Enhanced d-hydantoinase activity performance via immobilized cobalt ion affinity membrane and its kinetic study

Various immobilized metal ions affinity membranes (IMAMs) were prepared from the regenerated cellulose membrane (RC membrane) and chelated with various metal ions such as Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺. The D-hydantoin-hydrolyzing enzyme (DHTase) harboring a poly-His tagged residue was used as a model protein to be immobilized on the prepared IMAMs through the direct metal–protein interaction forces. The adsorption isotherm and the kinetic parameters V_max, K_m,app of DHTase on IMAMs were studied. The cobalt ions chelated IMAM (Co-IMAM) was found to yield the highest specific activity of DHTase. Under the immobilization condition, the cobalt ion chelated amount was 161.4 ± 4.7 μmol/disk with a DHTase activity of 4.1 ± 0.1 U/disk. As compared to the free DHTase, the immobilized DHTase membrane could achieve a broader pH tolerance and higher thermal stability. In addition, 98% of the residual activity could be retained for 7-times repeated use. Only little activity loss was observed within 36-day storage at 4 °C. This is the first report concerning about using cobalt ion as the effective chelated metal ion for simultaneous purification and immobilization operation.     

Disposable bioprocessing: The future has arrived

Increasing cost pressures are driving the rapid adoption of disposables in bioprocessing. While well ensconced in lab‐scale operations, the lower operating/ validation costs at larger scale and relative ease of use are leading to these systems entering all stages and operations of a typical biopharmaceutical manufacturing process. Here, we focus on progress made in the incorporation of disposable equipment with sensor technology in bioprocessing throughout the development cycle. We note that sensor patch technology is mostly being adapted to disposable cell culture devices, but future adaptation to downstream steps is conceivable. Lastly, regulatory requirements are also briefly assessed in the context of disposables and the Process Analytical Technologies (PAT) and Quality by Design (QbD) initiatives. Biotechnol. Bioeng. 2009;102: 348–356. © 2008 Wiley Periodicals, Inc.     

Sex differences in senescence: the role of intra-sexual competition in early adulthood

Males and females frequently differ in their rates of ageing, but the origins of these differences are poorly understood. Sex differences in senescence have been hypothesized to arise, because investment in intra-sexual reproductive competition entails costs to somatic maintenance, leaving the sex that experiences stronger reproductive competition showing higher rates of senescence. However, evidence that sex differences in senescence are attributable to downstream effects of the intensity of intra-sexual reproductive competition experienced during the lifetime remains elusive. Here, we show using a 35 year study of wild European badgers (Meles meles), that (i) males show higher body mass senescence rates than females and (ii) this sex difference is largely attributable to sex-specific downstream effects of the intensity of intra-sexual competition experienced during early adulthood. Our findings provide rare support for the view that somatic maintenance costs arising from intra-sexual competition can cause both individual variation and sex differences in senescence.     

Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.     

Multi-stage continuous high cell density culture systems: A review

A multi-stage continuous high cell density culture (MSC-HCDC) system makes it possible to achieve high productivity together with high product titer of many bioproducts. For long-term continuous operation of MSC-HCDC systems, the cell retention time and hydraulic retention time must be decoupled and strains (bacteria, yeast, plant, and animal cells) must be stable. MSC-HCDC systems are suitable for low-value high-volume extracellular products such as fuel ethanol, lactic acid or volatile fatty acids, and high-value products such as monoclonal antibodies as well as intracellular products such as polyhydroxybutyric acid (PHB), microbial lipids or a number of therapeutics. Better understanding of the fermentation kinetics of a specific product and reliable high-density culture methods for the product-generating microorganisms will facilitate timely industrialization of MSC-HCDC systems for products that are currently obtained in fed-batch bioreactors.     

A mechanistic model for gas–liquid mass transfer prediction in a rocking disposable bioreactor

Rocking disposable bioreactors are a newer approach to smaller-scale cell growth that use a cyclic rocking motion to induce mixing and oxygen transfer from the headspace gas into the liquid. Compared with traditional stirred-tank and pneumatic bioreactors, rocking bioreactors operate in a very different physical mode and in this study the oxygen transfer pathways are reassessed to develop a fundamental mass transfer (k_La) model that is compared with experimental data. The model combines two mechanisms, namely surface aeration and oxygenation via a breaking wave with air entrainment, borrowing concepts from ocean wave models. Experimental data for across the range of possible operating conditions (rocking speed, angle, and liquid volume) confirms the validity of the modeling approach, with most predictions falling within ±20% of the experimental values. At low speeds (up to 20 rpm) the surface aeration mechanism is shown to be dominant with a of around 3.5 hr⁻¹, while at high speeds (40 rpm) and angles the breaking wave mechanism contributes up to 91% of the overall (65 hr⁻¹). This model provides an improved fundamental basis for understanding gas–liquid mass transfer for the operation, scale-up, and potential design improvements for rocking bioreactors.     

A completely noninvasive method of dissolved oxygen monitoring in disposable small‐scale cell culture vessels based on diffusion through permeable vessel walls

Disposable cell culture vessels are extensively used at small scales for process optimization and validation, but they lack monitoring capabilities. Optical sensors that can be easily adapted for use in small‐scale vessels are commercially available for pH, dissolved oxygen (DO), and dissolved carbon dioxide (DCO₂). However, their use has been limited due to the contamination and compatibility issues. We have developed a novel solution to these problems for DO monitoring. Oxygen diffusion through permeable vessel wall can be exploited for noninvasive monitoring. An optical oxygen sensor can be placed outside the oxygen permeable vessel wall thereby allowing oxygen diffusing through the vessel wall to be detected by the sensor. This way the sensor stays separate from the cell culture and there are no concerns about contaminants or leachants. Here we implement this method for two cell culture devices: polystyrene‐made T‐75 tissue culture flask and fluorinated ethylene propylene (FEP)‐made Vuelife^® cell culture bag. Additionally, mammalian and microbial cell cultures were performed in Vuelife^® cell culture bags, proving that a sensor placed outside can be used to track changes in cell cultures. This approach toward noninvasive monitoring will help in integrating cell culture vessels with sensors in a seamless manner. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:172–177, 2014     

The Tetracycline Fermentation and Its Regulation

Abstract

The β-glucans different from cellulose are the most abundant class of polysaccharides. They are found in microorganisms and higher plants as structural entities of cell wall, as cytoplasmic and vacuolar reserve materials, and as extracellular substances. Enzyme systems capable to hydrolyze β-glucans are produced by different microorganisms. The occurrence and nature of β-glucanases and their substrates are reviewed. The regulation of biosynthesis of these enzymes, their properties, substrate and product specificities, mode of action and molecular cloning are described. The participation of β-glucanases in the morphogenetic events of yeast cell is presented. The role and synergism of different types of 1,3-β-glucanases in microbial cell wall lysis and the potential application for isolation of intracellular materials like proteins, carbohydrates, enzymes and as an analytical tool are discussed in the light of current knowledge.     

Modelling and simulation of continuous L (+) lactic acid production from sugarcane juice in membrane integrated hybrid-reactor system

Modelling and simulation was done for a two-stage membrane-integrated hybrid reactor system for continuous production of L (+) lactic acid under non-neutralizing conditions. The model captures microbial conversion of sugar cane juice to lactic acid under substrate–product inhibitions with downstream purification by nanofiltration. All the major phenomena and the governing parameters like fluid flow, feed dilution, substrate–product inhibitions, Donnan and steric effects during micro and nanofiltration for cell recycle, product separation and purification have been reflected in the modelling. The model describes a green, integrated continuous process of direct lactic acid production starting with a cheap, renewable carbon source. The highest lactic acid concentration achieved after the final stage of nanofiltration was 66.97 g/L at 13 kg/cm² operating pressure when the overall productivity reached 12.40 g/(L h). The developed model could successfully predict production, purification and transport of lactic acid through two stage membrane modules. Performance of the model was very good as indicated in the high overall correlation coefficient (R² > 0.980) and the low relative error (RE < 0.1).