On-line and real time cell counting and viability determination for animal cell process monitoring by in situ microscopy

International audienc

Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from G. stearothermoph

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from Geobacillus stearothermophilus with dual activity of β-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original β-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the Km value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process

Calpain Activity Zymography from Meat Sources as Quality Criterion

La falta de terneza es un problema presente en la carne bovina de buena calidad. La terneza de la carne se da por mecanismos poco conocidos y se han propuestos factores como componentes individuales y características de la especie, edad del animal, diferencias musculares, entre otros. Para un ablandamiento post mortem se deben romper las uniones de algunas proteínas estructurales por métodos físicos o por acción enzimática. Entre las principales enzimas endógenas que producen ablandamiento están las calpaínas. Las calpaínas son cisteín proteasas, encargadas de hidrolizar los enlaces peptídicos internos de las proteínas. Ellas pertenecen a una familia amplia de proteínas intracelulares citosólicas dependientes de calcio. El sistema calpaína (integrado por µ-calpaína, µ/m-calpaína, m-calpaína y la alpastatina, un inhibidor endógeno) es el principal responsable de la proteólisis post mortem. Con el objetivo de analizar y caracterizar bioquímicamente la actividad calpaína de fuentes cárnicas de diversos animales para el consumo humano, se empleó la técnica de zimografía para la detección de la actividad enzimática en geles de poliacrilamida. Las muestras, provenientes de tres tipos de carne, fueron obtenidas de una carnicería y de un matadero (res) y de un criadero rural (pollo y cerdo) y se homogeneizaron por dos métodos distintos. Luego, se concentraron cinco veces y se cuantificó la cantidad de proteínas totales. Se determinó la actividad proteolítica empleando caseína como sustrato y tirosina como patrón. La actividad obtenida (en nmol·min–1·mg–1) fue 0,78 para el homogenato de res; 0,34 para el de cerdo y 0,24 para el de pollo. Se realizó un análisis zimográfico para evidenciar la actividad calpaína, donde se visualizaron bandas correspondientes a masas moleculares de ~70 kDa para res, ~67 y 65 kDa para pollo y ~68 kDa para cerdo, respectivamente, comprobándose la autoproteólisis de la subunidad catalítica (80 kDa) de la calpaína. El método empleado resultó ser eficiente para estudiar la actividad calpaína como posible bioindicador de terneza de carnes para consumo [email protected] lack of tenderness is an important issue when purchasing high quality bovine meat. The mechanisms involved in meat tenderness are still not well understood and several factors have been proposed, like individual components and speciesrelated characteristics, age, differences in muscle types, etc. For post mortem tenderness, physical or enzymatic methods must be employed in order to break the union of some structural proteins. The main endogenous enzymes responsible for tenderness are cathepsin and calpain. Calpain is an important example of a cysteine protease, able to hydrolyze the internal peptide bonds of proteins. They form an ample family of calcium-dependent cytosolic intracellular proteins. The calpain system (composed of µ-calpain, µ/m-calpain, m-calpain and calpstatin, an endogenous inhibitor), is responsible for post mortem proteolysis (especially µ-calpain) and thus, also for meat tenderness. In order to analyze and characterize biochemically the calpain activity present in meat sources from several animals for human consumption, the zymography technique was used to detect enzymatic activity in polyacrylamide gels. Samples isolated from three types of meat were obtained from a local store and also from a slaughterhouse (beef) and from a local farm (chicken and pork). Meat sample were homogenized by two different methods. Then homogenates were concentrated five times and total amount of proteins was quantified. Proteolytic activity was quantified by photometry using casein as substrate. A standard curve was performed using tyrosine. Activity (nmol·min–1·mg–1) was determined to be 0.78 for beef homogenate, 0.34 for pork, and 0.24 for chicken. Calpain activity was also tested by zymography, resulting in active bands corresponding to ~70 kDa for beef, ~67 and 65 kDa for chicken and ~68 kDa for pork. Concurrently, the autoproteolysis of the catalytic subunit (80 kDa) was confirmed. The method used is efficient to study calpain activity as possible bioindicator of meat tenderness for human consumption

Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from G. stearothermophilus

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from Geobacillus stearothermophilus with dual activity of β-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original β-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the Km value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process

On-line and real time cell counting and viability determination for animal cell process monitoring by in situ microscopy.

International audienc

Optical Sampling in Situ Microscope for On-Line Monitoring of Animal Cell Cultures

International audienceCell concentration and cell vitality are key parameters to be monitored during cell cultivation processes. Common techniques used for these purposes are often based on sterile sampling and subsequent off line measurements. Extraction and preparation of samples is labour-intensive and risk-entailing. These disadvantages are avoided if the cell culture is directly inspected by using an in-situ technique, e.g. an in-situ microscope (ISM). An ISM delivers a wealth of image data which can be evaluated so as to provide automatic monitoring of the cell density and of morphological parameters as the cell-size. In-situ microscopy can either employ periodic opening and closing of a probe chamber inside the reactor or, alternatively, flash illumination and optical depth of field in order to define a virtual probe zone. Here, we describe optics and software of an advanced version of such an ISM with unprecedented resolution and frame rate. Fast collection of online-galleries of individual cell-portraits even at low cell concentrations enables online morphological analysis without sample extraction. Cell density data obtained by the ISM and traditional counting are shown in comparison, revealing the advantage of the ISM with respect to statistic deviations