Immunolocalization of a Recombinant Cellulase in Transgenic Tobacco Plants
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Journal Article Immunolocalization of a Recombinant Cellulase in Transgenic Tobacco Plants Get access H J Bae, H J Bae Laval University, Ste-foy, Quebec, Canada G1K 7P4 Search for other works by this author on: Oxford Academic Google Scholar H Chamberland, H Chamberland Laval University, Ste-foy, Quebec, Canada G1K 7P4 Search for other works by this author on: Oxford Academic Google Scholar S Laberge, S Laberge Centre de Recherches et de Agriculture et Agroalimentaire Canada G1V 2J3 Search for other works by this author on: Oxford Academic Google Scholar Y S Kim Y S Kim Chonnam National University, Kwangju, Korea 500-757 Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 8, Issue S02, 1 August 2002, Pages 1084–1085, https://doi.org/10.1017/S1431927602106982 Published: 01 August 2002The endoglucanase cDNA, Dvv-ENGase I, from western corn rootworm, Diabrotica virgifera virgifera LeConte was expressed using the GS115 methylotrophic strain of Pichia pastoris. The Dvv-ENGase I gene was cloned into the integrative plasmid pPICZαA under the control of AOX1, which is a methanol-inducible promoter. Positive clones were selected for their ability to produce the recombinant endoglucanase upon continuous methanol induction. The secreted recombinant insect endoglucanase Dvv-ENGase I has an apparent molecular mass of 29 kDa. The recombinant endo-1,4 -β-glucanase (ENGase) was able to digest the substrates: hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), and Whatman No. 1 filter paper. A higher accumulation of reducing sugar was evident when the P. pastoris expression medium contained HEC (1%) instead of CMC (1%). An enzymatic activity band was detected after performing electrophoretic separation under nondenaturing conditions. The biological activity of the recombinant Dvv-ENGase I was influenced by the presence of protease inhibitors in the culture medium.
Carboxymethyl cellulose
Western corn rootworm
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A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA, was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 °C) optima were determined.
Cloning (programming)
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A gene encoding cellulase BXC46 from Bursaphelenchus xylophilus was cloned by PCR,and showed 99% homology to the reported gene of cellulase BXC10(GenBank accession number FJ598020.1).The gene was cloned into pET-15b to construct expressing vector pET-15b-BXC.Then,this recombinant plasmid was introduced into E.coli BL21(DE3) to construct engineering bacterium.Recombinant cellulase was successfully expressed in engineering bacterium induced by IPTG as analyzed by SDS-PAGE.Recombinant protein was partially purified by Ni2+-NTA resin and showed good activity as determined by DNS method.The optimal temperature and pH of the recombinant cellulase were 45 ℃ and 4.0,respectively.
Cloning (programming)
Homology
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Cellulases are inherently interesting. Induction of such complex multiple synergistic components is efficiently accomplished though the substrate is extracellular and insoluble. Their study has been focused historically on applied aspects, initially on means of inhibiting their degradative action, with recent emphasis on obtaining enhanced yields/activities to promote efficient hydrolysis of cellulose to glucose syrups. Cloning of cellulases has opened new vistas for basic study. It facilitates purification of the individual components, a major advance in a notoriously difficult arena. It has aided development of the concept of the triple (binding, hinge and active site) domains of cellulase, besides allowing their further characterization via site specific mutagenesis. Evolutionary relationships have been clarified. The rDNA methodology also directly aids the application of cellulases through improvement and/or hyperproduction of specific components, thereby allowing them to be mixed optimally for routine and new applications.
Cloning (programming)
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Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.
Molecular mass
Specific activity
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Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus . Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L −1 . The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (K M
Enzyme Kinetics
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The heterologous expression of cellulase gene from Aspergillus sp. in E.coli strains of Rosetta
(DE3) and BL2 l (DE3) has been studied to compare the recombinant protein production and to
observe their activity, efficiency and production rate of recombinant cellulase. In previous study,
an improved protein quality has been detected by using Rosetta, therefore further works need to
be carried out to compare its protein production with BL2 l. The pFT AG-vector with the
recombinant cellulase was used for expression studies and inserted into both Rosetta and BL2 l.
Screening of cellullolyctic organism has failed to reveal the activity of recombinant cellulase as
there is no growth detected on the media, as well as no formation of halo zone. Other than
detection using the formation of halo zone, Bradford Assay and DNS assay was conducted to
determine the cellulase activity of the bacterial in liquid medium. Recombinant cellulase from
Rosetta was shown to have higher cellulase activity when the protein concentration was
calculated using the standard graph. It was due to the fact that Rosetta has rare codons
implemented inside them in order to yield higher protein production and better protein purity
Heterologous expression
Heterologous
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Strongylocentrotus purpuratus
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In order to improve the expression level of endoglucanase gene in E.coli,the expression conditions of the recombinant were studied in this experiment.The results showed that: the recombinant E.coli was incubated at 43 ℃ for 5 h,and when induced by 0.1 mmol/L IPTG or 0.1% lactose,its yield of endoglucanase can come to 141.22 U/mL which is about 2 times higher than that of the control.
Strain (injury)
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Recombinant cellulase, BL21-CodonPlus (DE3), Thermotoga naphthophila, Optimization of conditions
Thermotoga maritima
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