Isolation and characterization of novel bacterial strains exhibiting ligninolytic potential

Background

The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD⁺ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD⁺. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.

Results

The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.

Conclusion

We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.     

A New Green Species of Frog,Genus Eleutherodactylus,from Bolivia and Perú (Amphibia,Anura, Leptodactylidae)

Eleutherodactylus olivaceus sp. n., a member of the E. unistrigatus group is described. The new species is known from montane rainforests of the Departamento Cochabamba, Bolivia, as well as from lowland rainforest of the Departamento Madre de Dios, Perú. E. olivaceus is mainly characterized by its predominantly olive green dorsal color and a papilla on the tip of the snout. Advertisement call and habitat of E. olivaceus are described. The occurrence of Eleutherodactylus mendax in Bolivia is briefly discussed.     

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.     

Selenium in combination with a tomato lipid extract as a therapy for benign prostatic hyperplasia and its alterations in rats with induced BPH

Benign prostatic hyperplasia (BPH) is the most common adenoma in old men. Tomatoes are a rich source of bioactive compounds that, as well as selenium (Se), possess antioxidant and antiproliferative activity. The aim was to evaluate the therapeutic effect of Se in combination with a tomato extract in aged rats with BPH. Aged male Wistar rats were divided in the following groups (n = 10 rats/group): Control (C), BPH, BPH + Finasteride (BPH + F), BPH + Tomato Lipidic Extract (BPH + E), BPH + Selenium (BPH + S) and BPH plus E plus S (BPH + E + S). After 4 weeks of treatment, prostate weight, diuresis, antioxidants enzymes, prooxidants and inflammatory markers, growth factors and androgens were determined. BPH + E + S reduced prostate weight by 59.29% and inhibited growth by 99.35% compared to BPH + F which only decreased weight and inhibited growth by 15.31% and 57.54%, respectively. Prooxidant markers were higher with BPH + F (49.4% higher vs. BPH), but BPH + E + S decreased these markers (94.27% vs. BPH) and increased antioxidant activity. Finally, diuresis was higher with the BPH + E + S combination and markers of inflammation and growth factors were significantly lower with respect to BPH + F. Our findings provide a beneficial and protective therapeutic option of E + S directed against androgens, oxidative stress and inflammation that regulates cell proliferation in the prostate gland.     

From species to individuals: combining barcoding and microsatellite analyses from non-invasive samples in plant ecology studies

By means of DNA barcoding and microsatellite analyses, we studied the species and individuals of legitimate seed dispersers of the Mediterranean shrub Pistacia lentiscus, a keystone species that represents the main source of food in winter for frugivorous birds. We collected dropping of birds containing seeds, and after DNA extraction we amplified and sequenced a fragment of the mitochondrial COI gene. Through BLASTN queries of the sequenced fragments against registered sequences in the GenBank database we identified the bird species that are currently dispersing P. lentiscus seeds. Further, through the amplification of specific nuclear microsatellite loci we calculated standard genetic diversity parameters of the population of birds from the genus Sylvia (the blackcap and Sardinian warbler), the most important dispersers of P. lentiscus. Five bird species were identified as seed dispersers through their barcode match. Further, we found that S. melanocephala displayed slightly lower levels of genetic diversity than S. atricapilla. In this study we show how the genetic analyses of environmental faecal samples can be a useful and convenient tool for the study of plant-frugivore interactions through the ascertainment of the identity of the species involved and through the analyses of genetic variability of their populations.     

Domain structure characterization of the multifunctional alpha-aminoadipate reductase from Penicillium chrysogenum by limited proteolysis. Activation of alpha-aminoadipate does not require the peptidyl carrier protein box or the reduction domain

The alpha-aminoadipate reductase (alpha-AAR) of Penicillium chrysogenum, an enzyme that activates the alpha-aminoadipic acid by forming an alpha-aminoadipyl adenylate and reduces the activated intermediate to alpha-aminoadipic semialdehyde, was purified to homogeneity by immunoaffinity techniques, and the kinetics for alpha-aminoadipic acid, ATP, and NADPH were determined. Sequencing of the N-terminal end confirmed the 10 first amino acids deduced from the nucleotide sequence. Its domain structure has been investigated using limited proteolysis and active site labeling. Trypsin and elastase were used to cleave the multienzyme, and the location of fragments within the primary structure was established by N-terminal sequence analysis. Initial proteolysis generated two fragments: an N-terminal fragment housing the adenylation and the peptidyl carrier protein (PCP) domains (116 kDa) and a second fragment containing most of the reductive domain (28 kDa). Under harsher conditions the adenylation domain (about 64 kDa) and the PCP domain (30 kDa) become separated. Time-dependent acylation of alpha-AAR and of fragments containing the adenylation domain with tritiated alpha-aminoadipate occurred in vitro in the absence of NADPH. Addition of NADPH to the labeled alpha-AAR released most of the radioactive substrate. A fragment containing the adenylation domain was labeled even in absence of the PCP box. The labeling of this fragment (lacking PCP) was always weaker than that observed in the di-domain (adenylating and PCP) fragment suggesting that the PCP domain plays a role in the stability of the acyl intermediate. Low intensity direct acylation of the PCP box has also been observed. A domain structure of this multienzyme is proposed.     

IL28B SNP rs8099917 is strongly associated with pegylated interferon-α and ribavirin therapy treatment failure in HCV/HIV-1 coinfected patients

Recent genome-wide association studies report that the SNP rs8099917, located 8.9 kb upstream of the start codon of IL28B, is associated with both disease chronicity and therapeutic response to pegIFN-α and RBV in patients infected with genotype 1 HCV. To determine the effect of rs8099917 variation on the response of HCV to therapy, we genotyped this variant in a cohort of 160 HCV/HIV-1 coinfected patients in our clinic unit who received combined peg-IFN-α/RBV therapy. The rs8099917 T/G or G/G genotypes were observed in 56 patients (35%). Treatment failure occurred in 80% of G-allele carriers versus 48% of non-carriers (P<0.0001). This result reveals that the G allele was strongly associated with treatment failure in this patient cohort. Importantly, a highly significant association was found between the G-allele and response to therapy in HCV genotype 1-infected patients (P<0.0001) but not in HCV genotype 3-infected patients. Multivariate analysis (odds ratio; 95% confidence interval; P value) indicated that the rs8099917 TT genotype was a strong predictor of treatment success (5.83; 1.26-26.92; P = 0.021), independent of baseline plasma HCV-RNA load less than 500 000 IU/ml (4.85; 1.18-19.95; P = 0.025) and absence of advanced liver fibrosis (5.24; 1.20-22.91; P = 0.025). These results reveal the high prevalence of the rs8099917 G allele in HCV/HIV-1 coinfected patients as well as its strong association with treatment failure in HCV genotype 1-infected patients. rs8099917 SNP genotyping may be a valid pre-treatment predictor of which patients are likely to respond to treatment in this group of difficult-to-treat HCV/HIV-infected patients.