Refolding and characterisation of a heterologous expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.2)

Cloned Phanerochaete chrysosporium ME446 cbhI.2 cDNA was successfully expressed in Escherichia coli as an insoluble, internal, biologically inactive protein. In vitro chemical refolding restored the activity of the crude CBHI.2. However, this enzyme was active against 4-methylumbelliferyl-ß-Dcellobioside(MUC) and 4-methyllumbelliferyl-ß-D-lactopyranoside (MUL) substrates only. The crude enzyme lost almost 50% of its activity at 5 min at 100oC heat treatment whereas total inactivation was achieved at 30 min

Cloning and expression of Phanerochaete chrysosporium cellobiohydrolase (cbhI.2)

The Phanerochaete chrysosporium ME446 cbhI.2 cDNA was cloned and expressed in Escherichia coli using a pET system. Pulse-labelling revealed the expression of an induced protein of approximately 50- 60 kDa that reacted with anti-P. chrysosporium ME446 CBH antibodies. The expressed protein remained undegraded in vivo for 135 min but was biologically inactive in vitro

Lignocellulose biotechnology: issues of bioconversion and enzyme production

This review is written from the perspective of scientists working in lignocellulose bioconversion in a developing country and the aim of this review is to remind ourselves and other scientists working in related areas of lignocellulose research of the enormous economic potential of the bioprocessing of residual plant materials generally regarded as “waste”, and secondly to highlight some of the modern approaches which potentially could be used to tackle one of the major impediments, namely high enzyme cost, to speed-up the extensive commercialisation of the lignocellulose bioprocessing. Key words: lignocellulose, bioconversion, enzyme cost. African Journal of Biotechnology Vol. 2 (12), pp. 602-619, December 200

Characterisation of a chimeric Phanerochaete chrysosporium cellobiohydrolase expressed from Escherichia coli

The aim of this study was to purify and analyse a Phanerochaete chrysosporium cbhI.1 gene-product expressed as an inducible, secreted, heterologous protein from an Escerichia coli pGEXcbhI.1 clone. Using glutathione Sepharose 4B affinity chromatography, the expressed protein was purified from the supernatant of an induced E. coli transformed with pGEXcbhI.1 and ran as a single band on a Sodium dodecyl sulphate-polyacrylamide gel. The glutathione S-transferase (GST) fused CBHI.1 was approx-imately 80 kDa in size, approximately 2.2 kDa smaller than the theoretically predicted size. The purified protein exhibited time dependent hydrolytic reaction against carboxy-methyl-cellulose (CMC) and Avicel. On CMC the highest hydrolytic reaction occurred at 120 min. whereas for Avicel it was at 150 min. Optimum pH and temperature for activity of the protein against these cellulose substrates were pH 6 and 55oC, respectively, and the protein remained stable under these optimum conditions for 24 h. Key Words: Phanerochaete chrysosporium, cellobiohydrolase purification, heterologus expression. African Journal of Biotechnology Vol.3(7) 2004: 349-35

A Systematic Review and Meta-Analysis of Utility-Based Quality of Life in Chronic Kidney Disease Treatments

Background: Chronic kidney disease (CKD) is a common and costly condition to treat. Economic evaluations of health care often incorporate patient preferences for health outcomes using utilities. The objective of this study was to determine pooled utility-based quality of life (the numerical value attached to the strength of an individual's preference for a specific health outcome) by CKD treatment modality. Methods and Findings: We conducted a systematic review, meta-analysis, and meta-regression of peer-reviewed published articles and of PhD dissertations published through 1 December 2010 that reported utility-based quality of life (utility) for adults with late-stage CKD. Studies reporting utilities by proxy (e.g., reported by a patient's doctor or family member) were excluded. In total, 190 studies reporting 326 utilities from over 56,000 patients were analysed. There were 25 utilities from pre-treatment CKD patients, 226 from dialysis patients (haemodialysis, n = 163; peritoneal dialysis, n = 44), 66 from kidney transplant patients, and three from patients treated with non-dialytic conservative care. Using time tradeoff as a referent instrument, kidney transplant recipients had a mean utility of 0.82 (95% CI: 0.74, 0.90). The mean utility was comparable in pre-treatment CKD patients (difference = -0.02; 95% CI: -0.09, 0.04), 0.11 lower in dialysis patients (95% CI: -0.15, -0.08), and 0.2 lower in conservative care patients (95% CI: -0.38, -0.01). Patients treated with automated peritoneal dialysis had a significantly higher mean utility (0.80) than those on continuous ambulatory peritoneal dialysis (0.72; p = 0.02). The mean utility of transplant patients increased over time, from 0.66 in the 1980s to 0.85 in the 2000s, an increase of 0.19 (95% CI: 0.11, 0.26). Utility varied by elicitation instrument, with standard gamble producing the highest estimates, and the SF-6D by Brazier et al., University of Sheffield, producing the lowest estimates. The main limitations of this study were that treatment assignments were not random, that only transplant had longitudinal data available, and that we calculated EuroQol Group EQ-5D scores from SF-36 and SF-12 health survey data, and therefore the algorithms may not reflect EQ-5D scores measured directly. Conclusions: For patients with late-stage CKD, treatment with dialysis is associated with a significant decrement in quality of life compared to treatment with kidney transplantation. These findings provide evidence-based utility estimates to inform economic evaluations of kidney therapies, useful for policy makers and in individual treatment discussions with CKD patients. © 2012 Wyld et al

The phytochemical, antibacterial and antioxidant activity of five medicinal plants against the wound infecting bacteria

Leaf extracts of Senna italica, Ricinus communis, Lantana camara, Lippia javanica and Ziziphus  mucronata were screened for biological activity against bacteria which infect wounds. The leaves were extracted using different solvents of varying polarity (hexane, dichloromethane, acetone and methanol). Phytochemical analyses of the extracts were performed using thin layer chromatography (TLC). The extracts were loaded on TLC plates and developed in three solvent systems that is benzene/ethanol /ammonium solution (BEA), chloroform/ethyl acetate/formic acid (CEF) and ethyl acetate/methanol /water (EMW). Antibacterial activity of the plants was evaluated using micro-dilution and bioautography methods. The test organisms used were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis. Acetone extracts were chosen for antioxidant activity. Methanol was the best extractant, followed by acetone and dichloromethane (DCM). In the phytochemical analysis,  more compounds were observed on BEA, followed by EMW and CEF plates. Lantana camara had no  activity against any of the bacteria used. P. aeruginosa was the most resistant bacterium with only two plants active against it. E. faecalis and E. coli were sensitive to the extracts. More antibacterial  compounds were observed on BEA plates against all the test bacteria in bioautographic method. The Rf  values calculated from bioautography indicated that the selected plants have different active compounds. The most active compounds were from S. italica and Z. mucronata. BEA and EMW plates had good  antioxidant activity. No antioxidant activity was observed on the CEF plate. Most extracts were active against wound pathogens; their application on the wound area may prevent infection. Further studies are required to identify the active compounds in the plant extracts which showed significant anti-bacterial activities.Key words: Thin layer chromatography (TLC), plant extract, bacteria

A continuous flow-batch hybrid reactor for commodity chemical synthesis enabled by inline NMR and temperature monitoring

Inline, real time NMR and temperature measurements have been used to optimise the continuous flow synthesis of difluoromethyltrimethylsilane (TMSCF2H) by the reduction of the Ruppert-Prakash reagent (TMSCF3). These measurements were used to maximise the space-time-yield, while ensuring this exothermic process remains safe. In this way, a three-fold increase in space-time-yield was achieved compared to the reported batch procedure, isolating 25 g of pure TMSCF2H after 105 min

Does seasonal variation influence the phytochemical and antibacterial properties of Carpobrotus edulis?

Carpobrotus edulis L. (family Aizoaceae) has been used by locals over the years to treat microbial infections. Extracts of varying polarities were prepared from the leaf debris and filtrate of a spring andan autumn harvest of C. edulis. Thin layer chromatography was used to analyze the phytocompounds of the extracts as well as to assay the plant for antioxidant compounds. The spring harvest showedequal distribution of the phytochemicals within the leaf debris and the filtrate, but there was a high prevalence of phytocompounds within the leaf debris extracts of the autumn sample. An antioxidantcompound was intensely pronounced in the autumn extracts of intermediate polarity and in the polar extract. The plant was evaluated for antibacterial activity against Escherichia coli, Enterococcusfaecalis, Pseudomonas aeruginosa and Staphylococcus aureus by using a two-fold serial microdilution method as well as bioautography. The spring extracts were more potent against all test organisms,having MIC values that were lower than the autumn extracts. When taking the total activity of each extract into account, the autumn extracts showed higher efficacy than the extracts from the springsample. The antibacterial activity observed in the extracts of both seasons somewhat validated the ethnomedicinal use of C. edulis

In vitro evaluation of the antifungal activity of Sclerocarya birrea extracts against pathogenic yeasts

The antifungal activity of Sclerocarya birrea which is used in South African traditional medicine for the treatment of skin diseases was evaluated against three yeasts; Candida parapsilosis, Cryptococcusalbidus and Rhodoturula mucilaginosa. Barks of S. birrea were extracted with hexane, dichloromethane (DCM), chloroform, ethyl acetate, acetone, methanol and ethanol and tested against these three yeasts.The antifungal assay was performed by the microdilution technique and bioautography. Thin layer chromatography was used to analyze the phytocompounds of the extracts as well as to assay the plantfor antioxidant compounds. More compounds with antioxidant activity were observed in polar separation system, ethyl  cetate:methanol:water (EMW). All test organisms were resistant against all non-polar extracts. Acetone, ethanol and methanol S. birrea extracts had average MIC values of 0.39, 0.22 and 0.27 mg/ml, respectively. C. albidus was the most sensitive organism with an average MIC value of 0.17 mg/ml. Average total activity was highest for methanol (387 ml/g) followed by ethanol (363 ml/g) and acetone (299 ml/g) bark extracts. Acetone and methanolic bark extracts were more active in EMW system at Rf values of 0.07, 0.32 and 0.70 against C. parapsilosis. The results showed that the plant could be further explored for possible antifungal agents and provides preliminary scientific validation of the traditional medicinal use of this plant

Exploring Multistep Continuous-Flow Hydrosilylation Reactions Catalyzed by Tris(pentafluorophenyl) borane

Exploring the combination of continuous-flow processes with the boron Lewis acid catalyzed hydrosilylation of aldehydes and ketones has delivered a robust and generally applicable reaction protocol. Notably this approach permits ready access to high temperatures and pressures and thus allows improved reactivity of substrates that were previously recalcitrant under the traditional approach. Efforts to quench the output from the flow reactor with water showed surprising tolerance leading to the application of continuousflow systems in a multistep imine formation/hydrosilylation processes to generate the corresponding secondary amines from their aldehyde and aniline precursors