10 healthy control age and gender-matched subjects. Patients were of paranoid subtype, with duration of illness for at least 5 years. Nine patients were under haloperidol (1–2 mg daily dose) treatment and 26 (8/10/8) patients were under atypical treatment: quetiapine (300 mg daily dose), olanzapine (20 mg daily dose) or risperidone (2–4 mg daily dose), respectively.ResultsWe found a significant decrease in GPX specific activity and also a significant increase of MDA levels in schizophrenic patients, compared to age-matched control group, regardless of their type of treatment. Additionally, an increase in SOD specific activity was observed, mainly in the patients treated with haloperidol and quetiapine.ConclusionsFurther research is necessary in order to elucidate the effects of different antipsychotic agents on antioxidant enzymes.

In Vivo Digestion of a Thaumatin-Like Kiwifruit Protein in Rats

Food allergens must exhibit sufficient gastrointestinal stability to reach the intestinal mucosa where absorption and sensitization can occur. Therefore, investigation of protein stability within the gastrointestinal tract may provide a prospective test for the allergenic potential of novel proteins. The aim of this work was to examine the effect of the fruit matrix and purified pectin on the digestion in vivo of kiwifruit allergens in the rat gastrointestinal system. The major kiwi allergen, Act d 2, was quantified in several compartments of the gastrointestinal tract by a monoclonal antibody-based ELISA. Protein intactness was demonstrated by immunoblot. Under conditions of complex food digestion in vivo, a pepsin-labile protein survived passage from the stomach to the caecum during a 3-h period. Decay of Act d 2 in the rat gut exhibited an exponential pattern. Ingestion of kiwifruit was followed by a decrease in both total and specific pepsin activity. When purified, Act d 2 allergen was consumed together with pure apple pectin; both gastric acidity as well as specific and total pepsin activity declined and thus protected 23% of the ingested allergen from digestion for 90 min. In conclusion, ingestion of pectin-rich fruits and particularly pectin supplements may have a protective action on pepsin-labile allergens and prolong their survival in both gastric and duodenal juices, enabling efficient uptake and presentation to the immune system.

Structural and catalytic roles of amino acid residues located at substrate-binding pocket in Fibrobacter succinogenes 1,3-1,4-β-D -glucanase

We created 12 mutant enzymes (E11L, F40I, Y42L, N44L, N44Q, E47I, L62G, K64A, K64M, R137M, R137Q, and N139A) from the truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (TF-glucanase). The enzymes were used to investigate the structural and catalytic roles of specific amino acid residues located at the catalytic pocket and having direct interactions with glucose subsites of the product beta-1,3-1,4-cellotriose (CLTR). Fluorescence spectrometry showed no discernible changes in secondary structures among purified TF-glucanase and the mutants. Kinetic analyses showed E11L, F40I, Y42L, R137M, and R137Q with a >10-fold decrease of specific activity (11.2- to 67.4-fold), and E11L, N44Q, E47I, K64M, R137M, R137Q, and N139A with a 2.17- to 4.3-fold increase of K(m) value when compared with TF-glucanase. Notably, E11L, R137Q, R137M, F40I, and N139A showed the most significant decrease in catalytic efficiency relative to TF-glucanase, by 2155-, 84.9-, 48.5-, 41.1-, and 19.1-fold, respectively; the five mutants showed the greatest changes in comparative energy DeltaDeltaG(b), with values of 1.94 to 4.92 kcal/mol. Combined with results from kinetic and structure modeling analyses of all mutant enzymes and X-ray crystallography of F40I, we elucidate that Glu11, Phe40, Arg137, and Asn139 play a crucial role in the catalysis of TF-glucanase owing to their local and direct interaction through hydrogen bonds or van der Waals stacking interaction by aromatic rings onto the glucose subsites -3, -2, and -1 of CLTR/substrate. The overall globular structures in the wild-type and mutant F40I enzymes do not differ.

Evaluation of antioxidant enzymes activities and lipid peroxidation in schizophrenic patients treated with typical and atypical antipsychotics

Studies performed in schizophrenia patients have generally suggested the presence of a compromised antioxidant system, but this is not always consistent with specific observed parameters, which on the whole, show evidences of dysregulation. There are also controversies regarding the oxidative stress status in patients treated with typical vs. atypical antipsychotics. In this context, the aim of the present work was to evaluate the specific activity of some peripheral antioxidant defences like superoxide dismutase (SOD) and glutathione peroxidase (GPX) and the level of a lipid peroxidation maker (malondialdehyde—MDA), in schizophrenic patients treated with typical (haloperidol) or atypical (olanzapine, quetiapine and risperidone) antipsychotics, compared with age-matched healthy subjects. We found a significant decrease in GPX specific activity and also a significant increase of MDA levels in schizophrenic patients, compared to age-matched control group, regardless of their type of treatment. Additionally, an increase in SOD specific activity was observed, mainly in the patients treated with haloperidol and quetiapine. Further research is necessary in order to elucidate the effects of different antipsychotic agents on antioxidant enzymes and lipid peroxidation or possible interventions at the oxidative stress level in schizophrenic patients.

Organic Carbon Degradation in Anoxic Organic-Rich Shelf Sediments: Biogeochemical Rates and Microbial Abundance

Identifying and explaining bottlenecks in organic carbon mineralization and the persistence of organic matter in marine sediments remain challenging. This study aims to illuminate the process of carbon flow between microorganisms involved in the sedimentary microbial food chain in anoxic, organic-rich sediments of the central Namibian upwelling system, using biogeochemical rate measurements and abundances of Bacteroidetes, Gammaproteobacteria, and sulfate-reducing bacteria at two sampling stations. Sulfate reduction rates decreased by three orders of magnitude in the top 20 cm at one sampling station (280 nmol cm−3 d−1 – 0.1 nmol cm−3 d−1) and by a factor of 7 at the second station (65 nmol cm−3 d−1 – 9.6 nmol cm−3 d−1). However, rates of enzymatic hydrolysis decreased by less than a factor of three at both sampling stations for the polysaccharides laminarin (23 nmol cm−3 d−1– 8 nmol cm−3 d−1 and 22 nmol cm−3 d−1– 10 nmol cm−3 d−1) and pullulan (11 nmol cm−3 d−1– 4 nmol cm−3 d−1 and 8 nmol cm−3 d−1– 6 nmol cm−3 d−1). Increasing imbalance between carbon turnover by hydrolysis and terminal oxidation with depth, the steep decrease in cell specific activity of sulfate reducing bacteria with depth, low concentrations of volatile fatty acids (less than 15 μM), and persistence of dissolved organic carbon, suggest decreasing bioavailability and substrate limitation with depth.

Influence of partial substitution of dietary fish meal on the activity of digestive enzymes in the intestinal brush border membrane of gilthead sea bream, Sparus aurata and goldfish, Carassius auratus

The sustainable composition of diets of high nutritional value is of the utmost importance for intensive aquaculture. Digestion and absorption of nutrients depend on the activity of the digestive enzymes, in particular those located in the brush border membrane of enterocytes, which are responsible for the final stages of breaking down and absorption of nutrients. In the present study, the substitution of fish meal by lupin or rapeseed meal in the diet was evaluated on gilthead sea bream ( Sparus aurata) and goldfish ( Carassius auratus). The objectives were to compare the activities of intestinal brush border enzymes in both species fed the control and experimental diets. When gilthead sea bream were fed the vegetable diets, significantly lower activities compared with the control group were observed for alkaline phosphatase and γ-glutamyl transpeptidase, but these differences were not significant in goldfish. Maltase activity was found decreased in the group fed lupin meal, both in sea bream and in goldfish. However, in spite of these differences in enzyme activities, growth characteristics of the fishes were similar with the three diets. It seemed that both fish were able to adapt to partial substitution of fish meal, but it remains to investigate the mechanism for compensating the decrease in specific enzymatic activity in the enterocytes of carnivorous gilthead sea bream.

Assessing the use of cutinase reversed micellar catalytic system for the production of biodiesel from triglycerides

AbstractBACKGROUND: This work evaluates the use of a reversed micellar catalytic system containing a lipolytic biocatalyst, cutinase, to promote the reaction of triglycerides with methanol, ethanol or butanol for the production of biodiesel in isooctane.RESULTS: The specific activity of cutinase increased with increase of the alcohol chain length, and methanol showed a significant toxicity toward the enzyme leading to a specific activity decrease for high concentrations of this alcohol, which did not occur when ethanol or butanol were used. The composition of the mixture was analyzed and it was shown that, although the reaction could proceed without water, the presence of water allowed higher reaction rates and higher conversions, probably because water helps to maintain both the micellar structure and lipase conformation. Excess water, however, had a deleterious effect on conversion.CONCLUSION: The influence of water and methanol, apart from micelle structure, was found to be linked to partition effects of diglyceride and monoglyceride, which may also have a significant impact on the reaction rate. Copyright © 2010 Society of Chemical Industry

Cholesterol 7α-hydroxylase (CYP7A1) activity is modified after chronic ingestion of depleted uranium in the rat

Depleted uranium (DU) is a radioactive heavy metal derived from the nuclear energy production. Its wide use in civilian and military items increases the risk of its environmental dissemination, and thus the risk of internal contamination of populations living in such contaminated territories. Previous studies have shown that vitamin D and cerebral cholesterol metabolisms were affected following chronic ingestion of DU. Even more than the brain, the liver is a crucial organ in cholesterol homeostasis since it regulates cholesterol distribution and elimination at body level. The aim of this work was to assess the impact of a low-level chronic ingestion of DU on hepatic cholesterol metabolism. Rats were contaminated with DU in their drinking water at a concentration of 40 mg/l for 9 months. The major effect induced by DU was a decrease of CYP7A1 specific activity (−60%) correlated with a matching decrease of its product 7α-hydroxycholesterol in the plasma. Hepatic gene expression of transporters ABC A1, ABC G5, ABC G8 and of nuclear receptor RXR was increased, whereas that of catabolism enzyme CYP7B1 was decreased. Thus, after a chronic ingestion of DU, rats experience a modulation of cholesterol catabolism but overcome it, since their cholesterolemia is preserved and no pathology is declared.

Esterase activity ofBordetella pertussis CyaC-acyltransferase against synthetic substrates: implications for catalytic mechanismin vivo

Adenylate cyclase-hemolysin toxin (CyaA) produced from the human respiratory tract pathogen Bordetella pertussis requires fatty-acyl modification by CyaC-acyltransferase to become an active toxin. Previously, the recombinant CyaA pore-forming (CyaA-PF) fragment expressed in Escherichia coli was shown to be hemolytically active upon palmitoylation in vivo by cosynthesized CyaC. Here, the 21-kDa CyaC enzyme separately expressed in E. coli as an inclusion body was solubilized in 8 M urea and successfully refolded into an enzymatically active monomer. In addition to the capability of activating CyaA-PF in vitro, CyaC showed esterase activity against p-nitrophenyl acetate (pNPA) and p-nitrophenyl palmitate (pNPP), with preferential hydrolysis toward pNPP when compared with chymotrypsin. A homology-based CyaC structure suggested a conceivable role of a catalytic triad including Ser(30), His(33) and Tyr(66) in substrate catalysis. Alanine substitutions of these individual residues caused a drastic decrease in specific activities of all three mutant enzymes (S30A, H33A and Y66A) toward pNPP, signifying that CyaC-acyltransferase shares a similar mechanism of hydrolysis with a serine esterase in which Ser(30) is part of the catalytic triad.

Control of post-harvest pericarp browning of litchi (Litchi chinensis Sonn)

Pericarp browning is the major post-harvest problem of litchi (Litchi chinensis Sonn) fruit, resulting in reduced commercial value of the fruit. Control of post-harvest pericarp browning of fruit using 9 different post-harvest treatments were studied. The treated fruits were packed in transparent perforated (0.2% ventilation) low density polythene bags (100 gauge). On storage, pericarp browning increased irrespective of treatments with the decrease in pericarp specific activity, total pericarp phenol and total anthocyanin. Anthocyanin degradation index and polymeric colour increased during storage. Pre-cooled (10°C) fruits treated with 0.6% sodium metabisulphite solution for 10 min, air dried followed by dipping in 2% HCl for 5 min and packing in perforated LDPE bags recorded the lowest polyphenol oxidase specific activity (2.2 units/mg protein) with maximum retention of total anthocyanin (47.3 mg/100g) leading to the lowest pericarp browning after 9 days of storage with attractive red colour, freshness and enhanced shelf life of 9 days at ambient conditions (27.7 ± 1.2°C, RH 78 ± 4%).

Extraction, partial characterization and susceptibility to Hg2+ of acid phosphatase from the microalgae Pseudokirchneriella subcapitata

Pseudokirchneriella subcapitata is a unicellular green algae widely distributed in freshwater and soils. Due to its cosmopolitan characteristic, its use is recommended by national and international protocols in ecotoxicity studies. The alteration of phosphatase activities by agriculture pollutants like heavy metals has been extensively used as a biomarker in risk assessment and biomonitoring. In this study, we compared the extraction of acid phosphatase from P. subcapitata by different procedures and we studied the stability, substrates specificity, kinetics and the effect of Hg2+ in the crude extract. The freezing and thawing technique associated with probe sonication was the most suitable method of extraction. The enzyme was stable when frozen at -20ºC for at least six months, showed an optimum pH of 5 and a Km value of 0.27 mM for p-nitrophenylphosphate (pNPP) as substrate. Some natural organic substrates were cleaved by a similar extent as the synthetic substrate pNPP. Short term exposure (24 hours) to Hg2+ had little effect but inhibition of the specific activity was observed after 7 days with EC50 (concentration of Hg2+ that promotes 50% decrease of specific activity) value of 12.63 μM Hg2+.