Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity
Abstract Background The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an...
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BMC
2018-02-01
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| Series: | BMC Biotechnology |
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| Online Access: | http://link.springer.com/article/10.1186/s12896-018-0420-7 |
| _version_ | 1811292119125458944 |
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| author | Anil S. Prajapati Vishakha A. Pawar Ketankumar J. Panchal Ankit P. Sudhir Bhaumik R. Dave Darshan H. Patel R. B. Subramanian |
| author_facet | Anil S. Prajapati Vishakha A. Pawar Ketankumar J. Panchal Ankit P. Sudhir Bhaumik R. Dave Darshan H. Patel R. B. Subramanian |
| author_sort | Anil S. Prajapati |
| collection | DOAJ |
| description | Abstract Background The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an important role in substrate binding through steric hindrance. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates. Results Wild type and mutated genes were cloned and overexpressed in BL21. Optimum pH and temperature of rBAxn exhibited pH 9.0 and 50 °C respectively and it was stable up to 215 h. Along with the physical properties of rBAxn, kinetic parameters (K m 19.34 ± 0.72 mg/ml; k cat 6449.12 ± 155.37 min− 1 and k cat /K m 333.83 ± 6.78 ml min− 1 mg− 1) were also compared with engineered enzymes. Out of five mutations, W63A, Y128A and W144A lost almost 90% activity and Y124A and W187A retained almost 40–45% xylanase activity. Conclusions The site-specific single mutation, led to alteration in substrate specificity from xylan to CMC while in case of double mutant the substrate specificity was altered from xylan to CMC, FP and avicel, indicating the role of aromatic residues on substrate binding, catalytic process and overall catalytic efficiency. |
| first_indexed | 2024-04-13T04:39:32Z |
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| id | doaj.art-58425ae359bc4c278b4185b92367fba1 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-13T04:39:32Z |
| publishDate | 2018-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-58425ae359bc4c278b4185b92367fba12022-12-22T03:02:02ZengBMCBMC Biotechnology1472-67502018-02-0118111010.1186/s12896-018-0420-7Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificityAnil S. Prajapati0Vishakha A. Pawar1Ketankumar J. Panchal2Ankit P. Sudhir3Bhaumik R. Dave4Darshan H. Patel5R. B. Subramanian6P. G. Department of Biosciences, UGC-Centre of advanced studies, Satellite campus, Sardar Patel UniversityP. G. Department of Biosciences, UGC-Centre of advanced studies, Satellite campus, Sardar Patel UniversityP. G. Department of Biosciences, UGC-Centre of advanced studies, Satellite campus, Sardar Patel UniversityP. G. Department of Biosciences, UGC-Centre of advanced studies, Satellite campus, Sardar Patel UniversityP. G. Department of Biosciences, UGC-Centre of advanced studies, Satellite campus, Sardar Patel UniversityP. D. Patel Institute of Applied Sciences, Charotar University of Science and Technology (CHARUSAT)P. G. Department of Biosciences, UGC-Centre of advanced studies, Satellite campus, Sardar Patel UniversityAbstract Background The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an important role in substrate binding through steric hindrance. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates. Results Wild type and mutated genes were cloned and overexpressed in BL21. Optimum pH and temperature of rBAxn exhibited pH 9.0 and 50 °C respectively and it was stable up to 215 h. Along with the physical properties of rBAxn, kinetic parameters (K m 19.34 ± 0.72 mg/ml; k cat 6449.12 ± 155.37 min− 1 and k cat /K m 333.83 ± 6.78 ml min− 1 mg− 1) were also compared with engineered enzymes. Out of five mutations, W63A, Y128A and W144A lost almost 90% activity and Y124A and W187A retained almost 40–45% xylanase activity. Conclusions The site-specific single mutation, led to alteration in substrate specificity from xylan to CMC while in case of double mutant the substrate specificity was altered from xylan to CMC, FP and avicel, indicating the role of aromatic residues on substrate binding, catalytic process and overall catalytic efficiency.http://link.springer.com/article/10.1186/s12896-018-0420-7XylanaseSite-directed mutagenesisProtein engineeringSubstrate specificity |
| spellingShingle | Anil S. Prajapati Vishakha A. Pawar Ketankumar J. Panchal Ankit P. Sudhir Bhaumik R. Dave Darshan H. Patel R. B. Subramanian Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity BMC Biotechnology Xylanase Site-directed mutagenesis Protein engineering Substrate specificity |
| title | Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity |
| title_full | Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity |
| title_fullStr | Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity |
| title_full_unstemmed | Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity |
| title_short | Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity |
| title_sort | effects of substrate binding site residue substitutions of xyna from bacillus amyloliquefaciens on substrate specificity |
| topic | Xylanase Site-directed mutagenesis Protein engineering Substrate specificity |
| url | http://link.springer.com/article/10.1186/s12896-018-0420-7 |
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