Cu(II) binding to the λ6aJL2-R24G antibody light chain protein associated with light chain amyloidosis disease: The role of histidines.

Light chain amyloidosis is a conformational disease caused by the abnormal proliferation and deposition of antibody light chains as amyloid fibers in organs and tissues. The effect of Cu(II) binding to the model recombinant protein 6aJL2-R24G was previously characterized in our group, and we found an acceleration of the aggregation kinetics of the protein. In this study, in order to confirm the Cu(II) binding sites, histidine variants of 6aJL2-R24G were prepared and the effects of their interaction with Cu(II) were analyzed by circular dichroism, fluorescence spectroscopy, isothermal calorimetry titrations, and molecular dynamics simulations. Confirming our earlier work, we found that His8 and His99 are the highest affinity Cu(II) binding sites, and that Cu(II) binding to both sites is a cooperative event.

Multidomain chimeric enzymes as a promising alternative for biocatalysts improvement: a minireview.

Searching for new and better biocatalysts is an area of study in constant development. In nature, mechanisms generally occurring in evolution, such as genetic duplication, recombination, and natural selection processes, produce various enzymes with different architectures and properties. The recombination of genes that code proteins produces multidomain chimeric enzymes that contain two or more domains that sometimes enhance their catalytic properties. Protein engineering has mimicked this process to enhance catalytic activity and the global stability of enzymes, searching for new and better biocatalysts. Here, we present and discuss examples from both natural and synthetic multidomain chimeric enzymes and how additional domains heighten their stability and catalytic activity. Moreover, we also describe progress in developing new biocatalysts using synthetic fusion enzymes and revise some methodological strategies to improve their biological fitness.

Potencial de la levadura oleaginosa Clavispora lusitaniae Hi2 en la conversión de residuos agroindustriales a lípidos / Capacity of the oleaginous yeast Clavispora lusitaniae Hi2 to transform agroindustrial residues into lipids

AntecedentesLos lípidos obtenidos de microorganismos oleaginosos a partir de hidrolizados de residuos lignocelulósicos son una alternativa para la fabricación de biodiesel.ObjetivosAislar una levadura oleaginosa capaz de producir lípidos a partir de nejayote centrifugado (NC), hidrolizado de sólidos de nejayote (HSN) e hidrolizado de bagazo de caña de azúcar (HBC).MétodosPara identificar los aislamientos recuperados se secuenció el ADN ribosómico 26S. La capacidad metabólica se evaluó mediante tiras API20C AUX. La caracterización nutricional del NC, HSN y HBC se realizó cuantificando azúcares reductores, carbohidratos totales, almidón, proteína y nitrógeno total. La capacidad de producción de biomasa y lípidos de la cepa Clavispora lusitaniae Hi2 se evaluó mediante cinéticas de crecimiento en medios de cultivo formulados a partir de NC, HSN y HBC.ResultadosSe aislaron e identificaron seis cepas de levaduras oleaginosas, siendo C. lusitaniae Hi2 seleccionada para producir lípidos mediante el uso de nejayote. Dicha cepa puede utilizar glucosa, xilosa, arabinosa, galactosa y celobiosa como fuentes de carbono. Los cultivos de C. lusitaniae Hi2 en medio con NC y HSN (en relación 25:75) presentaron la mayor producción de biomasa, 5,6 ± 0,28 g/L; la mayor producción de lípidos, 0,99±0,09 g/L, se obtuvo con una relación 50:50 de estos residuos a las 20 h de incubación.ConclusionesLa utilización de NC, HSN y HBC para el crecimiento de C. lusitaniae Hi2 es una opción para el aprovechamiento de estos residuos y la generación de compuestos de interés biotecnológico. (AU)

BackgroundSingle-cell oils obtained from oleaginous microorganisms by using lignocellulosic waste hydrolysates are an alternative for producing biodiesel.AimsTo isolate a yeast strain able to produce lipids from centrifuged nejayote (CN), hydrolyzed nejayote solids (HNS) and hydrolyzed sugarcane bagasse (HSB).MethodsIn order to identify the yeasts recovered, 26S ribosomal DNA was sequenced. The metabolic profile was assessed by using API20C AUX strips. The nutritional characterization of CN, HNS and HSB was performed by quantifying reducing sugars, total carbohydrates, starch, protein and total nitrogen. The biomass and lipid production ability were evaluated by performing growth kinetics of Clavispora lusitaniae Hi2 in combined culture media.ResultsSix oleaginous yeast strains were isolated and identified, selecting C. lusitaniae Hi2 to study its lipids production by using nejayote. The C. lusitaniae Hi2 strain can use glucose, xylose, arabinose, galactose and cellobiose as carbon sources. Cultures of C. lusitaniae Hi2 presented the best biomass (5.6±0.28 g/L) and lipid production (0.99±0.09 g/L) at 20 h of incubation with the CN:HNS media in the 25:75 and 50:50 ratios, respectively.ConclusionsThe use of CN, HNS and HSB for the growth of C. lusitaniae Hi2 is an option to take advantage of these agro-industrial residues and generate compounds of biotechnological interest. (AU)

[Capacity of the oleaginous yeast Clavispora lusitaniae Hi2 to transform agroindustrial residues into lipids]. / Potencial de la levadura oleaginosa Clavispora lusitaniae Hi2 en la conversión de residuos agroindustriales a lípidos.

BACKGROUND: Single-cell oils obtained from oleaginous microorganisms by using lignocellulosic waste hydrolysates are an alternative for producing biodiesel. AIMS: To isolate a yeast strain able to produce lipids from centrifuged nejayote (CN), hydrolyzed nejayote solids (HNS) and hydrolyzed sugarcane bagasse (HSB). METHODS: In order to identify the yeasts recovered, 26S ribosomal DNA was sequenced. The metabolic profile was assessed by using API20C AUX strips. The nutritional characterization of CN, HNS and HSB was performed by quantifying reducing sugars, total carbohydrates, starch, protein and total nitrogen. The biomass and lipid production ability were evaluated by performing growth kinetics of Clavispora lusitaniae Hi2 in combined culture media. RESULTS: Six oleaginous yeast strains were isolated and identified, selecting C. lusitaniae Hi2 to study its lipids production by using nejayote. The C. lusitaniae Hi2 strain can use glucose, xylose, arabinose, galactose and cellobiose as carbon sources. Cultures of C. lusitaniae Hi2 presented the best biomass (5.6±0.28 g/L) and lipid production (0.99±0.09 g/L) at 20 h of incubation with the CN:HNS media in the 25:75 and 50:50 ratios, respectively. CONCLUSIONS: The use of CN, HNS and HSB for the growth of C. lusitaniae Hi2 is an option to take advantage of these agro-industrial residues and generate compounds of biotechnological interest.

Novel Thermotolerant Amylase from Bacillus licheniformis Strain LB04: Purification, Characterization and Agar-Agarose.

This study analyzed the thermostability and effect of calcium ions on the enzymatic activity of α-amylase produced by Bacillus licheniformis strain LB04 isolated from Espinazo Hot springs in Nuevo Leon, Mexico. The enzyme was immobilized by entrapment on agar-agarose beads, with an entrapment yield of 19.9%. The identification of the bacteria was carried out using 16s rDNA sequencing. The enzyme was purified through ion exchange chromatography (IEX) in a DEAE-Sephadex column, revealing a protein with a molecular weight of ≈130 kDa. The enzyme was stable at pH 3.0 and heat stable up to 80 °C. However, the optimum conditions were reached at 65 °C and pH 3.0, with a specific activity of 1851.7 U mg-1 ± 1.3. The agar-agarose immobilized α-amylase had a hydrolytic activity nearly 25% higher when compared to the free enzyme. This study provides critical information for the understanding of the enzymatic profile of B. licheniformis strain LB04 and the potential application of the microorganisms at an industrial level, specifically in the food industry.

Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F.

A Leuconostoc mesenteroides NRRL B-512 F levansucrase gene, (levS), was isolated, sequenced and cloned in Escherichia coli. The recombinant enzyme was shown to be a fructosyltransferase producing a polymer identified by (13)C-NMR as levan. Based on sequence analysis, we found that this levansucrase is a mosaic protein, bearing structural features of glucosyltransferases in the amino and carboxy terminal regions similarly to inulosucrase from Leuconostoc citreum. The phylogenetic analysis of the C-terminal region domain of levansucrases from L. mesenteroides demonstrates that they group together into a novel putative sub-family of genes and evolved long before all other glucosyltransferases, while their catalytic domain structure is species related.