Phytase activity in Cryptococcus laurentii ABO 510

Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayed temperature and pH optima of 62 degrees C and 5.0, respectively. The enzyme was thermostable at 70 degrees C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, D-glucose 6-phosphate, D-fructose 1,6-diphosphate and p-nitrophenyl phosphate (p-NPP), but its preferred substrate was phytic acid (K(m) of 21 microM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg(2+), Zn(2+), Cd(2+) and Ca(2+). These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.     

Randomized,Double-Blind,Crossover Trial of Amitriptyline for Analgesia in Painful HIV-Associated Sensory Neuropathy

We conducted a randomized, double-blind, placebo-controlled, crossover study at a single center in South Africa, to ascertain whether amitriptyline is an effective analgesic for painful HIV-associated sensory neuropathy of moderate to severe intensity in: i) antiretroviral drug naive individuals, and ii) antiretroviral drug users. 124 HIV-infected participants (antiretroviral drug naive = 62, antiretroviral drug users = 62) who met the study criteria for painful HIV-associated sensory neuropathy were randomized to once-daily oral amitriptyline (titrated to a median: interquartile range of 50: 25-50 mg) or placebo for six weeks, followed by a three-week washout period and subsequent treatment crossover. The primary outcome measure was change from baseline in worst pain intensity of the feet (measured by participant self-report using an 11-point numerical pain rating scale) after six weeks of treatment. 122 of 124 participants completed all study visits and were included in the analysis of the primary outcome. In the antiretroviral drug-naive group (n = 61) there was no significant difference in the mean change in pain score from baseline after six weeks of treatment with placebo or amitriptyline [amitriptyline: 2.8 (SD 3.3) vs. placebo: 2.8 (3.4)]. Similarly, there was no significant difference in the change in pain score after six weeks of treatment with placebo or amitriptyline in the antiretroviral drug-user group (n = 61) [amitriptyline: 2.7 (3.3) vs. placebo: 2.1 (2.8)]. Controlling for period effects and treatment order effects did not alter the outcome of the analyses. Nor did analyzing the intention-to-treat cohort (missing data interpolated using baseline observation carried forward) alter the outcome of the analyses. In summary, amitriptyline, at the doses used here, was no more effective than an inactive placebo at reducing pain intensity in individuals with painful HIV-associated sensory neuropathy of moderate to severe intensity, irrespective of whether they were on antiretroviral therapy or not.

Trial Registration

ISRCTN 54452526     

Black rhinoceros (Diceros bicornis) populations in northwestern Namibia are apparently not infected with piroplasms

Babesiosis is a potentially fatal disease in black rhinoceroses. Blood specimens collected from black rhinoceroses from Etosha National Park (n = 29) and Damaraland (n = 22), Namibia, were subjected to polymerase chain reaction using Theileria and Babesia genus-specific primers and reverse line blot, with negative results. The animals were sparsely infested with ticks. In the absence of suitable prophylactic measures, na?ve rhinoceroses would be at risk if translocated to Babesia-endemic areas.     

A Pilot Study Assessing the Impact of a Fortified Supplementary Food on the Health and Well-Being of Crèche Children and Adult TB Patients in South Africa

The South African population faces many of the global concerns relating to micronutrient deficiency and the impact this has on health and well-being. Moreover, there is a high prevalence of HIV infection, compounded by a high level of co-infection with TB.This pilot study evaluates the impact of a fortified supplementary food on the health and well-being of a cohort of crèche children, aged 3 to 6, and adult TB patients drawn from the Presidential Node of Alexandra, Johannesburg, South Africa. A further aim of this study was to evaluate the sensitivity and validity of non-invasive indicators of nutritional status in a field-based population sample.The investigational product, e’Pap, is supported by extensive anecdotal evidence that whole grain cereals with food-style nutrients constitute an effective supplementary food for those suffering from the effects of food insecurity, poor health and well-being, and coping with TB and HIV infection.The results indicate a beneficial effect of e’Pap for both study populations, and particularly for adult TB patients, whose baseline data reflected severe food insecurity and malnutrition in the majority of cases. There is evidence to suggest statistically significant improvements in key micronutrient levels, well-being and energy, hand-grip strength, the Bioelectrical Impedance Analysis (BIA) Illness Marker, and certain clinical indicators. Although Body Mass Index (BMI) and Mid Upper Arm Circumference (MUAC) are frequently used as standard measures to evaluate the efficacy of nutritional interventions, these indicators were not sufficiently sensitive in this study. Nor does weight gain necessarily indicate improved nutritional status. Hand-grip strength, lean body mass, and the BIA Illness Marker seem to be more useful indicators of change in nutritional status.     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

Comparative secretome analysis of <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> S4F8 and <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C30 during solid-state fermentation on sugarcane bagasse

Background

The lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF).

Results

Comparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Liquid chromatography tandem mass spectrometry analysis of the two fungal secretomes enabled the detection of 815 proteins in total, with 418 and 397 proteins being specific for S4F8 and Rut C30, respectively, and 174 proteins being common to both strains. In-depth analysis of the associated biological functions and the representation of glycoside hydrolase family members within the two secretomes indicated that the S4F8 secretome contained a higher diversity of main and side chain hemicellulases and β-glucosidases, and an increased abundance of some of these proteins compared with the Rut C30 secretome.

Conclusions

In SCB SSF, T. asperellum S4F8 produced a more complex lignocellulolytic cocktail, with enhanced hemicellulose and cellobiose hydrolysis potential, compared with T. reesei Rut C30. This bodes well for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail from T. asperellum for lignocellulosic feedstock hydrolysis.

     

Expression and evaluation of enzymes required for the hydrolysis of galactomannan

The cost-effective production of bioethanol from lignocellulose requires the complete conversion of plant biomass, which contains up to 30 % mannan. To ensure utilisation of galactomannan during consolidated bioprocessing, heterologous production of mannan-degrading enzymes in fungal hosts was explored. The Aspergillus aculeatus endo-β-mannanase (Man1) and Talaromyces emersonii α-galactosidase (Agal) genes were expressed in Saccharomyces cerevisiae Y294, and the Aspergillus niger β-mannosidase (cMndA) and synthetic Cellvibrio mixtus β-mannosidase (Man5A) genes in A. niger. Maximum enzyme activity for Man1 (374 nkat ml^?1, pH 5.47), Agal (135 nkat ml^?1, pH 2.37), cMndA (12 nkat ml^?1, pH 3.40) and Man5A (8 nkat ml^?1, pH 3.40) was observed between 60 and 70 °C. Co-expression of the Man1 and Agal genes in S. cerevisiae Y294[Agal-Man1] reduced the extracellular activity relative to individual expression of the respective genes. However, the combined action of crude Man1, Agal and Man5A enzyme preparations significantly decreased the viscosity of galactomannan in locust bean gum, confirming hydrolysis thereof. Furthermore, when complemented with exogenous Man5A, S. cerevisiae Y294[Agal-Man1] produced 56 % of the theoretical ethanol yield, corresponding to a 66 % carbohydrate conversion, on 5 g l^?1 mannose and 10 g l^?1 locust bean gum.     

Evaluation of a Real-Time PCR Test for the Detection and Discrimination of Theileria Species in the African Buffalo (Syncerus caffer)

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected.Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.     

Proteomic analysis reveals differential protein expression by Bacillus cereus during biofilm formation

Bacillus cereus, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods. B. cereus DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and L-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both sigma(B) and sigma(H), could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.