Login Register Cart 0
Generic placeholder image

Recent Patents on Biotechnology

Editor-in-Chief

ISSN (Print): 1872-2083
ISSN (Online): 2212-4012

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Random Mutagenesis of Thermophilic Xylanase for Enhanced Stability and Efficiency Validated through Molecular Docking

Author(s): Shweta Chauhan, Varun Jaiswal, Chandrika Attri and Amit Seth*

Volume 14, Issue 1, 2020

Page: [5 - 15] Pages: 11

DOI: 10.2174/1872208313666190719152056

Price: $65

Abstract

Background: Xylanases of thermophilic origin are more robust and stable and hence more suitable for industrial applications. The aim of the research was to develop a patent using a robust mutant exhibiting enhanced xylanase activity. The strain (Bacillus aestuarii SC-2014) subjected to mutagenesis is thermophilic in origin and hence it is envisioned that the enhancement of its catalytic potential will enhance its industrial applicability.

Objective: The main aim was to develop a stable and vigorous mutant having higher xylanase activity and improved thermostability.

Methods: The bacterial strain isolated from the Tattapani hot springs of Himachal Pradesh (India) was mutagenized by single separate exposure of Ethyl methane sulphonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (MNNG).

Results: A mutant library was generated and extensive screening led to the identification of the most potent mutant strain selected and designated as Bacillus sp. SC-2014 EMS200 (MTCC number 25046) which displayed not only enhanced xylanase activity and thermo stability but also appreciable genetic stability. This strain displayed a 3-fold increase in enzyme activity and simultaneously, a significant reduction in fermentation time from 72 h to 48 h was also observed. The xylanase gene from wild and mutant strain was cloned, sequenced and subjected to molecular docking. Two mutations H121D and S123T were present inside the binding pocket.

Conclusion: Mutation H121D made the binding pocket more acidic and charged, thus enhancing the xylanase activity for mutant protein. Mutations also resulted in charged amino acids (Y99K and H121D) which were identified as a probable cause for enhancing the thermostability of mutant protein.

Keywords: Xylanase, EMS, random mutagenesis, homology modelling, forward genetics, thermostability.

« Previous Next »
Graphical Abstract

[1]
Goswami GK, Rawat S. Microbial xylanase and their applications - A review. Int J Curr Res Acad Rev 2015; 3: 436-50.
[2]
Chauhan S, Seth CA, Seth A. Bioprospecting thermophilic microorganisms from hot springs of Western Himalayas for xylanase production and its statistical optimization by using response surface methodology. J Pure Appl Microbiol 2015; 9: 1417-28.
[3]
Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959; 31: 426-8.
[http://dx.doi.org/10.1021/ac60147a030]
[4]
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press 1989.
[5]
Huang J, Wang G, Xiao L. Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli. Bioresour Technol 2006; 97(6): 802-8.
[http://dx.doi.org/10.1016/j.biortech.2005.04.011] [PMID: 15951169]
[6]
Thompson JD, Gibson T, Higgins DG. Multiple sequence alignment using Clustal W and ClustalX. Curr Protoc Bioinformatics 2002; 2(2)
[http://dx.doi.org/10.1002/0471250953.bi0203s00]
[7]
Eswar N, Webb B, Marti-Renom M, Madhusudhan MS, Eramian D, Shen MY, et al. Comparative protein structure modeling using modeller. Curr Protoc Bioinfor 2006; 5(5.6) doi: 10.1002/0471250953.bi0506s15
[8]
Yu J, Zhou Y, Tanaka I, Yao M. Roll: a new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics 2010; 26(1): 46-52.
[http://dx.doi.org/10.1093/bioinformatics/btp599] [PMID: 19846440]
[9]
Wallace AC, Laskowski RA, Thornton JM. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 1995; 8(2): 127-34.
[http://dx.doi.org/10.1093/protein/8.2.127] [PMID: 7630882]
[10]
Hilhorst R, Gruppen H, Orsel C, Laane C, Schols HA, Voragen AGJ. Effects of xylanase and peroxidase on soluble and insoluble arabinoxylans in wheat bread dough. J Food Sci 2006; 67(2): 497-506.
[http://dx.doi.org/10.1111/j.1365-2621.2002.tb10626.x]
[11]
Primo-Martin C, Wang M, Lichtendonk WJ, Plijter JJ, Hamer RJ. An explanation for the combined effect of xylanase-glucose oxidase in dough systems. J Sci Food Agric 2005; 85(7): 1186-96.
[http://dx.doi.org/10.1002/jsfa.2107]
[12]
Sangkharak K, Vangsirikul P, Janthachat S. Strain improvement and optimization for enhanced production of cellulase in Cellulomonac Sp. TSU-03. Afr J Microbiol Res 2012; 6: 1079-84.
[13]
Chandra M, Kalra A, Sangwan NS, Gaurav SS, Darokar MP, Sangwan RS. Development of a mutant of Trichoderma citrinoviride for enhanced production of cellulases. Bioresour Technol 2009; 100(4): 1659-62.
[http://dx.doi.org/10.1016/j.biortech.2008.09.011] [PMID: 18951016]
[14]
Sriprang R, Asano K, Gobsuk J, Tanapongpipat S, Champreda V, Eurwilaichitr L. Improvement of thermostability of fungal xylanase by using site-directed mutagenesis. J Biotechnol 2006; 126(4): 454-62.
[http://dx.doi.org/10.1016/j.jbiotec.2006.04.031] [PMID: 16757052]
[15]
Zhang ZG, Yi ZL, Pei XQ, Wu ZL. Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis. Bioresour Technol 2010; 101(23): 9272-8.
[http://dx.doi.org/10.1016/j.biortech.2010.07.060] [PMID: 20691586]
[16]
Pratush A, Seth A, Bhalla TC. Generation of mutant of Rhodococcus rhodochrous PA-34 through chemical mutagenesis for hyperproduction of nitrile hydratase. Acta Microbiol Immunol Hung 2010; 57(2): 135-46.
[http://dx.doi.org/10.1556/AMicr.57.2010.2.6] [PMID: 20587386]
[17]
Pratush A, Seth A, Bhalla TC. Cloning, sequencing, and expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in E. coli. Appl Biochem Biotechnol 2012; 168(3): 465-86.
[http://dx.doi.org/10.1007/s12010-012-9790-9] [PMID: 22833401]
[18]
Pratush A, Seth A, Bhalla TC. Expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA-34 in Pichia pastoris. Biocatal Biotransform 2017; 35(1): 19-26.
[http://dx.doi.org/10.1080/10242422.2016.1247831]
[19]
Akira Abe, Shunichi Kosugi, Kentaro Yoshida, Satoshi Natsume, Hiroki Takagi, H,iroyuki Kanzaki, et al. Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol 2012; 30(2): 174-8.
[http://dx.doi.org/10.1038/nbt.2095] [PMID: 22267009]
[20]
Kumari R, Pramanik K. Improvement of multiple stress tolerance in yeast strain by sequential mutagenesis for enhanced bioethanol production. J Biosci Bioeng 2012; 114(6): 622-9.
[http://dx.doi.org/10.1016/j.jbiosc.2012.07.007] [PMID: 22867797]
[21]
Uchida N, Sakamoto T, Kurata T, Tasaka M. Identification of EMS-induced causal mutations in a non-reference Arabidopsis thaliana accession by whole genome sequencing. Plant Cell Physiol 2011; 52(4): 716-22.
[http://dx.doi.org/10.1093/pcp/pcr029] [PMID: 21398646]
[22]
Ross CD, Lim CU, Fox MH. Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry. Cytometry A 2005; 66(2): 85-90.
[http://dx.doi.org/10.1002/cyto.a.20168] [PMID: 16003719]
[23]
Murakami MT, Arni RK, Vieira DS, Degrève L, Ruller R, Ward RJ. Correlation of temperature induced conformation change with optimum catalytic activity in the recombinant G/11 xylanase A from Bacillus subtilis strain 168 (1A1). FEBS Lett 2005; 579(28): 6505-10.
[http://dx.doi.org/10.1016/j.febslet.2005.10.039] [PMID: 16289057]
[24]
Modolo LV, Escamilla-Treviño LL, Dixon RA, Wang X. Single amino acid mutations of Medicago glycosyltransferase UGT85H2 enhance activity and impart reversibility. FEBS Lett 2009; 583(12): 2131-5.
[http://dx.doi.org/10.1016/j.febslet.2009.05.046] [PMID: 19500551]
[25]
Kumwenda B, Litthauer D, Bishop OT, Reva O. Analysis of protein thermostability enhancing factors in industrially important thermus bacteria species. Evol Bioinform Online 2013; 9: 327-42.
[http://dx.doi.org/10.4137/EBO.S12539] [PMID: 24023508]
[26]
Okuma J. Thermostable xylanase belonging to GH family 10 US9732366B2. 2017.
[27]
Fenel F. Method to improve the stability and broaden the pH range of family G/11 xylanases US9481874B2. 2016.
[28]
Tan X. Genes with codon mutations encoding xylanase US10131895B2. 2018.
[29]
Sung WL. Modifications of xylanases to increase thermophilicity, thermostability and alkalophilicity US8927248B2. 2015.
[30]
Reisinger C. Polypeptides having xylanase activity with a high conversion rate of xylose containing polysaccharides US0327809A1. 2017.
[31]
Yanai H. Mutant xylanase, manufacturing method and use thereof and method for manufacturing saccharified lignocellulose US9567617B2. 2017.

Rights & Permissions Print Cite
Article Metrics
6
© 2024 Bentham Science Publishers | Privacy Policy