Expression, purification, and characterization of biol: A carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene biol. We have subcloned bioI and overexpressed the encoded protein, BioI. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography, Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coil acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis. In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production. (C) 2000 Academic Press.

Simultaneous steady state and dynamic design of process systems using structural indicators

The simultaneous design of the steady-state and dynamic performance of a process has the ability to satisfy much more demanding dynamic performance criteria than the design of dynamics only by the connection of a control system. A method for designing process dynamics based on the use of a linearised systems' eigenvalues has been developed. The eigenvalues are associated with system states using the unit perturbation spectral resolution (UPSR), characterising the dynamics of each state. The design method uses a homotopy approach to determine a final design which satisfies both steady-state and dynamic performance criteria. A highly interacting single stage forced circulation evaporator system, including control loops, was designed by this method with the goal of reducing the time taken for the liquid composition to reach steady-state. Initially the system was successfully redesigned to speed up the eigenvalue associated with the liquid composition state, but this did not result in an improved startup performance. Further analysis showed that the integral action of the composition controller was the source of the limiting eigenvalue. Design changes made to speed up this eigenvalue did result in an improved startup performance. The proposed approach provides a structured way to address the design-control interface, giving significant insight into the dynamic behaviour of the system such that a systematic design or redesign of an existing system can be undertaken with confidence.

Tidal fluctuations in a leaky confined aquifer: Dynamic effects of an overlying phreatic aquifer

Tidal fluctuations in a leaky confined coastal aquifer are damped significantly due to leakage into an overlying phreatic aquifer. Jiao and Tang [1999] presented an analytical solution to a simple model describing this phenomenon. Their solution assumes that the tidal fluctuations in the overlying phreatic aquifer are negligible (i.e,, a static phreatic aquifer), Here we examine dynamic effects of the overlying aquifer based on a new approximate analytical solution. The numerical results indicate that the dynamic effects can be significant for a relatively large leakage and a high transmissivity of the phreatic aquifer.

Using stochastic dynamic programming to determine optimal fire management for Banksia ornata

1. A model of the population dynamics of Banksia ornata was developed, using stochastic dynamic programming (a state-dependent decision-making tool), to determine optimal fire management strategies that incorporate trade-offs between biodiversity conservation and fuel reduction. 2. The modelled population of B. ornata was described by its age and density, and was exposed to the risk of unplanned fires and stochastic variation in germination success. 3. For a given population in each year, three management strategies were considered: (i) lighting a prescribed fire; (ii) controlling the incidence of unplanned fire; (iii) doing nothing. 4. The optimal management strategy depended on the state of the B. ornata population, with the time since the last fire (age of the population) being the most important variable. Lighting a prescribed fire at an age of less than 30 years was only optimal when the density of seedlings after a fire was low (< 100 plants ha(-1)) or when there were benefits of maintaining a low fuel load by using more frequent fire. 5. Because the cost of management was assumed to be negligible (relative to the value of the persistence of the population), the do-nothing option was never the optimal strategy, although lighting prescribed fires had only marginal benefits when the mean interval between unplanned fires was less than 20-30 years.

A 2D numerical model for simulating the physics of fault systems

Simulations provide a powerful means to help gain the understanding of crustal fault system physics required to progress towards the goal of earthquake forecasting. Cellular Automata are efficient enough to probe system dynamics but their simplifications render interpretations questionable. In contrast, sophisticated elasto-dynamic models yield more convincing results but are too computationally demanding to explore phase space. To help bridge this gap, we develop a simple 2D elastodynamic model of parallel fault systems. The model is discretised onto a triangular lattice and faults are specified as split nodes along horizontal rows in the lattice. A simple numerical approach is presented for calculating the forces at medium and split nodes such that general nonlinear frictional constitutive relations can be modeled along faults. Single and multi-fault simulation examples are presented using a nonlinear frictional relation that is slip and slip-rate dependent in order to illustrate the model.

Credit assignment: The memory representation of dynamic task events

The experiment examined the influence of memory for prior instances on aircraft conflict detection. Participants saw pairs of similar aircraft repeatedly conflict with each other. Performance improvements suggest that participants credited the conflict status of familiar aircraft pairs to repeated static features such as speed, and dynamic features such as aircraft relative position. Participants missed conflicts when a conflict pair resembled a pair that had repeatedly passed safely. Participants either did not attend to, or interpret, the bearing of aircraft correctly as a result of false memory-based expectations. Implications for instance models and situational awareness in dynamic systems are discussed.

Loose abrasive truing and dressing of resin bond diamond cup wheels for grinding fibre optic connectors

A loose abrasive lapping technology was developed for truing and dressing ultrafine diamond cup wheels for grinding spherical end faces of fibre optic connectors. The relative densities of exposed grits and grit pull-outs measured from wheel surfaces prepared using the loose abrasive lapping and the bonded abrasive dressing were compared. It was found that the lapping method with loose abrasives produced wheel surfaces with more exposed grits and less grit pull-outs, especially for finer grit size wheels. For dressing ultrafine grit size wheels, the particle size of the lapping paste should be smaller than the wheel grit size to achieve a better result. It is also found that the wheels dressed using the lapping method demonstrate an excellent grinding performance. (C) 2004 Elsevier B.V.. All rights reserved.

Dynamic strain measurement using piezoelectric polymer film

Piezoelectric polymers have been used to form the basis of dynamic strain gauges for the detection of stress waves. The linearity of response was tested using a split Hopkinson pressure bar arrangement. The results obtained illustrate the effectiveness of piezoelectric film strain gauges in the measurement of axial stress waves.

Analysis of dynamic process models for structural insight and model reduction .1. Structural identification measures

An important consideration in the development of mathematical models for dynamic simulation, is the identification of the appropriate mathematical structure. By building models with an efficient structure which is devoid of redundancy, it is possible to create simple, accurate and functional models. This leads not only to efficient simulation, but to a deeper understanding of the important dynamic relationships within the process. In this paper, a method is proposed for systematic model development for startup and shutdown simulation which is based on the identification of the essential process structure. The key tool in this analysis is the method of nonlinear perturbations for structural identification and model reduction. Starting from a detailed mathematical process description both singular and regular structural perturbations are detected. These techniques are then used to give insight into the system structure and where appropriate to eliminate superfluous model equations or reduce them to other forms. This process retains the ability to interpret the reduced order model in terms of the physico-chemical phenomena. Using this model reduction technique it is possible to attribute observable dynamics to particular unit operations within the process. This relationship then highlights the unit operations which must be accurately modelled in order to develop a robust plant model. The technique generates detailed insight into the dynamic structure of the models providing a basis for system re-design and dynamic analysis. The technique is illustrated on the modelling for an evaporator startup. Copyright (C) 1996 Elsevier Science Ltd

Analysis of dynamic models for structural insight and model reduction .2. A multi-stage compressor shutdown case-study

In this second paper, the three structural measures which have been developed are used in the modelling of a three stage centrifugal synthesis gas compressor. The goal of this case study is to determine the essential mathematical structure which must be incorporated into the compressor model to accurately model the shutdown of this system. A simple, accurate and functional model of the system is created via three structural measures. It was found that the model can be correctly reduced into its basic modes and that the order of the differential system can be reduced from 51(st) to 20(th). Of the 31 differential equational 21 reduce to algebraic relations, 8 become constants and 2 can be deleted thereby increasing the algebraic set from 70 to 91 equations. An interpretation is also obtained as to which physical phenomena are dominating the dynamics of the compressor add whether the compressor will enter surge during the shutdown. Comparisons of the reduced model performance against the full model are given, showing the accuracy and applicability of the approach. Copyright (C) 1996 Elsevier Science Ltd