Detection and Analysis of the Random Mutagenesis Site(s) of Xylanase Gene from Mutants of Bacillus subtilis subsp. spizizenii ATCC 6633

Aims: A total of five mutant strains of Bacillus subtilis subsp. spizizenii ATCC 6633 designated as the MXB 1, MXB 2, MXB 3, MXB 4 and MXB 5 were developed using random mutagenesis of ethyl methane sulfonate (EMS) and acridine orange (AO) in our previous study. Based on our present investigation, we identified, verified and sequenced xylanase gene of mutant strains of B. subtilis ATCC 6633 as the potent bacterial xylanase producers under submerged fermentation. Furthermore, amino acid analysis and comparison between the xylanases of the mutants and other xylanolytic bacteria were also elucidated. Overall, this study would provide gene and protein molecular information correlating nucleotide and amino acid structure related to the increased xylanase production by random mutagenesis. In respect to the objectives of this study, we compared the endoxylanase sequence of wild type B. subtilis ATCC 6633 with its mutants in order to determine their possible site(s) of mutagenesis and to analyse amino acid xylanase sequence of the mutants of B. subtilis ATCC 6633. Methodology: After the verification of xylanase production by all mutants of B. subtilis ATCC 6633 Original Research Article Ling Ho and Chinonso; BBJ, 12(1): 1-20, 2016; Article no.BBJ.23057 2 on the xylan agar using Congo-red staining in the previous study, xylanase gene of the mutants was amplified from the genomic DNA to detect the mutagenesis site(s) by synthesizing primers directed against the sequence of xylanase gene obtained from the wild type of Bacillus subtilis subsp. spizizenii ATCC 6633. Results: The comparison of xylanase genes from different mutants of B. subtilis and the wild type revealed the site(s) of mutagenesis. Interestingly, the mutations of the mutants of B. subtilis ATCC 6633 in this study were significantly reflected at the 5’ end of the mutants xylanase genes. The open reading frames (ORF) of the mutant xylanase genes ranged from 644 bp to 684 bp with translated encoding protein between 214 and 228 amino acid residues were obtained. On the other hand, predicted molecular mass from 23.94 kDa to 25.40 kDa and theoretical pI which ranged from 8.63 to 9.16 were attained from all of the mutant strains in this study. Based on the characteristics obtained, the mutant xylanases were suggested to belong to Glycosyl Hydrolase (GH) Family 11 with 98% homology to endo-1,4-beta-xylanase of B. subtilis subsp. spizizenii of W23. In fact, conserved regions, signal peptide, a cleavage site between the Ala28 and Ala29 residues and four Tyr residues specific to GH Family 11 xylanase were also observed and detected in all mutants. Indeed, two conserved glutamate residues of E94 and E183 that directly involved in the enzyme catalytic mechanism were also detected in the amino acid sequences of the mutants. The analysis of the deduced amino acid sequences revealed that the mutations in the signal peptide regions fostered increased hydrophobic core of xylanase residue. We suggested that these changes would probably be responsible for the increased extracellular xylanase yield in the mutants of B. subtilis. On the other hand, all the mutants of B. subtilis ATCC 6633 exhibited the tendency to be thermostable based on the Val, Ser and Thr frequency ratios which were almost identical to those of thermophiles. Furthermore, the increase of Thr to Ser ratio and presence of Arg residue found in the mutant strain of MXB 5 would enhance the polar interactions and hence improve the secondary structure stabilization that was usually one of the determining factors in the thermophilic proteins. Conclusion: In a nutshell, the properties of B. subtilis mutant xylanases particularly mutant MXB 5 revealed its relevant potential in the biotechnology applications in bio-bleaching, textile, paper and pulp industries that commonly require high temperature usage in xylanase applications.