A rapid assay for pectinesterase activity which can be used as a prescreen for pectinesterase inhibitors.

A rapid assay for inhibition of pectinesterase has been developed to facilitate large scale screening for possible inhibitors. The assay gives quantitative results which can be confirmed by other assay techniques. The assay uses an agar plate in which pectin and an indicator have been incorporated. The hydrolysis of pectin to pectinic acid by pectinesterase causes a lowering of the pH of the medium and produces a color change. The area of the color change produced by the hydrolysis of pectin is directly proportional to the activity of the pectinesterase.     

Visualization of enzyme-catalyzed reactions using pH indicators: rapid screening of hydrolase libraries and estimation of the enantioselectivity

The use of pH indicators to monitor hydrolase-catalyzed reactions is described. The formation of acid following an enzyme-mediated hydrolysis causes a drop in the pH that can be visualized by a change in the color of the indicator-containing solution. The best indicators are those showing a color transition within the operational pH range of the hydrolases, like bromothymol blue and phenol red. The enantioselectivity of lipases and esterases can be estimated using single isomers under the same conditions and comparing the color turnover for each one. The method has been tested to quickly evaluate the enantioselectivity of a lipase towards a set of ester substrates and applied to the hierarchical screening of a library of thermophilic esterases.     

A colorimetric assay for determination of methyl parathion using recombinant methyl parathion hydrolase

A simple, rapid and sensitive colorimetric dipstick assay for the detection of the organophosphorous insecticide methyl parathion (MPT) residue in vegetables was developed. The assay was based on the hydrolysis of MPT by a recombinant methyl parathion hydrolase (recMPH), the encoding gene of which was isolated from Burkholderia cepacia, a soil bacterium indigenous to Thailand. This reaction generates protons leading to a change in pH that correlates with the amount of MPH present. Hence, the pH indicator bromothymol blue was used to monitor the MPH hydrolysis as the associated color changes can be observed by the naked eye. The recMPH was immobilized on a PVDF membrane to establish a dipstick assay format. The assays could detect MPT residues in spiked vegetable samples at the concentration of 1 mg/L without using analytical instrumentation. The test is reusable and stable for up to 3 months in the absence of any preservatives.     

Determination of the enzymatic traits of anaerobic gram-negative nonsporulating bacteria

The method requiring the use of a semi-liquid medium with the reduced content of protein hydrolysates and with bromo-thymol blue added as indicator and permitting the determination of sugar fermentation by bacteroids and fusobacteria is presented. The determination is made by a change in the color of the indicator and the intensity of microbial growth (opacity) after the incubation of the inoculated medium under free access of air. A study has been made for comparing the above method with the electrometric method for the determination of fermentative features in anaerobic microorganisms.     

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK_a to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.     

Modification of the Microtiter Technique for Antimicrobial Drug Susceptibility Testing by Incorporation of Indicators

A pH indicator and dextrose were incorporated into growth media as a modification of microbial microtiter methods for determining the minimal inhibitory concentration of antimicrobial drugs. This modified method was tested to evaluate the ease of reading end points by changes in the indicator color. Application of the procedure to two media, three indicators, and eight species of bacteria indicated that definitive end points could be reached as a result of indicator color change caused by acid production during bacterial growth. This method is accurate and reproducible. It is a modification which eliminates a need for plating and facilitates the reading of minimal inhibitory concentration end points.     

Directed evolution of an esterase for the stereoselective resolution of a key intermediate in the synthesis of epothilones

The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester.     

Microspectrophotometric measurement of pH and pH effect on color of petal epidermal cells

A microspectrophotometric method has been developed for calorimetric pH determination using indicator dyes. Tissue samples as small as five cells were used. Measurements of standard buffered solutions of known pH were within a standard error of less than ± 0.04 pH units. Validity of the technique has previously been established by matching the pH values and absorption spectra of several model systems to that of living cells. A method for spectrophotometric pH determination of single cells is suggested. The pH change seemed to be the major factor in the color change in aging flowers. The epidermal pH, the absorption spectra of living tissue, the anthocyanidins, flavonols, and flavones present in more than 250 plants of many families were determined. These data indicate that pH is only one of numerous parameters determining flavonoid color in the living cell.     

Simultaneous sewage sludge digestion and metal leaching — effect of temperature

The effect of temperature on the simultaneous sewage sludge digestion and metal leaching process was studied in laboratory bioreactors of 20 l working volume. The results thus obtained showed that the process can be employed efficiently for metal solubilization, elimination of indicator microorganisms and sewage sludge stabilization at temperatures between 10°C and 30°C. Rates of pH reduction, sulfur oxidation, growth of thiobacilli, elimination of indicator microorganisms and solids degradation were found to decrease with temperature. Low metal solubilization efficiency was observed at 10°C; however, metals were solubilized to below the recommended level. The solubilization of organic matter and nutritive elements (N, P and K) was not significantly affected by the variation in temperature. The fertilizer value of sludge after leaching and digestion did not change significantly and remained the same irrespective of temperature. *** DIRECT SUPPORT *** AG903078 00005     

Development of a microbial time/temperature indicator prototype for monitoring the microbiological quality of chilled foods

A time/temperature indicator (TTI) system based on the growth and metabolic activity of a Lactobacillus sakei strain was developed for monitoring food quality throughout the chilled-food chain. In the designed system, an irreversible color change of a chemical chromatic indicator (from red to yellow) progressively occurs due to the pH decline that results from microbial growth and metabolism in a selected medium. The relation of the TTI response (color change) to the growth and metabolic activity (glucose consumption, lactic acid production, pH decrease) of L. sakei was studied. In addition, the temperature dependence of the TTI kinetics was investigated isothermally in the range of 0 to 16 degrees C and modeled with a system of differential equations. At all temperatures tested, the pH and color changes of the TTI system followed closely the growth of L. sakei, with the endpoint (the time at which a distinct visual color change to the final yellow was observed) of the TTI coinciding with a population level of 10(7) to 10(8) CFU/ml. The endpoint decreased from 27 days at 0 degrees C to 2.5 days at 16 degrees C, yielding an activation energy of 97.7 kJ/mol, which was very close to the activation energy of the L. sakei growth rate in the TTI substrate (103.2 kJ/mol). Furthermore, experiments conducted on the effect of the inoculum level showed a negative linear relationship between the level of L. sakei inoculated in the system medium and the endpoint of the TTI. For example, the endpoint at 8 degrees C ranged from 6 to 2 days for inoculum levels of 10(1) and 10(6) CFU/ml, respectively. This relationship allows the easy adjustment of the TTI endpoint at a certain temperature according to the shelf life of the food product of concern by using an appropriate inoculum level of L. sakei. The microbial TTI prototype developed in the present study could be used as an effective tool for monitoring shelf life during the distribution and storage of food products that are spoiled primarily by lactic acid bacteria or other bacteria exhibiting similar kinetic responses and spoilage potentials. Apart from the low cost, the main advantage of the proposed TTI is that its response closely matches the loss of the quality of a food product by simulating the microbial spoilage process in particular environments.     

Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.     

Calf thymus histone as a substrate for neutral and acid proteases of leucocyte lysosomes and other proteolytic enzymes

The use of calf thymus histone as a substrate has revealed a previously unknown neutral protease, optimally active at pH 7.2–7.3, in rabbit PMN lysosomes. The Sakaguchi reaction for the colorimetric determination of arginine has been modified, allowing a slow, linear development of color measurable at 500 nm over a period of up to 4 hr at 0–2°C. Specificity for arginine was shown since no other amino acid tested gave any color in the reaction. This new method has been used to measure arginine-reactive hydrolysis products released from histone by PMN lysosomes at neutral pH. Release of tyrosine, measured by the Folin method, was also used as an indicator of hydrolytic activity. Histone proved to be a useful substrate for the acid cathepsins of PMN, comparing favorably with hemoglobin, commonly used to measure such activity. Crystalline trypsin and chymotrypsin also hydrolyzed histone. The kinetics of arginine release by these enzymes over a period of 24 hr resembled those of neutral protease from PMN lysosomes.     

生物催化3-(4-氯苯基)-戊二腈去对称性水解合成光学纯巴氯芬的关键前体

研究了利用生物催化剂制备(S)-4-氰基-3-(4-氯苯基)-丁酸.以3-(4-氯苯基)-戊二腈为底物,采用苯酚-次氯酸钠法对实验室保藏的菌株进行筛选,得到一株产物立体选择性较高的菌株赤霉菌Gibberella intermedia WX12,并对其催化特性和发酵条件进行了初步研究.以30 g/L的乳糖和20 g/L的蛋白胨分别为碳、氮源,发酵培养96 h,收集的菌体在50 mmol/L磷酸缓冲液(pH 8.0)中30℃催化反应24 h,将3-(4-氯苯基)-戊二腈转化为4-氰基-3-(4-氯苯基)-丁酸,产率为90％.将产物化学转化为巴氯芬,手性HPLC分析表明水解产物构型是(S),其对映异构体过量值ee＞ 99％.该产物可以用来合成光学纯的(R)-和(S)-巴氯芬.     

TEMPORAL CHANGES IN PH WITHIN THE PHAGOCYTIC VACUOLE OF THE POLYMORPHONUCLEAR NEUTROPHILIC LEUKOCYTE

Although previous workers have established that the pH of the phagocytic vacuole of the polymorphonuclear (PMN) leukocyte changes from neutral to acid, the time course of conversion has not been investigated. The present experiments were initiated to study pH changes immediately after phagocytosis. Peritoneal exudates were induced in rats; 4 h later, yeast stained with pH indicators was injected intraperitoneally, and the exudate was retrieved at 30-s intervals and examined by light microscopy. Results revealed that (a) within 3 min, pH dropped to ~6.5, as indicated by the change in color of neutral red-stained yeast; (b) within 7–15 min, pH dropped progressively to ~4.0, as indicated by color change in bromcresol green-stained yeast; (c) pH did not fall below 4, since no color change was observed up to 24 h when bromphenol blue-stained yeast was used. The finding that intravacuolar acidity increases rapidly after phagocytosis is undoubtedly important with respect to PMN leukocyte function in killing and digesting microorganisms, for many PMN leukocyte granule enzymes (i.e., peroxidase and lysosomal enzymes) are activated at acid pH (~4.5). It follows that temporal changes in pH and maximal pH depression should be considered in studies of intraleukocytic microbicidal mechanisms, since a defect in these factors could result in impaired PMN leukocyte function.     

A simple method to determine urea concentration using intact Helicobacter pylori and BromoCresol Purple as a pH indicator

A modified method for urea quantification, by measuring the ammonia formed by urease, used the urease-positive Helicobacter pylori in place of purified urease with a pH indicator dye, BromoCresol Purple, to provide a color change. The color formed was stable for 20-min and could be read at 588-nm for urea quantification. Using this method, urea standard curves were linear up to 8.3-mM. As there was no need for centrifugation or precipitation, the assay was developed for use with 96-well microplates.     

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples.     

Feulgen Hydrolysis with Perchloric Acid

In tissue fixed with Carnoy's acetic alcohol (1:3), the hydrochloric acid hydrolysis performed as part of the Feulgen reaction is optimal for only a very short period of time. When 10% perchloric acid is used as the hydrolytic agent, the same color maximum is obtained, and the optimal hydrolysis time at 25°C. extends from 12 hours to 24 hours. During this time the intensity of color does not change. The events which take place during the period of suboptimal hydrolysis are the same as diose which take place during the corresponding period of hydrochloric acid hydrolysis. Part of the decrease in ultraviolet extinction of nuclei during the first 12 hours is due to the splitting off of purine bases from the desoxyribose nucleic acid. This is consistent with the increase in the amount of Feulgen dye bound by nuclei during this period of time. Between 12 hours and 24 hours no ultraviolet absorbing material is lost from nuclei, which is consistent with the fact that during this time the Feulgen color produced remains at a maximum.     

Chitinolytic activities in the gut of Aedes aegypti (Diptera:Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut.