Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening

Aims:  The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants.
Methods and Results:  The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1·2 g l⁻¹ of FaeB was selected (12-fold more than the A. niger overproducing strain).
Conclusions:  This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days.
Significance and Impact of the Study:  This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.     

Kinetic Behavior of Native Alkaline Phosphatase from Human Placenta

The kinetic behavior of human placental alkaline phosphatase, which catalyses the hydrolysis of p-nitrophenyl and of o-carboxyphenyl phosphates, was studied by means of graphical and non-linear regression statistical fitting analysis of data of rate versus substrate concentration. Non linear Lineweaver-Burk and Eadie-Hofstee plots and rational functions of degree 2:2 (F-test assessing the goodness of fit) show non-Michaelian kinetic behavior. In the same way, the behavior of the enzyme was also non-Michaelian in the simultaneous presence of these two substrates.

Norlaudanosoline is a key intermediate in the biosynthesis of the benzylisoquinoline alkaloids providing the benzyl-isoquinoline portion of the morphinan skeleton. This study examines a coupled reaction system for the production of norlaudanosoline from dopamine. In this coupled system, dopamine is enzymatically converted by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (dopaldehyde). In the presence of dopamine, this aldehyde undergoes a spontaneous Pictet-Spengler condensation to form norlaudanosoline. Three potential sources of MAO were investigated: a fungal source (Aspergillus niger), a bacterial source (Sarcina lutea) and a commercial source isolated from bovine plasma. Kinetic studies with dopamine as the substrate gave Michaelis constants (K_m) of 1.81 × 10^-5 M, 6.94 × 10^-3, and 1.61 × 10^-3 M for A. niger, S. lutea and bovine plasma oxidase, respectively. The reaction system is complicated because of the effect of the condensation reaction, so a more rigorous model was developed to account for this effect. The model was suitable for showing the effect of dopamine concentration on norlaudanosoline production alghough there were some model inadequacies. Using the model a forward rate constant for the Pictet-Spengler condensation was determined to be 6.8 × 10^-2 M^-1 s^-1 and the reverse reaction appears to be negligible. Overall conversion was 14% which is 20 times that achieved in an in situ reaction system using whole cells of Aspergillus niger.     

Vigilance towards raptors by nuclear species in bird mixed flocks in a Brazilian savannah

A nuclear or leader species is the one around which foraging activity is organized. In the campo-cerrado (Brazilian savannah) up to four bird species (Saltator atricollis, Cypsnagra hirundinacea, Mimus saturninus, and Neothraupis fasciata) may function as nuclear or leader species in mixed species flocks. The aim of this study was to assess the features shown by these nuclear species. I quantified parameters of sociality, communication and alertness of nuclear bird species in mixed flocks with different composition. Parameters related to sociality (mean intraspecific group size) and communication (frequency of contact calls) were not correlated with the leadership. On the other hand, the most alert species was in the front of a given mixed flock most of the time. The leader species spent more time in vigilance and gave most alarm calls due to approaching raptors earlier. The results of this study strongly suggest that the alertness of a species is the major character of nuclear bird species in mixed flocks of the campo-cerrado.     

Evaluation of population status and foraging ecology of Sable antelope (Hippotragus niger roosevelti,Heller, 1910) in Shimba Hills National Reserve,Kenya

The Sable antelope (Hippotragus niger roosevelti, Heller, 1910) is nationally endemic to Shimba Hills National Reserve (SHNR) in Kenya. In the past few decades, its population has declined considerably. Despite the alarming decline and resultant localised distribution, a little information exists on the species population status and foraging ecology. Different ecological research techniques were used to collect relevant data and information on the species population structure and feeding habits. The results showed that Sable population comprised of sex and age structures that are skewed towards females and adult, respectively, whereas young and subadult populations were not significantly different. Seasonal change did not have significant influence on the diversity of food plants selected by Sable. Although crude protein and phosphorous levels in Sable faecal samples differed significantly between the seasons, they were within the recommended minimum maintenance requirements for wild herbivores. The study concludes that Sable has good survival rate and potential to breed but lacks stability in the population. Additionally, Sable forage quality and availability may not be limiting its population growth. There is need to establish management strategies for improving reproduction in Sable and understand the level of competition of the species with other mega herbivores in SHNR.     

DNA-led rediscovery of the giant sable antelope in Angola

The giant sable antelope (Hippotragus niger variani), unique to Angola, was feared extinct after almost three decades of civil war. Comparisons of mitochondrial DNA sequences derived from dung samples recently collected in the field and from old museum specimens of certain provenance provide the first documented evidence to date that this enigmatic antelope has survived. Its DNA-led rediscovery in the former combat zone was subsequently confirmed by photographic evidence. The Angolan isolate constitutes a distinct monophyletic group that shows a dramatic population decline from historic levels. It represents a diagnosable conservation unit which is characterised by unique cranial morphological features (Blaine 1922 1922:317–339), a highly restricted range, and the presence of fixed genetic differences in all of its common relatives.     

Physical mapping of the mitochondrial DNA from Aspergillus niger

Abstract The mitochondrial DNA was isolated from Aspergillus niger WU-2223L, a citric acid-production strain, and characterized by restriction-endonuclease mapping. Cloned fragments which covered the total range of the mitochondrial DNA were assembled and utilized to construct the restriction-endonuclease map for nine restriction enzymes. This map showed that the mitochondrial DNA was a circular molecule of 32.6 kb.