Expression, purification, and molecular characterization of a full-length thermostable alkaline protease gene from Bacil
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ORIGINAL ARTICLE
Expression, purification, and molecular characterization of a full-length thermostable alkaline protease gene from Bacillus subtilis DMA-09 Dilara Abbas Bukhari 1 & Amina Barkat 1 & Abdul Rehman 2 Received: 1 June 2020 / Accepted: 16 September 2020 # Institute of Molecular Biology, Slovak Academy of Sciences 2020
Abstract The present study aim was to isolate bacterial strains, producing protease, from soil samples of different industrial wastes and dumping areas of slaughter houses. For this, 25 bacterial isolates forming clear zones in skimmed milk agar plates as a result of casein hydrolysis were selected. One bacterial isolate showing the highest protease activity (400 U/ml) was identified as Bacillus subtilis through 16S rRNA gene sequencing. Full-length alkaline protease gene was amplified, sequenced, cloned in expression vector pT7-7, and transformed in E. coli BL21C. Over-expression of protease gene was determined in isopropyl β-d-1thiogalactopyranoside (0.5 mM) at 37 °C for 3 h. Expressed alkaline protease was purified and 10 folds increased enzyme activity was determined as compared to the wild type B. subtilis DMA-09. The purified protease was detected on SDS-PAGE as a single band of 31 kDa and was stable at 90 °C and 85% enzyme activity was retained at 100 °C. The optimum enzyme activity was obtained at pH 10 and was enhanced in the presence of 5 mM each Mg2+ and Ca2+ while phenylmethylsulfonyl fluoride (PMSF) inhibited its activity at 0.5 mM. The expressed purified alkaline protease may find potential applications in food and biotechnological industries. Keywords Bacillus subtilis DMA-09 . alkaline protease . Cloning . Purification
Abbreviations SK Skimmed milk LBUV Luria–Bertani NaCl Sodium chloride DNA Deoxy ribonucleic acid IPTG Isopropyl-β-D-thiogalactopyranoside dNTPs deoxyribonucleotide triphosphates PCR Polymerase chain reaction Electronic supplementary material The online version of this article (https://doi.org/10.2478/s11756-020-00608-6) contains supplementary material, which is available to authorized users. * Abdul Rehman [email protected] Dilara Abbas Bukhari [email protected]
NCBI BLAST OD CM NaOH KCl MgCl2 EDTA PMSF pCMB DFP kDa ORF SDS PAGE mL M
National center for biotechnology information Basic local alignment search tool Optical density Carboxymethyl Sodium hydroxide Potassium chloride Magnesium chloride Ethylenediaminetetraacetic acid Phenylmethylsulfonyl fluoride p-chloromercuribenzoate Di-isopropyl fluorophosphates Kilodalton Open reading frame Sodium dodecyl sulphate Polyacrylamide gel electrophoresis Milliliter Molar
Amina Barkat [email protected] 1
Department of Zoology, Government College University Lahore, Lahore, Pakistan
2
Department of Microbiology and Molecular Genetics, University of the Punjab, New Campus, Lahore 54590, Pakistan
Introduction Proteases are degradative enzymes, which catalyze the hydrolysis of peptide bonds that link amino acids together in the
Biologia
polypeptide chain forming protein (Gupta and Lorenz 2010). Pr
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