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TABLE 1. Purification of recombinant xylanase produced in E. coli Production in E. coli and purification of xylanase _To elucidate the enzymatic activity of the xylanase, we expressed the gene in E. coli and purified the recombinant protein. High levels of recombinant protein were obtained in a soluble form. A total of 11450 nkat enzyme activity was obtained from 1L culture (Table 1). The purification pro- cedure involved a concentration step by ammonium sulphate pre- cipitation, and purification was performed by anion-exchange and hydrophobic interaction column chromatographies. The purified recombinant protein is shown by CBB stained SDS-PAGE (Fig. 3).

Table 1 Purification of recombinant xylanase produced in E. coli Production in E. coli and purification of xylanase _To elucidate the enzymatic activity of the xylanase, we expressed the gene in E. coli and purified the recombinant protein. High levels of recombinant protein were obtained in a soluble form. A total of 11450 nkat enzyme activity was obtained from 1L culture (Table 1). The purification pro- cedure involved a concentration step by ammonium sulphate pre- cipitation, and purification was performed by anion-exchange and hydrophobic interaction column chromatographies. The purified recombinant protein is shown by CBB stained SDS-PAGE (Fig. 3).

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FIG. 1. Phylogenetic tree based on the 16S rRNA sequence information. Calculations were performed using ClustalW program provided by DNA Data Bank of Japan (DDBJ). Segment: corresponding to an evolutionary distance of 0.01 are shown. Each name at the termini represents the species from which the 16S rRNA originated. The accession numbers for eact sequence are as follows: Bacillus subtilis strain R5, AB257199; B. subtilis strain 168, ALO09126; Bacillus subtilis strain PY79, EU081774; Bacillus amyloliquefaciens, NC_009725; Bacillu: polyfermenticus, AY149473; Bacillus pumilus, ABO20208; Bacillus cereus strain E33L, NC_006274; Bacillus cereus strain CCM2010, DQ207729; Bacillus anthracis, NC_005945; Bacillu: thuringienesis, NC_008600; Bacillus halodurans, NC_002570; Bacillus coagulans, AB362709; Bacillus clausii, NC_006582; Geobacillus stearothermophilus, AY608942; Geobacillu: kaustophilus, BA000043; Geobacillus thermoleovorans, Z26923. However, four of the above replacements (positions 141, 261, 480 and 498) did not have any effect on the encoded amino acid se- quence. The replacement of T at position 484 by A resulted in re- placement of serine by threonine. Both the amino acids (serine and threonine) belong to the same group having uncharged polar side chains with aliphatic hydroxyl groups which make them hydrophilic in nature. Therefore, this replacement is not expected to have a significant effect on the secondary and tertiary structures of the protein. A comparison of amino acid sequence of xylanase from strain R5 with B. subtilis strains 168 and BP-7 demonstrated that xylanase from strain R5 is more than 99 and 92% identical to those of strains 168 and BP-7, respectively (Fig. 2). The catalytic residues, E at position 106 and E at 200 (R5 numbering), are conserved in all the three sequences. A couple of non-polar amino acid residues in strains R5 and 168 are replaced by polar residues (143T and A193S, R5 numbering) in strain BP-7. Apart from these changes two charged residues in strains R5 and 168 are replaced by neutral aromatic residues (K127Y and H184Y, R5 numbering). Interestingly the amino acid sequence of xylanase from strain R5 was 100% identical to that of Bacillus pumilus and Bacillus cereus (accession numbers AAZ17390 and AAZ17391, respectively) xylanase sequences while it was 99.5% identical to that of B. subtilis strain 168 and B. aminoliquefaciens (accession numbers M263648 and AAZ17388, respectively). The 16S rRNA sequence of strain R5 was more homologous to B. subtilis strain 168 while xylanase sequence from this microorganism was identical to B. pumilus and B. cereus at the amino acid level. Unfor- tunately the xylanases from these microorganisms are not char- acterized in detail.
FIG. 1. Phylogenetic tree based on the 16S rRNA sequence information. Calculations were performed using ClustalW program provided by DNA Data Bank of Japan (DDBJ). Segment: corresponding to an evolutionary distance of 0.01 are shown. Each name at the termini represents the species from which the 16S rRNA originated. The accession numbers for eact sequence are as follows: Bacillus subtilis strain R5, AB257199; B. subtilis strain 168, ALO09126; Bacillus subtilis strain PY79, EU081774; Bacillus amyloliquefaciens, NC_009725; Bacillu: polyfermenticus, AY149473; Bacillus pumilus, ABO20208; Bacillus cereus strain E33L, NC_006274; Bacillus cereus strain CCM2010, DQ207729; Bacillus anthracis, NC_005945; Bacillu: thuringienesis, NC_008600; Bacillus halodurans, NC_002570; Bacillus coagulans, AB362709; Bacillus clausii, NC_006582; Geobacillus stearothermophilus, AY608942; Geobacillu: kaustophilus, BA000043; Geobacillus thermoleovorans, Z26923. However, four of the above replacements (positions 141, 261, 480 and 498) did not have any effect on the encoded amino acid se- quence. The replacement of T at position 484 by A resulted in re- placement of serine by threonine. Both the amino acids (serine and threonine) belong to the same group having uncharged polar side chains with aliphatic hydroxyl groups which make them hydrophilic in nature. Therefore, this replacement is not expected to have a significant effect on the secondary and tertiary structures of the protein. A comparison of amino acid sequence of xylanase from strain R5 with B. subtilis strains 168 and BP-7 demonstrated that xylanase from strain R5 is more than 99 and 92% identical to those of strains 168 and BP-7, respectively (Fig. 2). The catalytic residues, E at position 106 and E at 200 (R5 numbering), are conserved in all the three sequences. A couple of non-polar amino acid residues in strains R5 and 168 are replaced by polar residues (143T and A193S, R5 numbering) in strain BP-7. Apart from these changes two charged residues in strains R5 and 168 are replaced by neutral aromatic residues (K127Y and H184Y, R5 numbering). Interestingly the amino acid sequence of xylanase from strain R5 was 100% identical to that of Bacillus pumilus and Bacillus cereus (accession numbers AAZ17390 and AAZ17391, respectively) xylanase sequences while it was 99.5% identical to that of B. subtilis strain 168 and B. aminoliquefaciens (accession numbers M263648 and AAZ17388, respectively). The 16S rRNA sequence of strain R5 was more homologous to B. subtilis strain 168 while xylanase sequence from this microorganism was identical to B. pumilus and B. cereus at the amino acid level. Unfor- tunately the xylanases from these microorganisms are not char- acterized in detail.
‘IG. 3. CBB-stained SDS-PAGE of purified recombinant xylanase. Lane M, molecular nass markers; lane 1, total cell lysate of the host cells carrying the pT7-7 plasmid; lane ), total cell lysate of the host cells carrying the pT-xyl plasmid; lane 3, soluble fraction of he host cells carrying pT-xyl plasmid; lane 4, insoluble fraction of the host cells arrying pT-xyl plasmid; lane 5, ammonium sulphate precipitated sample after dialysis igainst 40 mM Tris-HCl; lane 6, eluate after ion exchange chromatography with fesource Q; lane 7, purified xylanase after hydrophobic column (Res ISO).
‘IG. 3. CBB-stained SDS-PAGE of purified recombinant xylanase. Lane M, molecular nass markers; lane 1, total cell lysate of the host cells carrying the pT7-7 plasmid; lane ), total cell lysate of the host cells carrying the pT-xyl plasmid; lane 3, soluble fraction of he host cells carrying pT-xyl plasmid; lane 4, insoluble fraction of the host cells arrying pT-xyl plasmid; lane 5, ammonium sulphate precipitated sample after dialysis igainst 40 mM Tris-HCl; lane 6, eluate after ion exchange chromatography with fesource Q; lane 7, purified xylanase after hydrophobic column (Res ISO).
FIG. 4. Effect of pH and temperature on the enzyme activity of recombinant xylanase (A) Activity as a function of pH. Activity was examined as described in the experimental section. The following buffers were used: sodium acetate buffer (open circles), sodium phosphate buffer (closed circles), and Tris buffer (open squares). (B) Effect o! temperature on xylanase activity. The enzyme activity was measured at various temperatures at pH 6.0 without addition of any metal ions in the reaction mixture.
FIG. 4. Effect of pH and temperature on the enzyme activity of recombinant xylanase (A) Activity as a function of pH. Activity was examined as described in the experimental section. The following buffers were used: sodium acetate buffer (open circles), sodium phosphate buffer (closed circles), and Tris buffer (open squares). (B) Effect o! temperature on xylanase activity. The enzyme activity was measured at various temperatures at pH 6.0 without addition of any metal ions in the reaction mixture.
FIG. 5. Effect of metal cations on the enzyme activity of recombinant xylanase. A chloride salt of each metal cation was used at a final concentration of 0.1 mM. The final concentration of EDTA, when used, was 5 mM. The activity was examined at 40 °C and PH 6.0 as described in the experimental section.
FIG. 5. Effect of metal cations on the enzyme activity of recombinant xylanase. A chloride salt of each metal cation was used at a final concentration of 0.1 mM. The final concentration of EDTA, when used, was 5 mM. The activity was examined at 40 °C and PH 6.0 as described in the experimental section.
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