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1.
Electron. j. biotechnol ; Electron. j. biotechnol;34: 29-36, july. 2018. ilus, tab, graf
Article de Anglais | LILACS | ID: biblio-1045993

RÉSUMÉ

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.


Sujet(s)
Bacillus/enzymologie , Cellulases/biosynthèse , Température , Stabilité enzymatique , Expression des gènes , Paroi cellulaire/enzymologie , Réaction de polymérisation en chaîne , Clonage moléculaire , Cellulases/isolement et purification , Cellulases/métabolisme , Escherichia coli/métabolisme , Cellules végétales/enzymologie , Concentration en ions d'hydrogène , Hydrolyse
2.
Article de Anglais | WPRIM | ID: wpr-780483

RÉSUMÉ

Aims@#In recent studies, xylanase is the valuable enzyme in paper industry, and the demand of this enzyme is increasing. The finding out of the new xylanases with good properties and applicable for paper industries from Indonesian biodiversity is still required. In this study, we isolated, cloned, and expressed the gene encoding alkalophilic xylanase gene from Indonesia indigenous Bacillus halodurans CM1 in Escherichia coli, and applied the recombinant xylanase in deinking process. @*Methodology and results@#The open reading frame of the gene consist of 1191 bp, with nucleotide identity 99% with that of B. halodurans C-125 has been obtained by using PCR. The gene was submitted into GenBank with accession number KU759320. The gene was then subcloned and expressed in E.coli using pET 21d(+) vector, and we found that only extracellular that have significant activity. The gene product had optimum activity at 65 °C and pH 9. After purification using Ni-NTA, the single band was obtained, and the molecular mass was about 45 kDa based on SDS/PAGE analyses. The recombinant xylanase had been applied in deinking process of old news paper at laboratory scale of a paper industry. This recombinant xylanase application showed better brightness and whiteness, as well as higher brightness and whiteness gain of of product compared to the commercial one when using the same raw material. @*Conclusion, significance and impact of study@#The study is the first example of the cloning of industrially important enzyme (xylanase) from B. halodurans CM1 and showed potential application of the recombinant enzyme in deinking process of waste paper.

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