Automated fast filtration and on‐filter quenching improve the intracellular metabolite analysis of microorganisms

To reliably determine intracellular metabolite concentrations in microorganisms, accurate sampling and sample inactivation strategies are crucial. Here, we present a method for automated fast filtration and on‐filter quenching of microbial samples to overcome metabolite leakage induced by cold shock and significantly reduce the sampling and treatment time compared to manual filtration methods. The whole process of sampling, sample filtration, filter wash, and quenching of the filter with liquid nitrogen was finished in less than 6–15 s, depending on the experimental setup. By integration into an automated fast sampling device, we compared our method to the conventional methanol quenching method and showed that intracellular amino acid contents in Escherichia coli were significantly increased (≥75%) with our fast filtration and on‐filter quenching method. Furthermore, we investigated different filter types for the fast filtration and the efficiency of metabolite extraction from cells held on filters. Additionally, we found that the fast filtration behaves considerably different during exponential and nonexponential growth, probably due to variations of cell morphologies. Overall, we demonstrated that the automation of the fast filtration method significantly reduces the time for filtration and quenching and hence enlarge the number of metabolites that can be quantified with this leakage‐free sampling method.     

Procedures for the automated analyses of proteoglycans and glycosaminoglycans

Methods for the automated analysis of hexose, uronic acid, and protein using the Technicon AutoAnalyzer II have been developed by modifying previously published procedures. A method of separating glucosamine and galactosamine, which is eminently suited to quantitating one in the presence of a large amount of the other, is reported. Procedures that can be recommended for determining the amino acid content and individual neutral sugars of proteoglycans or glycosaminoglycans are also described.     

Simple estimate of ureides in soybean tissue

Extracts of soybean tissue are treated with an acidic cation-exchange resin to remove amino acids. The sample is treated with hypochlorite, pH 4.0, to convert the amide from allantoic acid or allantoin to products that react with alkaline phenol to form indophenol. The procedure is inexpensive, sensitive, and rapid. The method may be adapted for automated analysis with the Technicon autoanalyzer.     

Amino acid and peptide ratio in protein hydrolysates for parenteral feeding

Using the method of amino acid analysis and routine methods of protein biochemistry, the ratio of amino acids and peptides in acid and enzyme protein hydrolyzates was determined. Depending on the production procedure, the hydrolyzates under study contained various amounts of free amino acids and peptides in which the number of amino acid residues varied from 2 to 7. Additional hydrolysis of these preparations by leucine aminopeptidase led to a decrease in the peptide content and to an increase in the amino acid content. This may have a beneficial effect on the quality of protein hydrolyzates.     

Determination of hexuronic acids in glycosaminoglycans by automated ion-exchange chromatography.

This report describes a procedure for analyzing glucuronic and iduronic acids using the Technicon automated sugar chromatography system. Glueronic and iduronic acids of standard samples of glycosaminoglycans have been analyzed after hydrolysis by formic acid. The method has been applied to quantitate uronic acids in chondroitin sulfates and dermatan sulfate mixtures obtained by Dowex 1 Cl^? column fractionation of glycosaminoglycans from aortas of different animal species. The results are in good agreement with those obtained by the gas-liquid chromatographic technique.     

Automated protein precipitation by filtration in the 96-well format

The use of automated protein precipitation by filtration in the 96-well format as a rapid sample preparation technique for high throughput bioanalysis using liquid chromatography tandem mass spectrometry is reported. A robotic sample processor is used to aspirate sequentially a plasma sample and acetonitrile separated by air gaps. These are then mixed by being dispensed into individual channels of a 96-well filter block. The resulting supernatant is separated from the precipitated plasma proteins by the application of gentle vacuum using a custom manifold. The filtered supernatants are collected into a deep well microtitre plate, evaporated to dryness using a heated 96-well dry down station and reconstituted in water prior to analysis. The efficiency of the extraction procedure is measured by the Lowry method for determining protein concentration. This method was used to optimise both the volume and the order of reagent addition, and to compare several prototype 96-well filter blocks. Using the optimised procedure a specific, precise and accurate method was developed for the β-agonist salbutamol in rabbit plasma with a calibration range of 1 to 100 ng/ml from 100 μl of sample.     

Automated assay of glycosidases

The Technicon Basic AutoAnalyzer sampler system was modified for simultaneous sampling of glycosidase(s) and substrate-buffer solutions. The inexpensive modification allows performance of automated enzyme analyses and enzyme kinetic studies with minimal consumption of substrate and/or enzyme.     

Sensitive automated methods for phosphate and (Na+ plus K+)-ATPase

Sensitive automated methods for phosphate and (Na⁺ + K⁺)-ATPase are presented. The automated systems use sampler and pump modules from a Technicon autoanalyzer along with a Gilford spectrophotometer. The phosphate assay has a molar absorbance of approximately 87,000 m^?1 cm^?1 640 nm. The method uses a single color reagent and has been used successfully with perchloric acid digests of phospholipids.The (Na⁺ + K⁺)-ATPase method is based on the phosphate method. The method is comparable to manual methods in the amount of ATP and enzyme used per assay. The blank due to ATP is low. The precision of the assay on replicate samples is usually within ±1%. The total time delay for a single assay is less than 9 min and the method can be operated at a rate of 30 samples/hr. The method has been particularly useful in enzyme purification work. Procedures for the use of the method in kinetic studies are described     

Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate

Sarcoplasmic proteins isolated from bovine livers were hydrolyzed using the enzyme thermolysin at 37 ?C for 2 h. The hydrolyzates were filtered through molecular weight cut off membranes (MWCO) and filtrates were obtained. The water activity (a_w) of unhydrolysed sarcoplasmic protein, full hydrolyzates, 10-kDa and 3-kDa filtrates were below the limit necessary for microbial growth. The antioxidant activities of both filtrates and fractions were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe²⁺ chelating ability assay. RP-HPLC was used for purification of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates. The peptidic content of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates were assessed using the Dumas method and peptide contents of each fraction were characterized using electrospray quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry with the resultant spectrum analysed using the software programmes Protein Lynx Global Server 2.4. and TurboSEQUEST. Similarities between the amino acid composition of characterized peptides from each fraction and previously reported antioxidant peptides were found. This study demonstrates that meat by-product such as liver can be utilised as raw material for the generation of bioactive peptides with demonstrated antioxidant activities in vitro using the enzyme thermolysin. It is significant as it presents a potential opportunity for meat processors to use their waste streams for the generation of bioactive peptides for potential functional food use.     

A moorable,automated plankton sampler

A moorable, automated plankton sampler is described, designed to obtain series of plankton samples over extended time periods in the absence of a surface vessel. The sampler consists of one or more net-containing boxes, a unit for generating as well as measuring water flow through the net boxes, a programmeable control unit, and a frame. Each net box contains several nets on a vinyl strip, used to move the nets from the storage chamber into the fishing position and then into the preservative chamber. Preservation of samples is in a formalin-brine mixture.Net boxes can be added and the number of nets per box changed; the prototype is described with 1 net box with 10 nets. Volume of water to be filtered, time between sample collections, number of net boxes and the number of nets in each box is programmed into the control unit before sampler deployment. Collections made with the sampler are compared with those made with a SCOR net and a pump.     

A deae-cellulose filter disk assay for aminoacyl-tRNA

A filtration assay is described for quantitation of aminoacylated tRNA. [³H]Aminoacyl-tRNA is adsorbed on DEAE-cellulose filter disks under conditions in which unreacted amino acid is removed by a 100-mm glycine-HCl (pH 2.3) wash. The assay is rapid, simple to perform, and linear over the range of 0.005–20 A₂₆₀ units/50-μl filtered aliquot. The assay is nondestructive to aa-tRNA, does not use strongly acidic solutions for removal of amino acid, and does not require carrier nucleic acid for accurate measurement of low aa-tRNA concentrations. It is a useful alternative to methods involving acid precipitation and ideally suited for measurement of low levels of highly purified aa-tRNA.     

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

For indirect DNA extraction for metagenomics studies, bacterial cells can be effectively separated from sample debris by using a simple size exclusion technique, such as filtration, and thereafter lysed. The requirement for the optimal recovery of cells in filtrates is critical to achieve sufficient DNA yield and a representative population. Particles smaller than the filter pore size are expected to be found in the filtrate, whereas particles larger than the filter pore sizes are excluded. However, this is not always the case. It is established that the membrane pore size influences filtration efficiency to some degree. In addition the physicochemical characteristics of the filter suspension and characteristics of the microbial cells being filtered influence the exclusion property of a membrane. This review provides an overview of membrane filtration techniques and the factors that affect filterability of bacteria cells through a filter membrane. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:853–866, 2015     

Active site stoichiometry of L-phenylalanine: tRNA ligase from Escherichia coli K(-10).

The existence of two active siter per molecule of L-phenylalanine:tRNA ligase from Escherichia coli K(-10) has been demonstrated by isolation of the E-aminoacyl adenylate and tel filtration and the nitrocellulose filter assay at pH 5.0 revealed the same stoichiometry for the E-tRNAPhe comples as protection against degradation by snake venom phosphodiesterase and equilibrium gel filtration at pH 7.5. Using a fluorescence titration technique, it was found that the dissociation constant for ligase-tRNAPhe complex is decreased 20-fold when the hydrogen ion concentration is changed from pH 6.0 to pH 5.0. The existence of two active sites binding the aminoacyl adenylate intermediate was demonstrated by gel filtration and retention on DEAE-cellulose filters. "Burst" experiments indicated that two sites were involved in a rapid ATP consumption at conditions of catalytic amino acid activation. Furthermore, it was observed that the activated amino acid could be transferred from both sites to cognate tRNA.     

Filtration Method for Bacteriophage Detection

A filtration method has been developed which can be used to detect and enumerate phage in low concentrations directly from solution without the need for prior concentration. In this method, a known volume of the phage solution is mixed with a suitable host solution. Samples are filtered through membrane filters; the filter is removed and incubated, and after 24 hr the resultant plaques are counted and the titer is calculated. Escherichia coli B and the coliphage T2 were used in these studies. Host cultures less than 12 hr old produced the best results. Approximately 10(10) host organisms must be present in the sample taken for filtration. To avoid phage reproduction, all steps prior to filtration must be done in less than 45 min. The method was compared with the soft-agar technique and was shown to be less precise but able to measure phage in lower concentrations.     

Some observations on air filtration

Summary 1. A method has been developed for testing the filtration efficiency of some filter materials. For each of the materials investigated — cotton wool, stillite and carbon — a suitable filter has been devised.2. The filtered air was analyzed as to its germ content with the aid of a set of 3 capillary impingers.3. The cotton wool filter gave on the whole satisfactory results provided that due attention was given to the packing of the filter and its sterilisation. Clear indications were obtained that the degree of the contamination of the air was of vital importance.4. The stillite filter proved to have the advantage of combining a high filtration efficiency with a low resistance to the passing air. Also for the stillite filter a critical degree of contamination of the air was established; on surpassing this degree the filtration effect was endangered.5. The carbon filter proved to be most efficient, but had a relatively low specific filtering capacity. It was found that the filtration result was depending on the height of the carbon column and on the velocity and the degree of contamination of the air.6. It should be stressed that in all experiments artificially contaminated air was used, and that the number of germs present in the air to be filtered was in all cases many times larger than that usually occurring in normal air.     

Use of Membrane Filter Technique in the Microbiological Control for the Brewing Industry

A physical method was developed involving serial filtration with membrane filters for separating yeast cells from bacteria. Such a method eliminates the need for antibiotics previously required to permit differential counting of such populations. All yeast cells filtered were successfully retained and cultivated on a 1.2-mu membrane filter by use of a synthetic medium. All bacteria filtered avoided entrapment on a 1.2-mu membrane filter and were successfully retained and cultivated on a 0.22-mu membrane filter with the same synthetic medium. Final filtrates from these serial filtrations were free from all yeast cells and bacteria when tested with Fluid Thioglycollate Medium.     

Rapid separation and quantitation of mixed microorganisms by filtration and bioluminescence

A membrane filtration/bioluminescence system was developed for the differentiation and quantitation of mixed populations of microorganisms. Samples containing microorganisms were filtered through two membrane filters of descending pore size. The microorganisms retained on the filter contain ATP that can be extracted and measured on the filter via the firefly luciferase-luciferin bioluminescence assay. Results, obtained in less than 20 min, show a good correlation (r greater than or equal to 0.95) between the light produced and the number of organisms in the sample. Using these techniques, Escherichia coli can be separated from yeast or mold and measured in samples containing both microorganisms. When lysostaphin is used to selectively lyse Staphylococci on the filter, the specific quantification of these bacteria among other microorganisms can also be accomplished. The filtration/bioluminescence technique offers the potential of being a rapid and sensitive method to differentiate and detect microorganisms, by selective sizing or lysing, in a variety of samples.     

A new approach for sample preparation of protein hydrolyzates for amino acid analysis

Sample preparations of protein hydrolyzates for amino acid analysis by ion-exchange chromatography has been accomplished without the removal of hydrochloric acid which was used for the hydrolysis. The technique involves partial neutralization of the available hydrochloric acid after hydrolysis with a solution which neutralizes and dilutes the sample hydrolyzate at the same time. The resulting sample solution which is employed for amino acid analysis produces an amino acid chromatogram having the same elution times and resolution as compared to a mixture of amino acids prepared in pH 2.2 sodium citrate buffer. Experimental data is also presented which shows that the amount of available hydrochloric acid in the final sample solution employed for amino acid analysis can affect both the resolution and elution time of many of the amino acids found in a protein hydrolyzate.     

Automated system for high-throughput protein production using the dialysis cell-free method

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14 h, including 8 h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).     

A sensitive automated method for adenosine triphosphatase kinetics.

An automated method for measuring adenosine triphosphatase (ATPase) activity is described. A modified version of a Technicon Autoanalyzer utilizing a sensitive colourimetric technique for determining inorganic phosphate concentrations (1 nmol/ml) allows investigations on enzymes of low specific activities. Dialysis may be used for measuring tissue homogenate activities or bypassed by examining purified enzyme preparations. When combined with a gradient apparatus, the proposed technique is particularly well suited for the study of enzyme kinetics in relation to cation or anion concentrations.