Search results for: zymogram

Authors: Nawel Boucherba, Azzedine Bettache, Abdelaziz Messis, Francis Duchiron, Said Benallaoua

Abstract:

The impetus for research in the field of bioseparation has been sparked by the difficulty and complexity in the downstream processing of biological products. Indeed, 50% to 90% of the production cost for a typical biological product resides in the purification strategy. There is a need for efficient and economical large scale bioseparation techniques which will achieve high purity and high recovery while maintaining the biological activity of the molecule. One such purification technique which meets these criteria involves the partitioning of biomolecules between two immiscible phases in an aqueous system (ATPS). The Production of xylanases is carried out in 500ml of a liquid medium based on birchwood xylan. In each ATPS, PEG 1000 is added to a mixture consisting of dipotassium phosphate, sodium chloride and the culture medium inoculated with the strain Jonesia denitrificans, the mixture was adjusted to different pH. The concentration of PEG 1000 was varied: 8 to 16 % and the NaCl percentages are also varied from 2 to 4% while maintaining the other parameters constant. The results showed that the best ATPS for purification of xylanases is composed of PEG 1000 at 8.33%, 13.14 % of K2HPO4, 1.62% NaCl at pH 7. We obtained a yield of 96.62 %, a partition coefficient of 86.66 and a purification factor of 2.9. The zymogram showed that the activity is mainly detected in the top phase.

Keywords: Jonesia denitrificans BN13, xylanase, aqueous two phases system, zymogram

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

Abstract:

The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcuslactis, recombinant, phytase, poultry

Procedia PDF Downloads 456

Authors: H. M. Takematsu, B. R. De Camargo, E. F. Noronha

Abstract:

The use of hydrolyzing enzymes for degradation of lignocellulosic biomass is of great concern for the production of second generation ethanol. Although many hollocelulases have already been described in the literature, much more has to be discovered. Therefore, the aim of this study to evaluate hollocelulase production of P. polonicum grown in liquid media containing sugarcane bagasse as the carbon source. From a collection of twenty fungi isolated from Cerrado biome soil, P. polonicum was molecular identified by sequencing of ITS4, βtubulin and Calmodulin genes, and has been chosen to be further investigated regarding its potential production of hydrolyzing enzymes. Spore suspension (1x10-6 ml-1) solution was inoculated in sterilized minimal liquid medium containing 0,5%(w/v) of non-pretreated sugarcane bagasse as the carbon source, and incubated in shaker incubator at 28°C and 120 rpm. The supernatant obtained, was subjected to enzymatic assays to analyze xylanase, mannanase, pectinase and endoglucanase activities. Xylanase activity showed better results (67,36 UI/mg). Xylanases bands were indicated by zymogram and SDS-PAGE, and one of them was partially purified and characterized. It showed maximum activity at 50 °C, was thermostable for 72h at 40°C, and pH5.0 was the optimum observed. This study presents P. polonicum as an interesting source of hollocelulases, especially xylanase, for lignocellulose bio-conversion processes with commercial use.

Keywords: sugarcane bagasse, Cerrado biome , hollocelulase, lignocellulosic biomass

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Authors: Amanda Gregorim Fernandes, Lorena Cardoso Cintra, Rosalia Santos Amorim Jesuino, Fabricia Paula De Faria, Marcio José Poças Fonseca

Abstract:

Bioethanol is one of the most promising biofuels and able to replace fossil fuels and reduce its different environmental impacts and can be generated from various agroindustrial waste. The Brazil is in first place in bioethanol production to be the largest producer of sugarcane. The bagasse sugarcane (SCB) has lignocellulose which is composed of three major components: cellulose, hemicellulose and lignin. Cellulose is a homopolymer of glucose units connected by glycosidic linkages. Among all species of Penicillium, Penicillium echinulatum has been the focus of attention because they produce high quantities of cellulase and the mutant strain 9A02S1 produces higher enzyme levels compared to the wild. Among the cellulases, the cellobiohydrolases enzymes are the main components of the cellulolytic system of fungi, and are also responsible for most of the potential hydrolytic in enzyme cocktails for the industrial processing of plant biomass and several cellobiohydrolases Penicillium had higher specific activity against cellulose compared to CBH I from Trichoderma reesei. This fact makes it an interesting pattern for higher yields in the enzymatic hydrolysis, and also they are important enzymes in the hydrolysis of crystalline regions of cellulose. Therefore, finding new and more active enzymes become necessary. Meanwhile, β-glycosidases act on soluble substrates and are highly dependent on cellobiohydrolases and endoglucanases action to provide the substrate in the hydrolysis of the biomass, but the cellobiohydrolases and endoglucanases are highly dependent β-glucosidases to maintain efficient hydrolysis. Thus, there is a need to understand the structure-function relationships that govern the catalytic activity of cellulolytic enzymes to elucidate its mechanism of action and optimize its potential as industrial biocatalysts. To evaluate the enzyme β-glucosidase of Penicillium echinulatum (PeBGL1) the gene was synthesized from the assembly sequence from a library in induction conditions and then the PeBGL1 gene was cloned in the vector pPICZαA and transformed into P. pastoris GS115. After processing, the producers of PeBGL1 were analyzed for enzyme activity and protein profile where a band of approximately 100 kDa was viewed. It was also carried out the zymogram. In partial characterization it was determined optimum temperature of 50°C and optimum pH of 6,5. In addition, to increase the secreted recombinant PeBGL1 production by Pichia pastoris, three parameters of P. pastoris culture medium were analysed: methanol, nitrogen source concentrations and the inoculum size. A 23 factorial design was effective in achieving the optimum condition. Altogether, these results point to the potential application of this P. echinulatum β-glucosidase in hydrolysis of cellulose for the production of bioethanol.

Keywords: bioethanol, biotechnology, beta-glucosidase, penicillium echinulatum

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