<i>In-Silico</i> Side-Directed Mutagenesis of Oxidoreductase from <i>Anoxybacillus sp.</i> SK3-4

Musa Ahmed Abubakar; Umar Shittu; Musa Hassan Muhammad; Saif Al-Sheyab Ahmad

doi:doi:10.11648/j.ijmb.20180302.14

| Peer-Reviewed

In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4

Musa Ahmed Abubakar, Umar Shittu, Musa Hassan Muhammad, Saif Al-Sheyab Ahmad

Published in International Journal of Microbiology and Biotechnology (Volume 3, Issue 2)

Received: 27 June 2018 Accepted: 11 July 2018 Published: 9 August 2018

Views: Downloads:

Download PDF

Share This Article

Twitter
Linked In
Facebook

Abstract

Precisely, mutagenesis can introduce mutations into the target gene by using mismatch primers which are partially complementary to the template strand of DNA using polymerase chain reaction (PCR). Oxidoreductase enzymes are generally proteins that involved in oxidation-reduction reactions in biological systems. For this study, primary sequence analyses of oxidoreductase protein from Anoxybacillus sp. SK3-4 was conducted with the aim of generating functional information and theoretically improve catalytic stability of the protein by in-silico mutagenesis. The primary sequence of a novel protein with 386 amino acid residues was analyzed using Expasy-tool for translation of the amino acid sequence into a nucleotide gene sequence. Important catalytic binding sites of the protein were predicted using 3DLigandSite program, Pheres2 and Protein Bioedit servers for generating functional information of the protein. Site-directed mutagenesis (SDM) was used against the novel protein (oxidoreductase), in which two site mutations were created based on rational design. Amino acids; leucine (L) and histidine (H), involved in substrate and metal binding sites in the protein were substituted for isoleucine (I) and arginine (R) i.e. L138I and H280R, to check for significant change in the functional stability of the protein, thereby increasing the efficiency of the enzyme to help speed up the rate of chemical reactions.

Published in	International Journal of Microbiology and Biotechnology (Volume 3, Issue 2)
DOI	10.11648/j.ijmb.20180302.14
Page(s)	51-56
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2018. Published by Science Publishing Group

Keywords

Anoxybacillus sp. SK3-4, Oxidoreductase, Catalytic Stability, Site-Directed Mutagenesis

References

[1]	Michael Mingfu Ling, Brian H. Robinson (1997). Approaches to DNA Mutagenesis: An Overview. Analytical Biochemistry 254, 157–178. Article No. AB972428.
[2]	Wu, S.-M., Blomberg, L. A., and Chan, W.-Y. (1996) BioTech niques 21, 356–362.
[3]	Chen, R. (1999). A general strategy for enzyme engineering. Trends Biotechnol. 17, 344–345.
[4]	Erlich, H. A. (1989). PCR Technology: Principles and Applications for DNA Ampliﬁcation, Stockton Press, New York.
[5]	Zoller, M. J. (1991). Curr. Opin. Biotechnol. 2, 526–531.
[6]	Smith, M. (1985). Annu. Rev. Genet. 19, 423–462.
[7]	Ridong Chen (2001). “Enzyme engineering: rational redesign versus directed evolution”. Trends in Biotechnology. Vol. 19 No.1.
[8]	Chai, Y. Y., et al. (2012). Isolation and characterization of pullulandegrading Anoxybacillus species isolated from Malaysian hot springs. Environmental technology. 33 (11): p. 1231- 1238.
[9]	Goh, K. M., et al. (2013). Recent discoveries and applications of Anoxybacillus. Applied microbiology and biotechnology. 97 (4): p. 1475-1488.
[10]	Jaeger K-E. (2004). Curr Opin Biotechnol15, 269-271.
[11]	Burk MJ. (2003). Adv Synth Catal345, 647-648
[12]	Kirk O, Borchert TV, and Fuglsang CC. (2002). Curr Opin Biotechnol13, 345-351.
[13]	Reid, M. F. & Fewson, C. A. (1994). Molecular characterization of microbial alcohol dehydrogenases, Crit. Rev. Microbiol. 20, 132-56.
[14]	Bruchhaus, I. & Tannich, E. (1994). Purification and molecular characterization of the NAD dependent acetaldehyde/ alcohol dehydrogenase from Entamoeba histolytica, Biochem. J. 303, 7432748.
[15]	Munro AW, Taylor P, and Walkinshaw MD. (2000). Curr Opin Biotechnol11, 369-376.
[16]	Clark, D. P. (1992) Evolution of bacterial alcohol metabolism, in the evolution of metabolic function (Mortlock, R. P., ed.) pp. 1052114, Tellford Press.
[17]	Liu, Y., Shen, W., Shi, G. Y., and Wang, Z. X. (2009). Role of the calcium-binding residues Asp231, Asp233,and Asp438 in alpha amylase of bacillusamyloliquefaciens as revealed by muattional analysis.curr Microbiol, 60, 162-166.
[18]	Obradors N., Elisa CABISCOL, J. AGUILAR1 and J. ROS (1998). Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase from Escherichia coli. Eur. J. Biochem. 258, 272-213.
[19]	Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC bioinformatics. 9 (1): p. 1.
[20]	Kamal Usman, Idris Shehu, Abdulrahman Mohmoud Dogara, Zainab Ladidi Idris (2017). In-Silico Characterization and Mutagenesis of βGlucosidase from Anoxybacillus sp. SK3-4. American Journal of Bioscience and Bioengineering. Vol. 5, No.1, pp. 1-7.doi: 10.11648/j.bio.20170501.11
[21]	Goh Mau Goh, et al., (2012). Trends and Tips in Protein Engineering; A Review. Journal of Technology (sciences and Engineering). 59 Suppl 1, 21–31.
[22]	Allendorf, F. W. (1983). Isolation, gene flow, and genetic differentiation among populations. Genetics and conservation. 18 (3): p. 51-65.
[23]	Campbell, W. H. (1994). Anal. Biochem. 221, 425–428.
[24]	Kobayashi, T., and Inouye, M. (1992). J. Mol. Biol. 226, 931– 933.
[25]	Tu, H. M., and Sun, S. S. M. (1996). BioTechniques 20, 352– 158.
[26]	Rouwendal, G. J. A., Wolbert, E. J. H., Zwiers, L.-H., and Springer, J. (1993). BioTechniques 15, 68–76.
[27]	Kamali N, Tavallaie M, Bambai B, Karkhane AA, Miri M. (2010). Site-directed mutagenesis enhances the activity of NADH-FMN oxidoreductase (DszD) activity of Rhodococcus erythropolis. Biotechnol Lett. 32(7):921-7.
[28]	Hurley, J. H. et al. (1996). Determinants of cofactor specificity in isocitrate dehyrdrogenase: structure of an engineered NADP+ → NAD+ specificity-reversal mutant. Biochemistry 35, 5670–5678.
[29]	Picard, V., Ersdal-Badju, E., Lu, A., and Bock, S. C. (1994). Nucleic Acids Res. 22, 2587–2591.
[30]	Ling, M., and Robinson, B. H. (1995). Anal. Biochem. 230, 167–3977.

Cite This Article

Plain Text BibTeX RIS

APA Style

Musa Ahmed Abubakar, Umar Shittu, Musa Hassan Muhammad, Saif Al-Sheyab Ahmad. (2018). In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4. International Journal of Microbiology and Biotechnology, 3(2), 51-56. https://doi.org/10.11648/j.ijmb.20180302.14

Copy | Download

ACS Style

Musa Ahmed Abubakar; Umar Shittu; Musa Hassan Muhammad; Saif Al-Sheyab Ahmad. In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4. Int. J. Microbiol. Biotechnol. 2018, 3(2), 51-56. doi: 10.11648/j.ijmb.20180302.14

Copy | Download

AMA Style

Musa Ahmed Abubakar, Umar Shittu, Musa Hassan Muhammad, Saif Al-Sheyab Ahmad. In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4. Int J Microbiol Biotechnol. 2018;3(2):51-56. doi: 10.11648/j.ijmb.20180302.14

Copy | Download

@article{10.11648/j.ijmb.20180302.14,
  author = {Musa Ahmed Abubakar and Umar Shittu and Musa Hassan Muhammad and Saif Al-Sheyab Ahmad},
  title = {In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4},
  journal = {International Journal of Microbiology and Biotechnology},
  volume = {3},
  number = {2},
  pages = {51-56},
  doi = {10.11648/j.ijmb.20180302.14},
  url = {https://doi.org/10.11648/j.ijmb.20180302.14},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijmb.20180302.14},
  abstract = {Precisely, mutagenesis can introduce mutations into the target gene by using mismatch primers which are partially complementary to the template strand of DNA using polymerase chain reaction (PCR). Oxidoreductase enzymes are generally proteins that involved in oxidation-reduction reactions in biological systems. For this study, primary sequence analyses of oxidoreductase protein from Anoxybacillus sp. SK3-4 was conducted with the aim of generating functional information and theoretically improve catalytic stability of the protein by in-silico mutagenesis. The primary sequence of a novel protein with 386 amino acid residues was analyzed using Expasy-tool for translation of the amino acid sequence into a nucleotide gene sequence. Important catalytic binding sites of the protein were predicted using 3DLigandSite program, Pheres2 and Protein Bioedit servers for generating functional information of the protein. Site-directed mutagenesis (SDM) was used against the novel protein (oxidoreductase), in which two site mutations were created based on rational design. Amino acids; leucine (L) and histidine (H), involved in substrate and metal binding sites in the protein were substituted for isoleucine (I) and arginine (R) i.e. L138I and H280R, to check for significant change in the functional stability of the protein, thereby increasing the efficiency of the enzyme to help speed up the rate of chemical reactions.},
 year = {2018}
}

Copy | Download

TY  - JOUR
T1  - In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4
AU  - Musa Ahmed Abubakar
AU  - Umar Shittu
AU  - Musa Hassan Muhammad
AU  - Saif Al-Sheyab Ahmad
Y1  - 2018/08/09
PY  - 2018
N1  - https://doi.org/10.11648/j.ijmb.20180302.14
DO  - 10.11648/j.ijmb.20180302.14
T2  - International Journal of Microbiology and Biotechnology
JF  - International Journal of Microbiology and Biotechnology
JO  - International Journal of Microbiology and Biotechnology
SP  - 51
EP  - 56
PB  - Science Publishing Group
SN  - 2578-9686
UR  - https://doi.org/10.11648/j.ijmb.20180302.14
AB  - Precisely, mutagenesis can introduce mutations into the target gene by using mismatch primers which are partially complementary to the template strand of DNA using polymerase chain reaction (PCR). Oxidoreductase enzymes are generally proteins that involved in oxidation-reduction reactions in biological systems. For this study, primary sequence analyses of oxidoreductase protein from Anoxybacillus sp. SK3-4 was conducted with the aim of generating functional information and theoretically improve catalytic stability of the protein by in-silico mutagenesis. The primary sequence of a novel protein with 386 amino acid residues was analyzed using Expasy-tool for translation of the amino acid sequence into a nucleotide gene sequence. Important catalytic binding sites of the protein were predicted using 3DLigandSite program, Pheres2 and Protein Bioedit servers for generating functional information of the protein. Site-directed mutagenesis (SDM) was used against the novel protein (oxidoreductase), in which two site mutations were created based on rational design. Amino acids; leucine (L) and histidine (H), involved in substrate and metal binding sites in the protein were substituted for isoleucine (I) and arginine (R) i.e. L138I and H280R, to check for significant change in the functional stability of the protein, thereby increasing the efficiency of the enzyme to help speed up the rate of chemical reactions.
VL  - 3
IS  - 2
ER  -

Copy | Download

Author Information

Musa Ahmed Abubakar

Department of Science Laboratory Technology, Kano State Polytechnic, Kano, Nigeria
Umar Shittu

Department of Biology, Isa Kaita College of Education, Dutsin-ma, Katsina, Nigeria
Musa Hassan Muhammad

Department of Microbiology & Biotechnology, Federal University Dutse, Gigawa, Nigeria
Saif Al-Sheyab Ahmad

Department of Applied Biological Sciences & Arts, Jordan University of Science and Technology, Irbid, Jordan

Download PDF

Sections

Plain Text BibTeX RIS

APA Style

Musa Ahmed Abubakar, Umar Shittu, Musa Hassan Muhammad, Saif Al-Sheyab Ahmad. (2018). In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4. International Journal of Microbiology and Biotechnology, 3(2), 51-56. https://doi.org/10.11648/j.ijmb.20180302.14

Copy | Download

ACS Style

Musa Ahmed Abubakar; Umar Shittu; Musa Hassan Muhammad; Saif Al-Sheyab Ahmad. In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4. Int. J. Microbiol. Biotechnol. 2018, 3(2), 51-56. doi: 10.11648/j.ijmb.20180302.14

Copy | Download

AMA Style

Musa Ahmed Abubakar, Umar Shittu, Musa Hassan Muhammad, Saif Al-Sheyab Ahmad. In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4. Int J Microbiol Biotechnol. 2018;3(2):51-56. doi: 10.11648/j.ijmb.20180302.14

Copy | Download

@article{10.11648/j.ijmb.20180302.14,
  author = {Musa Ahmed Abubakar and Umar Shittu and Musa Hassan Muhammad and Saif Al-Sheyab Ahmad},
  title = {In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4},
  journal = {International Journal of Microbiology and Biotechnology},
  volume = {3},
  number = {2},
  pages = {51-56},
  doi = {10.11648/j.ijmb.20180302.14},
  url = {https://doi.org/10.11648/j.ijmb.20180302.14},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijmb.20180302.14},
  abstract = {Precisely, mutagenesis can introduce mutations into the target gene by using mismatch primers which are partially complementary to the template strand of DNA using polymerase chain reaction (PCR). Oxidoreductase enzymes are generally proteins that involved in oxidation-reduction reactions in biological systems. For this study, primary sequence analyses of oxidoreductase protein from Anoxybacillus sp. SK3-4 was conducted with the aim of generating functional information and theoretically improve catalytic stability of the protein by in-silico mutagenesis. The primary sequence of a novel protein with 386 amino acid residues was analyzed using Expasy-tool for translation of the amino acid sequence into a nucleotide gene sequence. Important catalytic binding sites of the protein were predicted using 3DLigandSite program, Pheres2 and Protein Bioedit servers for generating functional information of the protein. Site-directed mutagenesis (SDM) was used against the novel protein (oxidoreductase), in which two site mutations were created based on rational design. Amino acids; leucine (L) and histidine (H), involved in substrate and metal binding sites in the protein were substituted for isoleucine (I) and arginine (R) i.e. L138I and H280R, to check for significant change in the functional stability of the protein, thereby increasing the efficiency of the enzyme to help speed up the rate of chemical reactions.},
 year = {2018}
}

Copy | Download

TY  - JOUR
T1  - In-Silico Side-Directed Mutagenesis of Oxidoreductase from Anoxybacillus sp. SK3-4
AU  - Musa Ahmed Abubakar
AU  - Umar Shittu
AU  - Musa Hassan Muhammad
AU  - Saif Al-Sheyab Ahmad
Y1  - 2018/08/09
PY  - 2018
N1  - https://doi.org/10.11648/j.ijmb.20180302.14
DO  - 10.11648/j.ijmb.20180302.14
T2  - International Journal of Microbiology and Biotechnology
JF  - International Journal of Microbiology and Biotechnology
JO  - International Journal of Microbiology and Biotechnology
SP  - 51
EP  - 56
PB  - Science Publishing Group
SN  - 2578-9686
UR  - https://doi.org/10.11648/j.ijmb.20180302.14
AB  - Precisely, mutagenesis can introduce mutations into the target gene by using mismatch primers which are partially complementary to the template strand of DNA using polymerase chain reaction (PCR). Oxidoreductase enzymes are generally proteins that involved in oxidation-reduction reactions in biological systems. For this study, primary sequence analyses of oxidoreductase protein from Anoxybacillus sp. SK3-4 was conducted with the aim of generating functional information and theoretically improve catalytic stability of the protein by in-silico mutagenesis. The primary sequence of a novel protein with 386 amino acid residues was analyzed using Expasy-tool for translation of the amino acid sequence into a nucleotide gene sequence. Important catalytic binding sites of the protein were predicted using 3DLigandSite program, Pheres2 and Protein Bioedit servers for generating functional information of the protein. Site-directed mutagenesis (SDM) was used against the novel protein (oxidoreductase), in which two site mutations were created based on rational design. Amino acids; leucine (L) and histidine (H), involved in substrate and metal binding sites in the protein were substituted for isoleucine (I) and arginine (R) i.e. L138I and H280R, to check for significant change in the functional stability of the protein, thereby increasing the efficiency of the enzyme to help speed up the rate of chemical reactions.
VL  - 3
IS  - 2
ER  -

Copy | Download