title: Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.
abstract: Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.
authors.affiliation: Institute of Industrial Biotechnology, GC University, Lahore, Pakistan.
authors.forename: Irfana
authors.initials: I
authors.lastname: Iqbal
authors.forename: Muhammad Nauman
authors.initials: MN
authors.lastname: Aftab
authors.forename: Mohammed
authors.initials: M
authors.lastname: Afzal
authors.forename: Asad
authors.initials: A
authors.lastname: Ur-Rehman
authors.forename: Saima
authors.initials: S
authors.lastname: Aftab
authors.forename: Asma
authors.initials: A
authors.lastname: Zafar
authors.forename: Zia
authors.initials: Z
authors.lastname: Ud-Din
authors.forename: Ateeque Rahman
authors.initials: AR
authors.lastname: Khuharo
authors.forename: Jawad
authors.initials: J
authors.lastname: Iqbal
authors.forename: Ikram
authors.initials: I
authors.lastname: Ul-Haq
mesh_terms: D020001:1-Butanol / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D020134:Catalytic Domain; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005285:Fermentation; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D057057:Serine Proteases / Q000737:chemistry; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D020001:1-Butanol; D010450:Endopeptidases; D057057:Serine Proteases; C087822:alkaline protease
keywords: Alkaline serine protease; Characterization; Geobacillus stearothermophilus; Purification; Thermostable