Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Ethnography of software maintenance activities in an industrial engineering setup

New technical and procedural interventions are less likely to be adopted in industry, unless they are smoothly integrated into the existing practices of professionals. In this paper, we provide a case study of the use of ethnographic methods for studying software bug-fixing activities at an industrial engineering conglomerate. We aimed at getting an in-depth understanding of software developers' everyday practices in bug-fixing related projects and in turn inform the design of novel productivity tools. The use of ethnography has allowed us to look at the social side of software maintenance practices. In this paper, we highlight: 1) organizational issues that influence bug-fixing activities; 2) social role of bug tracking systems, and; 3) social issues specific to different phases of bug-fixing activities.

Bone regeneration based on tissue engineering conceptions – a 21st century perspective

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteo- conductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineer- ing and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

Informing engineering education for sustainable development using a deliberative dynamic model for curriculum renewal

Literature from around the world clearly suggests that engineering education has been relatively slow to incorporate significant knowledge and skill areas, including the rapidly emerging area of sustainable development. Within this context, this paper presents the findings of research that questioned how engineering educators could consistently implement systematic and intentional curriculum renewal that is responsive to emerging engineering challenges and opportunities. The paper presents a number of elements of systematic and intentional curriculum renewal that have been empirically distilled from a qualitative multiple-method iterative research approach including literature review, narrative enquiry, pilot trials and peer-review workshops undertaken by the authors with engineering educators from around the world. The paper also presents new knowledge arising from the research, in the form of a new model that demonstrates a dynamic and deliberative mechanism for strategically accelerating for curriculum renewal efforts. Specifically the paper discusses implications of this model to achieve education for sustainable development, across all disciplines of engineering. It concludes with broader research and practice implications for the field of education research.

Engineering education for sustainable development : a review of international progress

Since the late 1980s there have been increasing calls around the world for embedding sustainability content throughout engineering curricula, particularly over the past decade. However in general there has been little by way of strategic or systematic integration within programs offered by higher education institutions(HEIs). Responding to a growing awareness towards the issues surrounding sustainability, a number of professional engineering institutions (PEIs) internationally have placed increasing emphasis on policies and initiatives relating to the role of engineering in addressing 21st Century challenges. This has resulted in some consideration towards integrating sustainable development into engineering curricula as envisaged by accreditation guidelines. This paper provides a global overview of such accreditation developments, highlighting emerging sustainability competencies (or ‘graduate attributes’) and places these in the context of relevant PEI declarations, initiatives, policies, codes of ethics and guideline publications. The paper concludes by calling for urgent action by PEIs, including strategic accreditation initiatives that promote timely curriculum renewal towards EESD.

Applying threshold learning theory to teach sustainable business practice in post-graduate engineering education

This paper presents the results of a qualitative action-research inquiry into how a highly diverse cohort of post-graduate students could develop significant capacity in sustainable development within a single unit (course), in this case a compulsory component of four built environment masters programs. The method comprised applying threshold learning theory within the technical discipline of sustainable development, to transform student understanding of sustainable business practice in the built environment. This involved identifying a number of key threshold concepts, which once learned would provide a pathway to having a transformational learning experience. Curriculum was then revised, to focus on stepping through these targeted concepts using a scaffolded, problem-based-learning approach. Challenges included a large class size of 120 students, a majority of international students, and a wide span of disciplinary backgrounds across the spectrum of built environment professionals. Five ‘key’ threshold learning concepts were identified and the renewed curriculum was piloted in Semester 2 of 2011. The paper presents details of the study and findings from a mixed-method evaluation approach through the semester. The outcomes of this study will be used to inform further review of the course in 2012, including further consideration of the threshold concepts. In future, it is anticipated that this case study will inform a framework for rapidly embedding sustainability within curriculum.

Using visualization tool to help engineering students learning dynamics

Dynamics is an essential core engineering subject. It includes high level mathematical and theoretical contents, and basic concepts which are abstract in nature. Hence, Dynamics is considered as one of the hardest subjects in the engineering discipline. To assist our students in learning this subject, we have conducted a Teaching & Learning project to study ways and methods to effectively teach Dynamics based on visualization techniques. The research project adopts the five basic steps of Action Learning Cycle. It is found that visualization technique is a powerful tool for students learning Dynamics and helps to break the barrier of students who perceived Dynamics as a hard subject.

Effect of trash on the coagulation and flocculation of sugarcane juice

The processing of juice expressed from whole green sugarcane crop (stalk and trash) leads to poor clarification performance, reduced sugar yield and poor raw sugar quality. The cause of these adverse effects is linked to the disproportionate contribution of impurities from the trash component of the crop. This paper reports on the zeta (ζ) potential, average size distribution (d50) and fractal dimension (Df) of limed juice particles derived from various juice types using laser diffraction and dynamic light scattering techniques. The influence of non-sucrose impurities on the interactive energy contributions between sugarcane juice particles was examined on the basis of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Results from these investigations have provided evidence (in terms of particle stability) on why juice particles derived from whole green sugarcane crop are relatively difficult to coagulate (and flocculate). The presence of trash reduces the van der Waals forces of attraction between particles, thereby reducing coagulation and flocculation processes. It is anticipated that further fundamental work will lead to strategies that could be adopted for clarifying juices expressed from whole green sugarcane crop.

Effect of trash on the coagulation and flocculation of sugarcane juice

The processing of juice expressed from whole green sugarcane crop (stalk and trash) leads to poor clarification performance, reduced sugar yield and poor raw sugar quality. The cause of these adverse effects is linked to the disproportionate contribution of impurities from the trash component of the crop. This paper reports on the zeta (?) potential, average size distribution (d50) and fractal dimension (Df) of limed juice particles derived from various juice types using laser diffraction and dynamic light scattering techniques. The influence of non-sucrose impurities on the interactive energy contributions between sugarcane juice particles was examined on the basis of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Results from these investigations have provided evidence (in terms of particle stability) on why juice particles derived from whole green sugarcane crop are relatively difficult to coagulate (and flocculate). The presence of trash reduces the van der Waals forces of attraction between particles, thereby reducing coagulation and flocculation processes. It is anticipated that further fundamental work will lead to strategies that could be adopted for clarifying juices expressed from whole green sugarcane crop.

A novel route in bone tissue engineering : magnetic biomimetic scaffolds

In recent years, interest in tissue engineering and its solutions has increased considerably. In particular, scaffolds have become fundamental tools in bone graft substitution and are used in combination with a variety of bio-agents. However, a long-standing problem in the use of these conventional scaffolds lies in the impossibility of re-loading the scaffold with the bio-agents after implantation. This work introduces the magnetic scaffold as a conceptually new solution. The magnetic scaffold is able, via magnetic driving, to attract and take up in vivo growth factors, stem cells or other bio-agents bound to magnetic particles. The authors succeeded in developing a simple and inexpensive technique able to transform standard commercial scaffolds made of hydroxyapatite and collagen in magnetic scaffolds. This innovative process involves dip-coating of the scaffolds in aqueous ferrofluids containing iron oxide nanoparticles coated with various biopolymers. After dip-coating, the nanoparticles are integrated into the structure of the scaffolds, providing the latter with magnetization values as high as 15 emu g�1 at 10 kOe. These values are suitable for generating magnetic gradients, enabling magnetic guiding in the vicinity and inside the scaffold. The magnetic scaffolds do not suffer from any structural damage during the process, maintaining their specific porosity and shape. Moreover, they do not release magnetic particles under a constant flow of simulated body fluids over a period of 8 days. Finally, preliminary studies indicate the ability of the magnetic scaffolds to support adhesion and proliferation of human bone marrow stem cells in vitro. Hence, this new type of scaffold is a valuable candidate for tissue engineering applications, featuring a novel magnetic guiding option.

Controlling microencapsulation and release of micronized proteins using poly(ethylene glycol) and electrospraying

The fabrication of tailored microparticles for delivery of therapeutics is a challenge relying upon a complex interplay between processing parameters and materials properties. The emerging use of electrospraying allows better tailoring of particle morphologies and sizes than current techniques, critical to reproducible release profiles. While dry encapsulation of proteins is essential for the release of active therapeutics from microparticles, it is currently uncharacterized in electrospraying. To this end, poly(ethylene glycol) (PEG) was assessed as a micronizing and solubilizing agent for dry protein encapsulation and release from electrosprayed particles made from polycaprolactone (PCL). The physical effect of PEG in protein-loaded poly(lactic-co-glycolic acid) (PLGA) particles was also studied, for comparison. The addition of 5–15 wt% PEG 6 kDa or 35 kDa resulted in reduced PCL particle sizes and broadened distributions, which could be improved by tailoring the electrospraying processing parameters, namely by reducing polymer concentration and increasing flow rate. Upon micronization, protein particle size was reduced to the micrometer domain, resulting in homogenous encapsulation in electrosprayed PCL microparticles. Microparticle size distributions were shown to be the most determinant factor for protein release by diffusion and allowed specific control of release patterns.

Rational design of artificial cellular niches for tissue engineering

Tissue Engineering is a promising emerging field that studies the intrinsic regenerative potential of the human body and uses it to restore functionality of damaged organs or tissues unable of self-healing due to illness or ageing. In order to achieve regeneration using Tissue Engineering strategies, it is first necessary to study the properties of the native tissue and determine the cause of tissue failure; second, to identify an optimum population of cells capable of restoring its functionality; and third, to design and manufacture a cellular microenvironment in which those specific cells are directed towards the desired cellular functions. The design of the artificial cellular niche has a tremendous importance, because cells will feel and respond to both its biochemical and biophysical properties very differently. In particular, the artificial niche will act as a physical scaffold for the cells, allowing their three-dimensional spatial organization; also, it will provide mechanical stability to the artificial construct; and finally, it will supply biochemical and mechanical cues to control cellular growth, migration, differentiation and synthesis of natural extracellular matrix. During the last decades, many scientists have made great contributions to the field of Tissue Engineering. Even though this research has frequently been accompanied by vast investments during extended periods of time, yet too often these efforts have not been enough to translate the advances into new clinical therapies. More and more scientists in this field are aware of the need of rational experimental designs before carrying out complex, expensive and time-consuming in vitro and in vivo trials. This review highlights the importance of computer modeling and novel biofabrication techniques as critical key players for a rational design of artificial cellular niches in Tissue Engineering.

Curricula integration : identifying and locating engineering education across the Australian curriculum

Science, technology, engineering and mathematics (STEM) has become an educational package emerging throughout the world (e.g. UK, China, US & Australia). Although science, technology and mathematics are taught in schools and engineering education occurs in universities, there appear to be few if any explicit engineering education programs in primary and junior secondary schools. A stronger inclusion of engineering education at these levels could assist students to make informed decisions about career opportunities in STEM-related fields. This paper suggests how engineering education can be integrated with other key learning areas such as English, mathematics, science, history and geography within the new Australian Curriculum.

Simulation of plant cell shrinkage during drying : A SPH–DEM approach

Plant based dried food products are popular commodities in global market where much research is focused to improve the products and processing techniques. In this regard, numerical modelling is highly applicable and in this work, a coupled meshfree particle-based two-dimensional (2-D) model was developed to simulate micro-scale deformations of plant cells during drying. Smoothed Particle Hydrodynamics (SPH) was used to model the viscous cell protoplasm (cell fluid) by approximating it to an incompressible Newtonian fluid. The visco-elastic characteristic of the cell wall was approximated to a Neo-Hookean solid material augmented with a viscous term and modelled with a Discrete Element Method (DEM). Compared to a previous work [H. C. P. Karunasena, W. Senadeera, Y. T. Gu and R. J. Brown, Appl. Math. Model., 2014], this study proposes three model improvements: linearly decreasing positive cell turgor pressure during drying, cell wall contraction forces and cell wall drying. The improvements made the model more comparable with experimental findings on dried cell morphology and geometric properties such as cell area, diameter, perimeter, roundness, elongation and compactness. This single cell model could be used as a building block for advanced tissue models which are highly applicable for product and process optimizations in Food Engineering.

Experimental and numerical analysis of the relationship between indoor and outdoor airborne particles in an operating room

This work investigated the impact of the HVAC filtration system and indoor particle sources on the relationship between indoor and outdoor airborne particle size and concentrations in an operating room. Filters with efficiency between 65% and 99.97% were used in the investigation and indoor and outdoor particle size and concentrations were measured. A balance mass model was used for the simulation of the impact of the surgical team, deposition rate, HVAC exhaust and air change rates on indoor particle concentration. The experimental results showed that high efficiency filters would not be expected to decrease the risk associated with indoor particles larger than approximately 1 µm in size because normal filters are relatively efficient for these large particles. A good fraction of outdoor particles were removed by deposition on the HVAC system surfaces and this deposition increased with particle size. For particles of 0.3-0.5 µm in diameter, particle reduction was about 23%, while for particles >10 µm the loss was about 78%. The modelling results showed that depending on the type of filter used, the surgical team generated between 93-99% of total particles, while the outdoor air contributed only 1-6%.