Bacterial strain which was used as a host for recombinant plasmid propagation was E. coli DH5 α, whereas for subcloning and expression was B. subtilis DB104 ; Plasmid used in this experiment was pSKE194 an E.coli-Bacillus shuttle vector (both bacterial strain and plasmid were precious gift from Prof Meinhardt, Muenster University). For regenerating Bacillus protoplasts containing desired recombinant plasmid the 1.5% (w/v) agar containing regeneration medium DM3 was used (Chang and Cohen 1984), and for selection of positive Bacillus transformant, LB medium containing erythromycin 5 µg mL-1 were used. LB agar medium containing ampicillin (100 µg mL-1) or erythromycin (5 µg mL-1) and 0.7% (1 w /v) Oat spelt xylan was used for confirmation of the gene expression.
Subcloning of Xylanase Gene into Bacillus-E. coli Shuttle Vector. All genetic engineering experiments were carried out based on standard protocols (Sambrook and Russel 2001). The inserted endoxylanase gene (xyn11) with confirmed DNA sequence as reported previously was cut from previously reported recombinant pGEM-T easy plasmid (Helianti et al. 2010) by digesting with KpnI and Bgl II. Then it was ligated into pSKE194 vector at the Kpn I and BamH I sites. The ligation was transformed into E. coli DH5, and the positive clone harbouring recombinant pSKE 194 plasmid with clear zone was checked in Oat spelt xylan-LB agar medium to observe the xylanase activity by the xylan–Congo red clearance plate assay. The recombinant plasmid pSKE194-xynAQ1 was then extracted from E.coli, and then transformed to B. subtilis DB104 using protoplast transformation. Bacillus protoplasting and transformation has been carried out based on Chang and ...
... middle of paper ...
...um albumin (BSA) was used as the standard protein (Bradford 1976).
Analyses of Fermentation Product and Enzymatics Hydrolyses of Corn Cobs Xylan using Thin Layer Chromatography Analyses
The supernatant of fermentation broth in Erlenmeyer flask with determined concentration of
corncobs and TLW was taken after 0, 8, 24, and 32 h cultivation, and they were spotted each 0.75 L on Thin Layer Chromatography (TLC) plates coated with 0.2 mm thick layer of silica gel G. Xylooligosaccharides standard were also treated with the same manner. Standards that have been used were xylose, xylobiose, xylotriose, xylotetraose. The plate was developed with a solvent system of butanol, acetic acid, and water. Staining mixture was anilyn, diphenylamin, acetone, and phosphate acid with composition 0.4 ml; 0.4gr; 20 ml; 3 ml, respectively. The TLC plate was dried until the spots appeared.
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