الفهرس 
Production of Polyhydroxybutyrate by Locally Isolated Bacterial Strain, Cloning and Expression of the Corresponding Genes into Escherichia coli
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Abstract
Polyhydroxybutyrate (PHB) is¬ a natural biodegradable polymer which can act as a good substitute to petrochemical-based plastics. The screening was done using 38 bacterial isolates where 15 isolates were found to be positive for PHA production. The bacterial isolate 6N-NRC was found to be the best producer of PHA. The extracted PHA was examined using NMR which confirmed that it belongs to polyhydroxybutyrate (PHB) category. Molecular characterization of the selected bacterial isolate (6N-NRC) revealed that it is Bacillus aryabhattai. This isolate was submitted to the NCBI Gene Bank with nucleotide sequence database under accession no. MH997667.1. The optimization of fermentation processes and strains for production of PHB from economic carbon source is required for the competition with synthetic plastics. Shaken flask experiments were conducted where the culture medium and growth parameters were optimized using one factor at a time as well as multifactorial experimental design (Plackett-Burman and Box-Behnken). Beet molasses was utilized as an economic substrate for the production of PHB and several methods were applied to make it bioavailable for the local bacterial isolate, Bacillus aryabhattai MH997667.1. Beet molasses (30 g/l) as the carbon source and ammonium chloride (0.75 g/l) as the nitrogen source were observed to be the best sources of nutrients for the optimum production of PHB. Incubation period 36 h, pH 8.0 and temperature 30ºC were found to be the best conditions for obtaining optimum PHB yield of 3.799 g/l. Batch fermentation was conducted using 5L-bench top bioreactor in order to control the process parameters. The optimum agitation speed was 250 rpm. The highest yield of PHB (2.3 g/l) was obtained at 24 h fermentation period. Logistic equation and Luedeking-Piret equation were used to describe biomass growth and PHB production accurately. MATLAB software 2019 was used to simulate the experimental process and the simulated data showed a good fit with the experimental results obtained during the first 24 h of PHB production at 250 rpm. The present work also aimed to study the genes responsible for the production of polyhydroxybutyrate (PHB) like acetoacetyl-CoA reductase (phbB) and polyhydroxybutyrate synthase (phbC). Appropriate primers were designed for phbB and phbC PCR approach. The phbB (744 bp) and phbC (1089 bp) genes were successfully isolated, identified and cloned with the pET-29a(+) plasmid vector. Transformation of Escherichia coli BL21 was performed. The amplification of the phbB and phbC genes using specific primers of pET-29a(+) plasmid was done. Based on DNA sequencing, the open reading frame of phbB sequence was found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of B. aryabhattai (GenBank accession no. CP024035.1), while the open reading frame of phbC sequence was found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of B. aryabhattai (GenBank accession no. CP024035.1). The analysis of the recombinant proteins from E. coli BL21 recombinant colony by tricine-polyacrylamide gel electrophoresis clarified that the expressed phbB and phbC genes in E. coli BL21 strain showed distinct bands of molecular weights 26.3 KD and 37.5 KD, respectively.
Keywords:
Polyhydroybutyrate, Bacillus aryabhattai, Beet molasses, Cloning, Expression, Polyhydroxybutyrate genes, Escherichia coli.