Response Surface Methodology for the production of endopolygalacturonase by a novel Bacillus licheniformis

Saqib Hussain Hadri, Muhammad Javaid Asad, Muhammad Zeeshan Hyder, Syed Muhammad Saqlan Naqvi, Tariq Mukhtar, Raja Tahir Memhmood, J. H. David Wu


Background: Polygalacturonase is one of the most important commercial pectinase. The production cost and the mesophilic nature of the present polygalacturonase is a big problem in its application in the juice industry. A lot of work is going on for the isolation of thermophilic bacterial strains which can utilize pectin as the only carbon source.

Methods: Bacterial strains were isolated from rotten fruits and vegetables and cultured at 50 – 70oC. The strains were than screened for endopolygalacturonase activity and identified on the basis of 16S rRNA sequence. Different growth parameters for the production of endopolygalacturonase by Bacillus licheniformis IEB-8 were optimized using Response Surface Methodology under Center Composite Design using JMP-12 software. Endopolygalacturonase was purified in two steps; ammonium sulfate precipitation and then by size exclusion column chromatography.

Results: Only four strains, IEB-8, IEB-11, IEB-12 and IEB-13 showed growth above 60oC. Among these four, only IEB-8 was found to be endopolygalacturonase positive, which was identified as Bacillus licheniformis by 16S rRNA gene sequencePurification fold of 2.57 and 7.48 in the specific activity were achieved using ammonium sulfate precipitation and gel filtration chromatography respectively. Molecular weight of the purified endopolygalacturonase was found to be 42 kDa. The purified endopolygalacturonase showed an optimum pH of 7 and optimum temperature of 55oC.

Conclusion: Bacillus licheniformis IEB-8 is a novel bacteria which can efficiently be utilized in the industry for the production of endopolygalacturonase very cheaply. Furthermore, the high optimum working temperature of endopolygalacturonase, increases its significance for its industrial applications.

Keywords: Endopolygalacturonase; Bacillus licheniformis; Thermophilic; Response Surface Methodology; Ammonium sulfate precipitation

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