Search

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Campylobacter jejuni infection poses a serious global threat to public health. The increasing incidence and antibiotic resistance of this bacterial infection have necessitated the adoption of various strategies to curb this trend, primarily through developing new drugs with new mechanisms of action. The enzyme malate:quinone oxidoreductase (MQO) has been shown to be essential for the survival of several bacteria and parasites. MQO is a peripheral membrane protein that catalyses the oxidation of malate to oxaloacetate, a crucial step in the tricarboxylic acid cycle. In addition, MQO is involved in the reduction of the quinone pool in the electron transport chain and thus contributes to cellular bioenergetics. The enzyme is an attractive drug target as it is not conserved in mammals. As a preliminary step in assessing the potential application of MQO from C. jejuni (CjMQO) as a new drug target, we purified active recombinant CjMQO and conducted, for the first time, biochemical analyses of MQO from a pathogenic bacterium. Our study showed that ferulenol, a submicromolar mitochondrial MQO inhibitor, and embelin are nanomolar inhibitors of CjMQO. We showed that both inhibitors are mixed-type inhibitors versus malate and noncompetitive versus quinone, suggesting the existence of a third binding site to accommodate these inhibitors; indeed, such a trait appears to be conserved between mitochondrial and bacterial MQOs. Interestingly, ferulenol and embelin also inhibit the in vitro growth of C. jejuni, supporting the hypothesis that MQO is essential for C. jejuni survival and is therefore an important drug target., Frontiers in Molecular Biosciences, 10, art. no. 1095026; 2023

Anthocyanidin and flavonol glycosides have been linked to the health-promoting effects associated with apple consumption. However, very few enzymes involved in flavonoid glycosylation have been characterised to date. Here, we present the identification and phylogenetic analysis of 234 putative glycosyltransferases involved in flavonoid biosynthesis, and detail the biochemical and structural characterisation of MdUGT78T2 as a strict galactosyltransferase involved in the formation of quercetin-3-O-galactoside and cyanidin-3-O-galactoside, the major glycoconjugates of flavonoids in apple. The enzyme is also active on other flavonoids but with a lower catalytic efficiency. Our data, complemented with gene expression analysis suggest that MdUGT78T2 synthesises the glycoconjugates at both the early and late stages of fruit development. This newly discovered type of catalytic activity can potentially be exploited for in vitro modification of flavonoids to increase their stability in food products and to modify apple fruits and other commercial crops through breeding approaches to enhance their health benefits., QC 20230630

Campylobacter jejuni infection poses a serious global threat to public health. The increasing incidence and antibiotic resistance of this bacterial infection have necessitated the adoption of various strategies to curb this trend, primarily through developing new drugs with new mechanisms of action. The enzyme malate:quinone oxidoreductase (MQO) has been shown to be essential for the survival of several bacteria and parasites. MQO is a peripheral membrane protein that catalyses the oxidation of malate to oxaloacetate, a crucial step in the tricarboxylic acid cycle. In addition, MQO is involved in the reduction of the quinone pool in the electron transport chain and thus contributes to cellular bioenergetics. The enzyme is an attractive drug target as it is not conserved in mammals. As a preliminary step in assessing the potential application of MQO from C. jejuni (CjMQO) as a new drug target, we purified active recombinant CjMQO and conducted, for the first time, biochemical analyses of MQO from a pathogenic bacterium. Our study showed that ferulenol, a submicromolar mitochondrial MQO inhibitor, and embelin are nanomolar inhibitors of CjMQO. We showed that both inhibitors are mixed-type inhibitors versus malate and noncompetitive versus quinone, suggesting the existence of a third binding site to accommodate these inhibitors; indeed, such a trait appears to be conserved between mitochondrial and bacterial MQOs. Interestingly, ferulenol and embelin also inhibit the in vitro growth of C. jejuni, supporting the hypothesis that MQO is essential for C. jejuni survival and is therefore an important drug target., Frontiers in Molecular Biosciences, 10, art. no. 1095026; 2023

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical a/b-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal b-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. Copyright © 2023 Distaso et al.

Bacterial Salmonella infections and helminthic schistosomiasis are classified under Neglected Tropical Diseases (NTD). Biochemical characterization was carried out on Salmonella serovars in Kwande LGA, Benue State, Nigeria. The aim was to determine the prevalence of the infection and mixed cases of schistosomiasis. The best biochemical test for the detection of Salmonella was also targeted.  A total of 180 subjects were randomly investigated from five different locations (Iange, Sati, Kyogyen, Ikov Sati and Adikpo). Two specimens (stool and urine) were collected from each subject. Preparation and culture of specimens followed standard practices. Salmonella cases were diagnosed on stool samples using six biochemical tests. Schistosoma haeamatobium and S.mansoni were diagnosed using urine and faecal specimens respectively. Average prevalence of Salmonella infection in Kwande LGA was 13.9%  (8.3% female, 5.6% male) consisting of 44% S. typhi and 56% of other serovars. Infection was associated with location (?2 = 145.79, p=0.000). Ikov Sati had the highest cases (65%) followed by Adikpo (15%). No cases was recorded at Kyogyen. Infection was most common among farmers (8.3% prevalent), thus associated with occupation type (?2 =52.63, p=0.000). Among the five biochemical tests, catalase test was the best in the identification of Salmonella serovars followed by hydrogen sulphide and citrate. Among the 180 subjects studied, six mixed infection cases of Salmonella-Schistosomiasis were recorded with a prevalence of 3% unassociated age, sex and location. However, farmers and school children recorded 2 cases each while okada riders and civil servants had a single case each. Based on organism type, S.typhi-S.mansoni-S.haematobium mixed infection had only one case. Co-infection of S.typhi-S.mansoni also had a single case. Salmonella-S.haematobium co-infection cases were four (2.2% prevalent). From all indications, Salmonella cases in the study area were high. Farming pra

Bacterial Salmonella infections and helminthic schistosomiasis are classified under Neglected Tropical Diseases (NTD). Biochemical characterization was carried out on Salmonella serovars in Kwande LGA, Benue State, Nigeria. The aim was to determine the prevalence of the infection and mixed cases of schistosomiasis. The best biochemical test for the detection of Salmonella was also targeted.  A total of 180 subjects were randomly investigated from five different locations (Iange, Sati, Kyogyen, Ikov Sati and Adikpo). Two specimens (stool and urine) were collected from each subject. Preparation and culture of specimens followed standard practices. Salmonella cases were diagnosed on stool samples using six biochemical tests. Schistosoma haeamatobium and S.mansoni were diagnosed using urine and faecal specimens respectively. Average prevalence of Salmonella infection in Kwande LGA was 13.9%  (8.3% female, 5.6% male) consisting of 44% S. typhi and 56% of other serovars. Infection was associated with location (?2 = 145.79, p=0.000). Ikov Sati had the highest cases (65%) followed by Adikpo (15%). No cases was recorded at Kyogyen. Infection was most common among farmers (8.3% prevalent), thus associated with occupation type (?2 =52.63, p=0.000). Among the five biochemical tests, catalase test was the best in the identification of Salmonella serovars followed by hydrogen sulphide and citrate. Among the 180 subjects studied, six mixed infection cases of Salmonella-Schistosomiasis were recorded with a prevalence of 3% unassociated age, sex and location. However, farmers and school children recorded 2 cases each while okada riders and civil servants had a single case each. Based on organism type, S.typhi-S.mansoni-S.haematobium mixed infection had only one case. Co-infection of S.typhi-S.mansoni also had a single case. Salmonella-S.haematobium co-infection cases were four (2.2% prevalent). From all indications, Salmonella cases in the study area were high. Farming pra

Bacterial Salmonella infections and helminthic schistosomiasis are classified under Neglected Tropical Diseases (NTD). Biochemical characterization was carried out on Salmonella serovars in Kwande LGA, Benue State, Nigeria. The aim was to determine the prevalence of the infection and mixed cases of schistosomiasis. The best biochemical test for the detection of Salmonella was also targeted.  A total of 180 subjects were randomly investigated from five different locations (Iange, Sati, Kyogyen, Ikov Sati and Adikpo). Two specimens (stool and urine) were collected from each subject. Preparation and culture of specimens followed standard practices. Salmonella cases were diagnosed on stool samples using six biochemical tests. Schistosoma haeamatobium and S.mansoni were diagnosed using urine and faecal specimens respectively. Average prevalence of Salmonella infection in Kwande LGA was 13.9%  (8.3% female, 5.6% male) consisting of 44% S. typhi and 56% of other serovars. Infection was associated with location (?2 = 145.79, p=0.000). Ikov Sati had the highest cases (65%) followed by Adikpo (15%). No cases was recorded at Kyogyen. Infection was most common among farmers (8.3% prevalent), thus associated with occupation type (?2 =52.63, p=0.000). Among the five biochemical tests, catalase test was the best in the identification of Salmonella serovars followed by hydrogen sulphide and citrate. Among the 180 subjects studied, six mixed infection cases of Salmonella-Schistosomiasis were recorded with a prevalence of 3% unassociated age, sex and location. However, farmers and school children recorded 2 cases each while okada riders and civil servants had a single case each. Based on organism type, S.typhi-S.mansoni-S.haematobium mixed infection had only one case. Co-infection of S.typhi-S.mansoni also had a single case. Salmonella-S.haematobium co-infection cases were four (2.2% prevalent). From all indications, Salmonella cases in the study area were high. Farming pra

This study investigates the amino acid profiles of various species of Paramphistomum, a group of parasitic flatworms known to affect livestock. By analyzing the amino acid composition, we aim to better understand the nutritional and metabolic needs of these parasites. Our findings reveal distinct amino acid profiles across different Paramphistomum species, which could have implications for developing targeted treatments and control measures. This research provides valuable insights into the biochemical characteristics of these parasites and underscores the importance of amino acid profiling in parasitology.

This study investigates the amino acid profiles of various species of Paramphistomum, a group of parasitic flatworms known to affect livestock. By analyzing the amino acid composition, we aim to better understand the nutritional and metabolic needs of these parasites. Our findings reveal distinct amino acid profiles across different Paramphistomum species, which could have implications for developing targeted treatments and control measures. This research provides valuable insights into the biochemical characteristics of these parasites and underscores the importance of amino acid profiling in parasitology.

2S albumins are relevant and often major allergens from several tree nuts and seeds, affecting mainly children and young people. The present study aims to assess how the structural features of 2S albumins could affect their immunogenic capacity, which is essential to comprehend the role of these proteins in food allergy. For this purpose, twelve 2S albumins were isolated from their respective extracts by chromatographic methods and identified by MALDI-TOF massspectrometry. Their molecular and structural characterization was conducted by electrophoretic, spectroscopic and in silico methods, showing that these are small proteins that comprise a wide range of isoelectric points, displaying a general high structure stability to thermal treatment. Despite low amino acid sequence identity, these proteins share structural features, pointing conformational epitopes to explain cross-reactivity between them. Immunoblotting with allergic patients’ sera revealed those possible correlations between evolutionarily distant 2S albumins from different sources. The availability of a well-characterized panel of 2S albumins from plant-derived sources allowed establishing correlations between their structural features and their allergenic potential, including their role in cross-reactivity processes, Ministerio de Economia y ́Competitividad, FEDER funds, Ministerio de Educación, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes., QC 20201228

The present study determines the best conditions for the fermentation of Schinus molle drupes by the combination of different types of hydrolysis with the search for an adequate yeast strain. Schinus molle seed residues from an essential oil extraction plant (EOEP) have a high potential for ethanol production. Native yeast strains were isolated from the residues and were used to ferment the lignocellulosic residues, along with baker’s yeast (Saccharomyces cerevisiae) at 30 °C and pH 5.5 for comparison. Morphological and biochemical characterizations were carried out on the isolated yeast strains. Thermogravimetric and high-performance liquid chromatography analyses were done on the S. molle seeds (fresh and residue) to determine the ethanol production potential. The followed methodology included increasing the sugar content by hydrolysis with chemical (sulphuric acid, acetic acid, and sodium hydroxide), physical (thermal, vacuum, and ultrasound), and enzymatic treatments (amyloglucosidase and α-amylase). Once the optimum combination of yeast-hydrolysis was determined, a comparison of the greenhouse gas emissions between the original and proposed processes was done. The fermentation of the residues might replace methane from uncontrolled decomposition and reduce the solid residues in 50%/day, hence the EOEP global warming potential is reduced by 47%. The yearly income was estimated to increase by USD 2592.50 from 6302.6 L of ethanol produced from the residues., QC 20191105

The main feature of Naja nigricollis venom is its high proteolytic activity responsible for most of local and systemic effects observed during envenomation by this snake. In this present work we reported the purification and characteristics metalloprotease from Naja nigricollis venom by combination of two step chromatography; a gel filtration (sephadexG-75) and an ion – exchange(DEAE–sephadex –A-50). Ethanolic extraction of the leaf of Annona senegalensis was carried out. An hemorrhagic Naja nigricollis metalloprotease(NNMP) was partially purified from Naja negricolis venom using sephadex G-75 and DEAE-sephadex A-50 column chromatography. Purification of the hemmoraghic metalloprotease was about 11-fold with a total recovery of 5.8% from the crude Naja negricollis venom. Biochemical characterization of NNMP show a Km of 10.5M and Vmax  of 45.5 unitsmg-1. The hemorrhagin show optimal stability at PH 5.5 acetate buffer and thermally stable at 400C. NNMP has activation energy as 0.25kjmol-1 and it caused hemorrhage when injected intra-dermally in albino mice suggesting hemorrghic metalloprotease. NNMP hemorrhagic activities were highly inhibited by ethylenediaminetetraacetic acid (EDTA) and the ethanolic leaf extract of Annona senegalensis.

The main feature of Naja nigricollis venom is its high proteolytic activity responsible for most of local and systemic effects observed during envenomation by this snake. In this present work we reported the purification and characteristics metalloprotease from Naja nigricollis venom by combination of two step chromatography; a gel filtration (sephadexG-75) and an ion – exchange(DEAE–sephadex –A-50). Ethanolic extraction of the leaf of Annona senegalensis was carried out. An hemorrhagic Naja nigricollis metalloprotease(NNMP) was partially purified from Naja negricolis venom using sephadex G-75 and DEAE-sephadex A-50 column chromatography. Purification of the hemmoraghic metalloprotease was about 11-fold with a total recovery of 5.8% from the crude Naja negricollis venom. Biochemical characterization of NNMP show a Km of 10.5M and Vmax  of 45.5 unitsmg-1. The hemorrhagin show optimal stability at PH 5.5 acetate buffer and thermally stable at 400C. NNMP has activation energy as 0.25kjmol-1 and it caused hemorrhage when injected intra-dermally in albino mice suggesting hemorrghic metalloprotease. NNMP hemorrhagic activities were highly inhibited by ethylenediaminetetraacetic acid (EDTA) and the ethanolic leaf extract of Annona senegalensis.

The main feature of Naja nigricollis venom is its high proteolytic activity responsible for most of local and systemic effects observed during envenomation by this snake. In this present work we reported the purification and characteristics metalloprotease from Naja nigricollis venom by combination of two step chromatography; a gel filtration (sephadexG-75) and an ion – exchange(DEAE–sephadex –A-50). Ethanolic extraction of the leaf of Annona senegalensis was carried out. An hemorrhagic Naja nigricollis metalloprotease(NNMP) was partially purified from Naja negricolis venom using sephadex G-75 and DEAE-sephadex A-50 column chromatography. Purification of the hemmoraghic metalloprotease was about 11-fold with a total recovery of 5.8% from the crude Naja negricollis venom. Biochemical characterization of NNMP show a Km of 10.5M and Vmax  of 45.5 unitsmg-1. The hemorrhagin show optimal stability at PH 5.5 acetate buffer and thermally stable at 400C. NNMP has activation energy as 0.25kjmol-1 and it caused hemorrhage when injected intra-dermally in albino mice suggesting hemorrghic metalloprotease. NNMP hemorrhagic activities were highly inhibited by ethylenediaminetetraacetic acid (EDTA) and the ethanolic leaf extract of Annona senegalensis.

The present work has been undertaken for remediating phthalate exposure in the environment. The microbial strain was isolated by enrichment culture technique from the rubbish dump space close to Patna that was contaminated with phthalates for higher degradation ability. The isolated microbial strain T7 was designated as Bacillus cereus after Gram-staining, biochemical characterization, 16S-rRNA sequence and phylogenetic studies. The isolate had the power to utilize 25O μg/ml Di (2-ethyl Hexyl Phthalate) (DEHP) dose taken from 10 mg/ml (DEHP) stock solution within the growth medium. The optimum pH and temperature for DEHP degradation were 8.5 at 37 ̊C. The isolated bacterial strain T7 may allow up to 10% NaCl in minimal salt medium that was enrich with DEHP. The metabolic end product obtained after LCMS was bis [3-(oxolan-2-yl) propyl] nonanedioate having chemical formula C23H40O6. This work provides some new proof for soil rectification by Bacillus species.

The main feature of Naja nigricollis venom is its high proteolytic activity responsible for most of local and systemic effects observed during envenomation by this snake. In this present work we reported the purification and characteristics metalloprotease from Naja nigricollis venom by combination of two step chromatography; a gel filtration (sephadexG-75) and an ion – exchange(DEAE–sephadex –A-50). Ethanolic extraction of the leaf of Annona senegalensis was carried out. An hemorrhagic Naja nigricollis metalloprotease(NNMP) was partially purified from Naja negricolis venom using sephadex G-75 and DEAE-sephadex A-50 column chromatography. Purification of the hemmoraghic metalloprotease was about 11-fold with a total recovery of 5.8% from the crude Naja negricollis venom. Biochemical characterization of NNMP show a Km of 10.5M and Vmax  of 45.5 unitsmg-1. The hemorrhagin show optimal stability at PH 5.5 acetate buffer and thermally stable at 400C. NNMP has activation energy as 0.25kjmol-1 and it caused hemorrhage when injected intra-dermally in albino mice suggesting hemorrghic metalloprotease. NNMP hemorrhagic activities were highly inhibited by ethylenediaminetetraacetic acid (EDTA) and the ethanolic leaf extract of Annona senegalensis.

The main feature of Naja nigricollis venom is its high proteolytic activity responsible for most of local and systemic effects observed during envenomation by this snake. In this present work we reported the purification and characteristics metalloprotease from Naja nigricollis venom by combination of two step chromatography; a gel filtration (sephadexG-75) and an ion – exchange(DEAE–sephadex –A-50). Ethanolic extraction of the leaf of Annona senegalensis was carried out. An hemorrhagic Naja nigricollis metalloprotease(NNMP) was partially purified from Naja negricolis venom using sephadex G-75 and DEAE-sephadex A-50 column chromatography. Purification of the hemmoraghic metalloprotease was about 11-fold with a total recovery of 5.8% from the crude Naja negricollis venom. Biochemical characterization of NNMP show a Km of 10.5M and Vmax  of 45.5 unitsmg-1. The hemorrhagin show optimal stability at PH 5.5 acetate buffer and thermally stable at 400C. NNMP has activation energy as 0.25kjmol-1 and it caused hemorrhage when injected intra-dermally in albino mice suggesting hemorrghic metalloprotease. NNMP hemorrhagic activities were highly inhibited by ethylenediaminetetraacetic acid (EDTA) and the ethanolic leaf extract of Annona senegalensis.

Pearl millet is a staple food and fodder crop of marginal areas of sub-Saharan Africa, South Asia and the Americas. Although pearl millet is stable food for 70 % of Namibian population, its productivity is very low (low yield, susceptible to diseases/insect and prolonged reproductive cycle) and has a limited genetic diversity. For the purpose of widening gene diversity, developing high yielding and drought resistant pearl millet, the Kangara and Okashana 2 were mutated using Colbalt-60 gamma irradiation source with various dose levels. However, all sixth generation of Okashana 2 putative mutant derivatives used in this study were all produced using 350 Gray while for Kangara mutants were produced using 400 Gray. A total of 32 genotypes including two parental lines were collected from the three agro-ecological zones (Research Stations). In addition to two parental lines, stratified sampling technique was used to randomly select further 12 samples from 30 putative mutants for Okashana 2 and Kangara that offered higher average grain yields. Such technique used to subsample was applied in order to select putative mutants that shown phenotypically high yielding trait. This study aimed to examine the genetic diversity existing in the Namibian pearl millet putative mutant lines using 19 simple sequence repeats and 23 biochemical markers. Genetic characterization was studied using conventional PCR and separated using gel electrophoresis while biochemical parameters used dumas method (crude protein), fiber extractor (crude fibre, ADF and NDF), fat solvent extractor (fat), ICP spectrophotometer (minerals) and UV/VIS spectrometer (phytate). A total of 108 putative bands were generated from 11 SSR markers whereas the remaining eight markers were not effective in amplifying any mutant line. Amplified fragment size ranged from 90 to 1150 bp and the 11 SSR loci had a 63.6 % polymorphism. Polymorphic information content (PIC) varied from 0.139 to 0.872, with a mean of 0.379. Cluste

Massive cultivation of microalgae for the production of alternative biofuels, value added compounds with chemical or pharmaceutical applications and as a food source for a growing world population remains one of the most promising fields of bioengineering and biotechnology. However, much work must be done still in research and development to achieve all the possibilities offered by this renewable source. Thus, this review intends to show the principles concerning the cultivation of microalgae: its importance, technological problems still to be solved, all aspects related to their types and systems of cultivation, growth phases, kinetics equations for cell duplication, important aspects related to the selection and isolation of strains, scaling, biochemical characterization methods (determination of chlorophylls, proteins, lipids, carbohydrates, silica, etc.) and finally the most relevant microalgae species., El cultivo masivo de microalgas para la producción de biocombustible alternativo al petróleo, compuestos de valor añadido (CAVA) con aplicaciones químicas o farmacéuticas y como fuente de alimento para una población mundial creciente, sigue siendo uno de los campos más prometedores de la bioingeniería y biotecnología. Sin embargo, todavía queda mucho trabajo por realizar en investigación y desarrollo para optar a todas las magníficas posibilidades que ofrece esta fuente renovable. De este modo, en esta revisión se pretende mostrar los principios referentes al cultivo de microalgas: su importancia, los problemas tecnológicos que todavía quedan por solucionar, todos los aspectos relacionados con sus tipos y sistemas de cultivo, las fases del crecimiento, las ecuaciones de la cinética de duplicación celular, características importantes referentes a la selección y aislamiento de cepas, escalado, métodos de caracterización bioquímica (determinación de clorofilas, proteínas, lípidos, carbohidratos, sílice, etc.) y, finalmente, las especies de microalgas más relevantes.

Pearl millet is a staple food and fodder crop of marginal areas of sub-Saharan Africa, South Asia and the Americas. Although pearl millet is stable food for 70 % of Namibian population, its productivity is very low (low yield, susceptible to diseases/insect and prolonged reproductive cycle) and has a limited genetic diversity. For the purpose of widening gene diversity, developing high yielding and drought resistant pearl millet, the Kangara and Okashana 2 were mutated using Colbalt-60 gamma irradiation source with various dose levels. However, all sixth generation of Okashana 2 putative mutant derivatives used in this study were all produced using 350 Gray while for Kangara mutants were produced using 400 Gray. A total of 32 genotypes including two parental lines were collected from the three agro-ecological zones (Research Stations). In addition to two parental lines, stratified sampling technique was used to randomly select further 12 samples from 30 putative mutants for Okashana 2 and Kangara that offered higher average grain yields. Such technique used to subsample was applied in order to select putative mutants that shown phenotypically high yielding trait. This study aimed to examine the genetic diversity existing in the Namibian pearl millet putative mutant lines using 19 simple sequence repeats and 23 biochemical markers. Genetic characterization was studied using conventional PCR and separated using gel electrophoresis while biochemical parameters used dumas method (crude protein), fiber extractor (crude fibre, ADF and NDF), fat solvent extractor (fat), ICP spectrophotometer (minerals) and UV/VIS spectrometer (phytate). A total of 108 putative bands were generated from 11 SSR markers whereas the remaining eight markers were not effective in amplifying any mutant line. Amplified fragment size ranged from 90 to 1150 bp and the 11 SSR loci had a 63.6 % polymorphism. Polymorphic information content (PIC) varied from 0.139 to 0.872, with a mean of 0.379. Cluste

Pearl millet is a staple food and fodder crop of marginal areas of sub-Saharan Africa, South Asia and the Americas. Although pearl millet is stable food for 70 % of Namibian population, its productivity is very low (low yield, susceptible to diseases/insect and prolonged reproductive cycle) and has a limited genetic diversity. For the purpose of widening gene diversity, developing high yielding and drought resistant pearl millet, the Kangara and Okashana 2 were mutated using Colbalt-60 gamma irradiation source with various dose levels. However, all sixth generation of Okashana 2 putative mutant derivatives used in this study were all produced using 350 Gray while for Kangara mutants were produced using 400 Gray. A total of 32 genotypes including two parental lines were collected from the three agro-ecological zones (Research Stations). In addition to two parental lines, stratified sampling technique was used to randomly select further 12 samples from 30 putative mutants for Okashana 2 and Kangara that offered higher average grain yields. Such technique used to subsample was applied in order to select putative mutants that shown phenotypically high yielding trait. This study aimed to examine the genetic diversity existing in the Namibian pearl millet putative mutant lines using 19 simple sequence repeats and 23 biochemical markers. Genetic characterization was studied using conventional PCR and separated using gel electrophoresis while biochemical parameters used dumas method (crude protein), fiber extractor (crude fibre, ADF and NDF), fat solvent extractor (fat), ICP spectrophotometer (minerals) and UV/VIS spectrometer (phytate). A total of 108 putative bands were generated from 11 SSR markers whereas the remaining eight markers were not effective in amplifying any mutant line. Amplified fragment size ranged from 90 to 1150 bp and the 11 SSR loci had a 63.6 % polymorphism. Polymorphic information content (PIC) varied from 0.139 to 0.872, with a mean of 0.379. Cluste

Pearl millet is a staple food and fodder crop of marginal areas of sub-Saharan Africa, South Asia and the Americas. Although pearl millet is stable food for 70 % of Namibian population, its productivity is very low (low yield, susceptible to diseases/insect and prolonged reproductive cycle) and has a limited genetic diversity. For the purpose of widening gene diversity, developing high yielding and drought resistant pearl millet, the Kangara and Okashana 2 were mutated using Colbalt-60 gamma irradiation source with various dose levels. However, all sixth generation of Okashana 2 putative mutant derivatives used in this study were all produced using 350 Gray while for Kangara mutants were produced using 400 Gray. A total of 32 genotypes including two parental lines were collected from the three agro-ecological zones (Research Stations). In addition to two parental lines, stratified sampling technique was used to randomly select further 12 samples from 30 putative mutants for Okashana 2 and Kangara that offered higher average grain yields. Such technique used to subsample was applied in order to select putative mutants that shown phenotypically high yielding trait. This study aimed to examine the genetic diversity existing in the Namibian pearl millet putative mutant lines using 19 simple sequence repeats and 23 biochemical markers. Genetic characterization was studied using conventional PCR and separated using gel electrophoresis while biochemical parameters used dumas method (crude protein), fiber extractor (crude fibre, ADF and NDF), fat solvent extractor (fat), ICP spectrophotometer (minerals) and UV/VIS spectrometer (phytate). A total of 108 putative bands were generated from 11 SSR markers whereas the remaining eight markers were not effective in amplifying any mutant line. Amplified fragment size ranged from 90 to 1150 bp and the 11 SSR loci had a 63.6 % polymorphism. Polymorphic information content (PIC) varied from 0.139 to 0.872, with a mean of 0.379. Cluste

Massive cultivation of microalgae for the production of alternative biofuels, value added compounds with chemical or pharmaceutical applications and as a food source for a growing world population remains one of the most promising fields of bioengineering and biotechnology. However, much work must be done still in research and development to achieve all the possibilities offered by this renewable source. Thus, this review intends to show the principles concerning the cultivation of microalgae: its importance, technological problems still to be solved, all aspects related to their types and systems of cultivation, growth phases, kinetics equations for cell duplication, important aspects related to the selection and isolation of strains, scaling, biochemical characterization methods (determination of chlorophylls, proteins, lipids, carbohydrates, silica, etc.) and finally the most relevant microalgae species., El cultivo masivo de microalgas para la producción de biocombustible alternativo al petróleo, compuestos de valor añadido (CAVA) con aplicaciones químicas o farmacéuticas y como fuente de alimento para una población mundial creciente, sigue siendo uno de los campos más prometedores de la bioingeniería y biotecnología. Sin embargo, todavía queda mucho trabajo por realizar en investigación y desarrollo para optar a todas las magníficas posibilidades que ofrece esta fuente renovable. De este modo, en esta revisión se pretende mostrar los principios referentes al cultivo de microalgas: su importancia, los problemas tecnológicos que todavía quedan por solucionar, todos los aspectos relacionados con sus tipos y sistemas de cultivo, las fases del crecimiento, las ecuaciones de la cinética de duplicación celular, características importantes referentes a la selección y aislamiento de cepas, escalado, métodos de caracterización bioquímica (determinación de clorofilas, proteínas, lípidos, carbohidratos, sílice, etc.) y, finalmente, las especies de microalgas más relevantes.

Pearl millet is a staple food and fodder crop of marginal areas of sub-Saharan Africa, South Asia and the Americas. Although pearl millet is stable food for 70 % of Namibian population, its productivity is very low (low yield, susceptible to diseases/insect and prolonged reproductive cycle) and has a limited genetic diversity. For the purpose of widening gene diversity, developing high yielding and drought resistant pearl millet, the Kangara and Okashana 2 were mutated using Colbalt-60 gamma irradiation source with various dose levels. However, all sixth generation of Okashana 2 putative mutant derivatives used in this study were all produced using 350 Gray while for Kangara mutants were produced using 400 Gray. A total of 32 genotypes including two parental lines were collected from the three agro-ecological zones (Research Stations). In addition to two parental lines, stratified sampling technique was used to randomly select further 12 samples from 30 putative mutants for Okashana 2 and Kangara that offered higher average grain yields. Such technique used to subsample was applied in order to select putative mutants that shown phenotypically high yielding trait. This study aimed to examine the genetic diversity existing in the Namibian pearl millet putative mutant lines using 19 simple sequence repeats and 23 biochemical markers. Genetic characterization was studied using conventional PCR and separated using gel electrophoresis while biochemical parameters used dumas method (crude protein), fiber extractor (crude fibre, ADF and NDF), fat solvent extractor (fat), ICP spectrophotometer (minerals) and UV/VIS spectrometer (phytate). A total of 108 putative bands were generated from 11 SSR markers whereas the remaining eight markers were not effective in amplifying any mutant line. Amplified fragment size ranged from 90 to 1150 bp and the 11 SSR loci had a 63.6 % polymorphism. Polymorphic information content (PIC) varied from 0.139 to 0.872, with a mean of 0.379. Cluste

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial beta-glucosidases (BGL) with significant beta-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesizemore complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific beta-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal beta-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 degrees C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are beta-D-Galp-(1 -> 6)-D-Lac (6GALA) and beta-D-Galp-(1 -> 3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated., QC 20150331

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for ∼1.5% of most prokaryotic genomes and are estimated to be involved in ∼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.

Aeromonas spp. are regularly present in water, and in some cases, they cause diseases in fish and amphibians. Humans are infected by ingestion of the bacteria through contaminated food or water. Objective of this paper was to examine the presence of Aeromonas species in trout samples and to investigate the biochemical characteristics of isolated strains. In microbiological analysis of 120 trout samples, 12 bacteria strains of Aeromonas genus were isolated, of which seven (58.3%) belonged to strains of A. hydrophila group 1, and five (41.6%) to strains of A. hydrophila group 2. By biochemical characterization of isolated strains it was established that they were oxidase positive, catalase positive, arginine positive, ornitin positive, H2S negative, urease negative, tryptophan desaminase negative, gelatine negative, fermenting mannitol, inositol, rhamnose and saccharose, the are mobile, hemolytic and hydrolyze starch. In regard to other biochemical characteristics, it was established that 11 (91.7%) of 12 studied strains of A.hydrophila had β-galactosidase, they were citrate and indole positive, sorbitole negative, arabinose positive, produce NO2 and reduce it to N2. Also 10 (83.3%) of 12 studied strains were Voges-Proskauer positive and melibiosis negative, whereas eight (66.7%) of 12 studied strains had lysine-decarboxylase positive reaction and were amygdalin positive. In 58.3 % of strains (seven of 12 strains) were established to ferment glucose., Aeromonas spp. redovno su prisutni u vodi, i u izvesnim slučajevima, uzrokuju oboljenje riba i vodozemaca. Čovek se infi cira ingestijom ovih bakterija putem kontaminirane hrane ili vode. Cilj ovog rada bio je da se ispita prisustvo Aeromonas vrsta u uzorcima pastrmke i ispitaju biohemijske karakteristike izolovanih sojeva. Mikrobiološkom analizom 120 uzoraka pastrmke, izolovano je 12 sojeva bakterija iz roda Aeromonas, od kojih je sedam (58,3%) pripadalo sojevima A. hydrophila grupa 1, a pet (41,6%) je pripadalo sojevima A. hydrophila grupa 2. Biohemijskom karakterizacijom izolovanih sojeva utvrđeno je da su oksidaza pozitivni, katalaza pozitivni, arginin pozitivni, ornitin pozitivni, H2S negativni, ureaza negativni, triptofan dezaminaza negativni, želatin negativni, fermentuju manitol, inositol, rhamnosu i saharozu, pokretni su, hemolitični i hidrolizuju skrob. Od ostalih biohemijskih karatkeristika utvrđeno je da 11 (91,7%) od 12 ispitanih sojeva A. hydrophila poseduje β-galaktosidazu, zatim citrat i indol su pozitivni, sorbitol negativni, arabinoza pozitivni, produkuju NO2 i vrši njegovu redukciju do N2, zatim da je 10 (83,3%) od 12 ispitanih sojeva Voges-Proskauer pozitivno i negativno na melibiozu, dok je osam (66,7%) od 12 sojeva dalo lizin-dekarboksilaza pozitivnu reakciju i bilo je amigdalin pozitivno. Kod 58,3% sojeva (sedam od 12 sojeva) utvrđeno je da fermentuju glukozu.

Aeromonas hydrophila spp. is commonly present in water and in certain incidences they might cause diseases in fish and amphibians. Humans are infected by ingestion of these bacteria with contaminated food or water. Aeromonas hydrophila spp. might cause gastro-intestinal disorders and poisoning in humans. One of the major issues associated with Aeromonas hydrophila and incidence of gastro-intestinal diseases is accurate identification of aeromonades at the species level. Therefore, there is a need for a clear phenotypic scheme and biochemical characterization of the mentioned species. Objective of this paper was to study the presence of Aeromas hydrophila in trout samples and to test biochemical characteristics of isolated strains. For isolation of Aeromonas hydrophila species, 120 samples of rainbow trout, commercially available, were studied. Aeromonas hydrophila was isolated from fish muscle tissue and skin according to "Microbiological Methods for the Meat Industry", second edition (1991) Biochemical studies were performed on Aeromonas hydrophila medium, followed by oxidase test (+), catalase test (+), staining according to Gram (-) and vibriostat test agar (0/129, vibriostatic), as well as biochemical tests API 20E and API 20NE. From the 120 studied rainbow trout samples, 12 strains of Aeromonas hydrophila were isolated, out of which seven strains (58.3%) of Aeromonas hydrophila belonged to group 1, whereas five strains (41,6%) belonged to Aeromonas hydrophila group 2. Out of 12 isolated strains, 11 (91.7%) had β-galactosidase. They were citrate and indol positive, sorbitol negative, arabinose positive, NO2 - producing and reducing it to N2. Then (83.3%) of 12 twelve isolated strains were Voges-Proskauer positive and melibiose negative, whereas eight (66.7%) were amigdalin and lysine-decarboxylase positive. It was established that seven of 12 twelve strains (58.3%) ferment glucose. The obtained results demonstrate the need to supplement phenotypic schemes for id

A low molecular mass alkaliphilic extra-cellular lipase of Bacillus cereus MTCC 8372 was purified 35-fold by hydrophobic interaction (Octyl-Sepharose) chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 8 kDa. It is a homopentamer of 40 kDa as revealed by native-PAGE. The lipase was optimally active at 55 °C and retained approximately half of its original activity after 40 min incubation at 55 °C. The enzyme was maximally active at pH 8.5. Mg 2+ , Cu 2+ , Ca 2+ , Hg 2+ , Al 3+ and Fe 3+ at 1 mM enhanced hydrolytic activity of the lipase. Interestingly, Hg 2+ ions synergized and Zn 2+ and Co 2+ ions antagonized the lipase activity. Among surfactants, Tween 80 promoted the lipase activity. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was highly specific towards p -nitrophenyl palmitate and showed a V max and K m of 0.70 mmol.mg −1 .min −1 and 32 mM for hydrolysis of p NPP.

English: The construction and screening of gene libraries prepared from DNA directly isolated from environmental samples is a recent and powerful tool for the discovery of new enzymes of biotechnological interest (Gabor et al., 2004). Standard methods based on the screening of isolated microorganisms are inherently limited to the tiny fraction of cultivable microbial species (<1%); environmental gene banks in principle provide access to the entire sequence space present in nature (Handelsman et al., 1998). Environmental libraries allow the screening of functional classes of genes from thousands of organisms and research in this area will provide an essential backdrop for understanding evolution and biochemical pathways (Rajendran et al., 2008). The metagenomics approach has been shown to be an efficient method for obtaining novel biocatalysts and useful genes from uncultured microorganisms from diverse environments. Before proceeding to the metagenome analysis, we constructed genomic libraries from a South African deep mine isolate Geobacillus thermoleovorans GE-7. The library was screened for lipolytic activity on LB tributyrin (TLB). Active clones were sequenced using 454 technology, and the sequencing results revealed the presence of the lipA and GDSL lipases, of which the latter has not yet been characterized in this organism. This family displays the characteristic G-D-S-L motif instead of the conventional G-X-S-X-G (Xdenotes any amino acid) motif. GDSL lipases are hydrolytic enzymes with multifunctional properties such as broad substrate specificity and regiospecificity. They have potential for use in the hydrolysis and synthesis of ester compounds that are of interest in pharmaceutical, food, biochemical and the biological sector (Akoh et al., 2004; Lämmle et al., 2007). In addition, genes associated with fatty acid degradation, different glycolytic activities, lipolytic activity, spore germination, proper protein folding, antibiotic resistance and the cell w, Afrikaans: Die konstruksie en sifting (‘screening’) van genoombiblioteke wat voorberei is vanaf DNS, geïsoleer direk uit die omgewing is ‘n eietydse en kragtige tegniek vir die ontdekking van nuwe ensieme met biotegnologiese waarde (Gabor et al., 2004). Standaard metodes gebaseer op die sifting van geïsoleerde mikro-organismes is inherent beperk tot die klein breukdeel van kweekbare mikrobiese spesies (<1%). Omgewings genoombiblioteke, daarenteen, lewer meer toegang tot die totale DNS basispaar opeenvolging wat daardie omgewing bied (Handelsman et al. 1998). Omgewings genoombiblioteke bied die navorser die geleentheid om te sif vir funksionele klasse van ensieme vanaf duisende organismes en navorsing in hierdie studieveld sal bydra tot ons huidige insig en begrip van die evolusie van metaboliese we¸ (Short, 1997; Rajendran et al., 2008). Dit is bewys dat die metagenoom aanslag ‘n effektiewe tegniek is vir die isolering van nuwe biokataliste en bruikbare gene van onkweekbare mikro-organismes afkomstig vanaf diverse omgewings. Voordat daar met die metagenoomanalise begin is, het ons ‘n genoombiblioteek berei uit ‘n bakteriese isolaat afkomstig van ’n diep myn: Geobacillus thermoleovorans GE-7. Die biblioteek is gesif vir lipolitiese aktiwiteit m.b.v. LB tributirien plate (TLB). Klone wat aktiwiteit getoon het, se DNS basispaar opeenvolging is bepaal d.m.v. 454 tegnologie en die data het getoon dat die lipA en GDSL lipases teenwoordig was. Die laasgenoemde lipase is nog nie gekaraktiriseer in hierdie organisme nie. Hierdie lipase familie vertoon die kenmerkende G-D-S-L aminosuur motief in plaas van die konvensionele G-X-S-X-G motief (X = dui enige amino suur aan).GDSL lipases is hidrolitiese ensieme met multifunksionele eienskappe soos bv. ‘n wye substraatspesifisiteit en regiospesifisiteit. Hierdie ensieme het ook die potensiaal om die hidrolise en sintese van ester verbindings te bewerkstellig wat van belang is in die farmaseutiese, voedsel, biochemiese en biologiese, National Research Foundation (NRF), Ernst, Ethel Erickson Trust, Metagenomics Platform

In order to biochemically characterise ZP soya bean genotypes the analyses of the main chemical composition (contents of ash, fibre, total proteins, oil), contents of protein fraction and trypsin inhibitor, as well as, the activity of enzymes urease, lipoxygenase 1 (LOX-1) and peroxidase (POD) in grain of five ZP soya bean cultivars (Lana, Lidija, Bosa, Nena and ZPS 015) were performed. As a check and in order to compare results, the analyses of grain of cultivars not derived at the Maize Research Institute, Zemun Polje, were also carried out. According to the protein content, the cultivar Nena stands out among ZP soya bean genotypes. The content of the grain total protein and water soluble proteins amounted to 43.34% and 34.97%, respectively. The cultivar Lana was also one of the best analyzed ZP genotypes. A genotype with recessive genes for the Kunitz trypsin inhibitor was derived by conventional selection methods and therefore, grain of this cultivar has an extremely low content of the total trypsin inhibitor amounting, on the average, to 14. 1mg g-1. It is interesting that the highest, i.e. lowest LOX-1 activities were detected in the varieties Lana and Nena, respectively. According to the POD activity, the analyzed soya bean cultivars can be classified into the following three groups: cultivars with high activity (EpEp genotypes), cultivars with low activity (epep genotypes), while the cultivar Bosa, heterozygous for this trait, belongs to the third group (Epep)., U cilju biohemijske karakterizacije ZP genotipova soje izvršena je analiza osnovnog hemijskog sastava (pepeo, celuloza, ukupni proteini, ulje), sadržaja proteinskih frakcija, i tripsin inhibitora, kao i aktivnosti enzima ureaze, lipoksigenaze 1 (LOX-1) i peroksidaze (POD) u zrnu pet ZP sorti soje (Lana, Lidija, Bosa, Nena i ZPS 015). Kao kontrola, i radi uporedivosti rezultata, izvršene su i analize zrna sorti soje koje nisu selekcionisane u Institutu za kukuruz. Među ZP genotipovima soje, prema sadržaju proteina, ističe se sorte Nena. Sadržaj ukupnih proteina u zrnu ove sorte iznosio je 43,34%, dok je sadržaj proteina rastvorljivih u vodi bio u prošeku 35,0%. Od analiziranih ZP genotipova može se izdvojiti sorta Lana. Klasičnim metodama selekcije stvorenje genotip sa recesivnim genima za Kunitz tripsin inhibitor, pa se zrno ove sorte odlikuje izuzetno niskim sadržajem ukupnog tripsin inhibitora, u prošeku 14,1mgg-1. Zanimljivo je da je u zrnu soje sorte Lana zabeležena najviša, a u zrnu sorte Nena najniža aktivost LOX-1. Ispitivane sorte soje, prema aktivnosti POD, mogu se podeliti u tri grupe. Sorte sa visokom aktivnošću (EpEp genotipovi), sorte sa niskom aktivnošću (epep genotipovi), a u treću grupu spada sorta Bosa koja je heterozigotna za ovo svojstvo (Epep).

In order to biochemically characterise ZP soya bean genotypes the analyses of the main chemical composition (contents of ash, fibre, total proteins, oil), contents of protein fraction and trypsin inhibitor, as well as, the activity of enzymes urease, lipoxygenase 1 (LOX-1) and peroxidase (POD) in grain of five ZP soya bean cultivars (Lana, Lidija, Bosa, Nena and ZPS 015) were performed. As a check and in order to compare results, the analyses of grain of cultivars not derived at the Maize Research Institute, Zemun Polje, were also carried out. According to the protein content, the cultivar Nena stands out among ZP soya bean genotypes. The content of the grain total protein and water soluble proteins amounted to 43.34% and 34.97%, respectively. The cultivar Lana was also one of the best analyzed ZP genotypes. A genotype with recessive genes for the Kunitz trypsin inhibitor was derived by conventional selection methods and therefore, grain of this cultivar has an extremely low content of the total trypsin inhibitor amounting, on the average, to 14. 1mg g-1. It is interesting that the highest, i.e. lowest LOX-1 activities were detected in the varieties Lana and Nena, respectively. According to the POD activity, the analyzed soya bean cultivars can be classified into the following three groups: cultivars with high activity (EpEp genotypes), cultivars with low activity (epep genotypes), while the cultivar Bosa, heterozygous for this trait, belongs to the third group (Epep)., U cilju biohemijske karakterizacije ZP genotipova soje izvršena je analiza osnovnog hemijskog sastava (pepeo, celuloza, ukupni proteini, ulje), sadržaja proteinskih frakcija, i tripsin inhibitora, kao i aktivnosti enzima ureaze, lipoksigenaze 1 (LOX-1) i peroksidaze (POD) u zrnu pet ZP sorti soje (Lana, Lidija, Bosa, Nena i ZPS 015). Kao kontrola, i radi uporedivosti rezultata, izvršene su i analize zrna sorti soje koje nisu selekcionisane u Institutu za kukuruz. Među ZP genotipovima soje, prema sadržaju proteina, ističe se sorte Nena. Sadržaj ukupnih proteina u zrnu ove sorte iznosio je 43,34%, dok je sadržaj proteina rastvorljivih u vodi bio u prošeku 35,0%. Od analiziranih ZP genotipova može se izdvojiti sorta Lana. Klasičnim metodama selekcije stvorenje genotip sa recesivnim genima za Kunitz tripsin inhibitor, pa se zrno ove sorte odlikuje izuzetno niskim sadržajem ukupnog tripsin inhibitora, u prošeku 14,1mgg-1. Zanimljivo je da je u zrnu soje sorte Lana zabeležena najviša, a u zrnu sorte Nena najniža aktivost LOX-1. Ispitivane sorte soje, prema aktivnosti POD, mogu se podeliti u tri grupe. Sorte sa visokom aktivnošću (EpEp genotipovi), sorte sa niskom aktivnošću (epep genotipovi), a u treću grupu spada sorta Bosa koja je heterozigotna za ovo svojstvo (Epep).

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a beta-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

A leaf cDNA library from hybrid aspen, Populus tremula x tremuloides, was constructed. From this two different cDNA clones, denoted CAla and CAlb, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced. Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the beta-CA family, and have identities around 70% to other dicotyledonous plant CAs. The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucleotides differ. Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolated cDNA clones are alleles. Northern blot hybridization revealed a CA transcript of ca. 1300 bases, 140 bases shorter than in pea. Western and northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid aspen CA is expressed specifically in the leaf under the growth conditions used. Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa. The enzyme was over-expressed in Escherichia coli, and purified by affinity chromatography. Biochemical characterization showed that the protein structure and the CO2-hydration activity are similar to the pea enzyme. Molecular characterization of a CA from a perennial plant has not previously been performed, and it demonstrates that both the structure and activity of hybrid aspen CA resembles CAs from annual plants.