Effect of exchange of amino acid residues of the surface region of the PST-01 protease on its organic solvent-stability.

Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Biochemical and Biophysical Research Communications (Impact Factor: 2.28). 08/2007; 358(4):1028-33. DOI: 10.1016/j.bbrc.2007.05.047
Source: PubMed

ABSTRACT The PST-01 protease from an organic solvent tolerant Pseudomonas aeruginosa has high stability and activity in the presence of various organic solvents. The structure gene of the PST-01 protease was amplified by the error-prone PCR method. The mutated proteases were incubated in the presence of acetonitrile. By measuring remaining activities, two kinds of mutated PST-01 proteases of which the stabilities were changed were selected. These mutations hardly changed the profile of the activity and stability at various pHs. Their activity and stability at higher temperatures were slightly lower than those of the wild-type PST-01 protease. The stabilities of the mutated enzymes in the presence of various organic solvents were greatly reduced. In both the mutated PST-01 proteases, amino acids located at the surface of the enzyme had been substituted.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
    The Protein Journal 04/2014; DOI:10.1007/s10930-014-9560-3 · 1.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: One of the major barriers to the use of enzymes in industrial biotechnology is their insufficient stability under processing conditions. The use of organic solvent systems instead of aqueous media for enzymatic reactions offers numerous advantages, such as increased solubility of hydrophobic substrates or suppression of water-dependent side reactions. For example, reverse hydrolysis reactions that form esters from acids and alcohols become thermodynamically favorable. However, organic solvents often inactivate enzymes. Industry and academia have devoted considerable effort into developing effective strategies to enhance the lifetime of enzymes in the presence of organic solvents. The strategies can be grouped into three main categories: (i) isolation of novel enzymes functioning under extreme conditions, (ii) modification of enzyme structures to increase their resistance toward nonconventional media, and (iii) modification of the solvent environment to decrease its denaturing effect on enzymes. Here we discuss successful examples representing each of these categories and summarize their advantages and disadvantages. Finally, we highlight some potential future research directions in the field, such as investigation of novel nanomaterials for immobilization, wider application of computational tools for semirational prediction of stabilizing mutations, knowledge-driven modification of key structural elements learned from successfully engineered proteins, and replacement of volatile organic solvents by ionic liquids and deep eutectic solvents.
    ACS Catalysis 10/2013; 3(12):2823–2836. DOI:10.1021/cs400684x · 7.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Haloperoxidases are oxygenases that catalyze the halogenation of a range of organic compounds without the need for additional high-cost cofactors. Thus, haloperoxidases with high activity and stability are desired for industrial application. In this study, a directed evolution approach was adopted to improve the thermostability of the homodimeric BPO-A1 haloperoxidase from Streptomyces aureofaciens. Among 1000 mutant BPO-A1 haloperoxidases, 2 mutants HT177 and HT507, having higher thermostabilities than the wild-type BPO-A1 haloperoxidase, were obtained by directed evolution. The residual activities of mutants HT177 and HT507 were 2.3- and 5.1-fold higher than that of wild-type BPO-A1, respectively, after incubation at 80 °C for 1 h. In addition, mutant HT177 showed higher stability in organic solvents, such as methanol, ethanol, dimethyl sulfoxide, and N,N-dimethylformamide, than the wild-type BPO-A1 haloperoxidase. Furthermore, mutant HT507 showed higher specific activity. Based on the evaluation of single-amino-acid-substituted mutants, stabilization of the α-helix conformation, substitution of amino acid residues located at the surface of the protein molecule, and enhancement of the interaction between subunits may account for the improvement in thermostability, organic solvent stability, and specific activity. Consequently, the thermostability, organic solvent stability, and specific activity of S. aureofaciens BPO-A1 haloperoxidase were successfully improved by a directed evolution approach.
    Journal of Biotechnology 11/2014; 192. DOI:10.1016/j.jbiotec.2014.10.030 · 2.88 Impact Factor