Overproduction of Xylanase from Mutants of Bacillus subtilis with Barley Husk as the Prime Carbon Source under Submerged Fermentation after Random Mutagenesis Using Ethyl Methane Sulfonate (EMS) and Acridine Orange (AO)

Aims: Xylanase (EC 3.2.1.8) also known as endo-1,4-β-xylanohydrolase is a type of hydrolytic enzyme participated in the hydrolysis of hemicelluloses particularly in xylan to generate xylose and xylo-oligosaccharides. Due to its enormous potentials, xylanase is frequently used in biobleaching of kraft pulp, clarification of fruit juice, extraction of plant oils, processing of animal feeds, softening of fruits, degradation of agricultural wastes and plant fibers and manufacturing of chemicals including biofuel, ethanol and xylitol. These applications of xylanase avoid the use of chemicals that are expensive, mutagenic and highly non-biodegradable. Interestingly, in recent years, the applications of xylanase in biobleaching and bioprocessing of paper pulp have gained numerous attentions and interests in the industry over the world. Therefore, couple of lignocellulolytic substrate as the alternative cheap carbon source and strain improvement for overproduction of microbial xylanase is implemented as a more potent approach in improving its yield and productivity in submerged fermentation. As a result, the main aim of the present study was primarily involved in the overproduction of xylanase by five mutant strains of Bacillus subtilis subsp. spizizenii ATCC 6633 designated as the MXB 1, MXB 2, MXB 3, MXB 4 and MXB 5 in submerged fermentation using barley husk as the prime carbon source.

Methodology: In order to attain the mutants, B. subtilis was subjected to random mutagenesis using ethyl methane sulfonate (EMS) and acridine orange (AO) in the earlier study before screened for the overproduction of xylanase in the present investigation.

Results: Based on the present investigation, mutant strains of B. subtilis ATCC 6633 were identified as the potent xylanase producers using cheap agro-industrial residue of barley husk as the sole carbon source under submerged fermentation. Furthermore, extracellular protein production and profile of medium pH during growth of wild type and mutants of B. subtilis under submerged fermentation were also elucidated. Based on the result findings, the time course of xylanase biosynthesis by the mutants of B. subtilis revealed that the enzyme production was initiated from the logarithmic to stationary growth phase whereby the maximum xylanase activity was achieved after 24 h of fermentation. In fact, all mutant strains of B. subtilis were successfully synthesized relatively higher production of xylanase than their parental wild type in submerged fermentation using barley husk as the prime carbon source. Notably, the maximum xylanase activity of 1.76±0.02 U/mL was attained by the mutant MXB 4 of B. subtilis which was approximately 29.4% increase in xylanase activity than the wild type with 1.36±0.003 U/mL. Furthermore, MXB 1, MXB 2, MXB 3 and MXB 5 also exhibited comparatively higher maximum xylanase activity of 1.64±0.009 U/mL, 1.73±0.05 U/mL, 1.74±0.02 U/mL and 1.66±0.02 U/mL compared to their parental wild type. Indeed, the statistical single factor analysis of variance (ANOVA) on xylanase production revealed there was significant difference in the mean of the xylanase production by the wild type and mutant strains of B. subtilis (p<0.05). On the other hand, the total maximum extracellular protein production was achieved by mutant strains of MXB 2 and MXB 4 with 0.82±0.02 mg/mL and 0.82±0.03 mg/mL, respectively. Both demonstrated increment of 49.1% in protein production than the wild type which possessed relatively lower concentration of 0.55±0.01 mg/mL. Besides that, the profile of medium pH on xylanase activity by mutants and wild type of B. subtilis was also elucidated in the present study. The highest xylanase activity was attained at slight acidic pH of 6.1±0.2 as shown by the mutant MXB 4 in comparison with wild type at pH 6.47±0.3.

Conclusion: In a nutshell, the result findings suggested the mutant strains of B. subtilis ATCC 6633 particularly MXB 4 as the most potent xylanase producer under submerged fermentation using barley husk as the prime carbon source. Mutant MXB 4 of B. subtilis is anticipated to be beneficial in various xylanase applications especially in the processing of animal feeds and food industry.