Genetic Transformation of Bacillus licheniformis by Gene Responsible for α-Amylase Production in Media Contains Sugar Crops Wastes | ||||
| Journal of Agricultural Chemistry and Biotechnology | ||||
| Article 8, Volume 9, Issue 12, December 2018, Page 311-317 PDF (7.78 MB) | ||||
| Document Type: Original Article | ||||
| DOI: 10.21608/jacb.2018.37056 | ||||
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| Authors | ||||
| K. Khaled1; M. Abd El-Aziz2; Omnia Badr3 | ||||
| 1Genet. Dep. Fac. of Agric., Beni-Suef Univ., Egypt | ||||
| 2Genet. Dep. Fac. of Agric., Mansoura Univ., Egypt | ||||
| 3Sugar Crops Institute, ARC, Egypt | ||||
| Abstract | ||||
| The importance of amylase enzymes is attributed to their ability to catalyze hydrolysis of starch and polysaccharides into maltose, glucose and/or maltodextrin for industrial processes. Their thermophilic characteristics are necessary to maintain stability under different conditions. Amylases are produced by, fungi, higher plants and animals. In the present study, thermophilic Bacillus (Bacillus stearothermophilus) isolated from beet pulp during sugar processing was used for cloning α-amylase gene into Bacillus licheniformis. The cloned and sequenced gene was 1827 bp in length. It has been registered in the GenBank (accession no. LC259133.1). This thermophilic amylase, with a molecular weight of 60 kD, was expressed and purified from the recombinant strain and matched to the α-amylase family protein of Bacillus stearothermophilus (GenBank accession no. M57457.1) using the NCBI database. The amino acid sequence of the cloned recombinant enzyme was 98% similar to that of the Bacillus stearothermophilus enzyme in the database. Bacillus strains were cultured and the activity of the enzyme was determined. The purified amylase on sugarcane bagasse and/or sugarbeet pulp media was highly active. Moreover, the modified B. licheniformis surpassed the other strain in cell mass amount and amylase activity on all media. These results confirmed that the gene transferred into B. licheniformis effectively increased amylase activity in this bacterium, while sugarcane wastes increased cell mass amount and enzyme activity. Also, thermostability of α-amylase was not clearly different between modified and original B. licheniformis. These provided evidence that gene transfer to B. licheniformis effectively increased its amylase activity but did not affect its thermostability. The enzyme produced in this study have high thermostability even 90 °C, high thermostability range allows it to be utilized in industrial applications, because their high yield, as well as their time and cost saving. | ||||
| Keywords | ||||
| amylase; Bacillus; Sugar Crops wastes; Sugarcane; Sugarbeet, Cloning, thermostability | ||||
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