Effect of exchange 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: 2.3). 08/2007; 358(4):1028-33. DOI: 10.1016/j.bbrc.2007.05.047
Source: PubMed


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.

6 Reads
  • Source
    • "Published studies of the mechanism of adaptation of enzymes to function in organic solvent are relatively few. Ogino et al. [116,117] investigated the mechanism of organic solvent tolerance in a Pseudomonas aeruginosa PST-01 protease by site-directed and random mutagenesis. They reported that the disulfide bonds and amino acid residues located on the surface of the molecule play important roles in organic solvent stability of the enzymes. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology.
    Aquatic Biosystems 02/2012; 8(1):4. DOI:10.1186/2046-9063-8-4
  • Source
    • "Ghorbel et al. isolated a protease from Bacillus cereus BG1 which retained 89.5 of its original activity, after 15-min incubation at 55°C, in the presence of 2 mM Ca2+; meanwhile, no activity was detected in the absence of Ca2+ [12]. An organic solvent-stable protease from Pseudomonas aeruginosa PST-01 was reported to be stable at the temperature below 50°C [23]. A solvent stable protease, from Pseudomonas aeruginosa PseA retained 80% of its initial activity after heating, for 30 min at 55°C [11]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Many researchers have reported on the optimization of protease production; nevertheless, only a few have reported on the optimization of the production of organic solvent-tolerant proteases. Ironically, none has reported on thermostable organic solvent-tolerant protease to date. The aim of this study was to isolate the thermostable organic solvent-tolerant protease and identify the culture conditions which support its production. The bacteria of genus Bacillus are active producers of extra-cellular proteases, and the thermostability of enzyme production by Bacillus species has been well-studied by a number of researchers. In the present study, the Bacillus subtilis strain Rand was isolated from the contaminated soil found in Port Dickson, Malaysia. A thermostable organic solvent-tolerant protease producer had been identified as Bacillus subtilis strain Rand, based on the 16S rRNA analysis conducted, as well as the morphological characteristics and biochemical properties. The production of the thermostable organic solvent-tolerant protease was optimized by varying various physical culture conditions. Inoculation with 5.0% (v/v) of (AB600 = 0.5) inoculum size, in a culture medium (pH 7.0) and incubated for 24 h at 37 degrees C with 200 rpm shaking, was the best culture condition which resulted in the maximum growth and production of protease (444.7 U/ml; 4042.4 U/mg). The Rand protease was not only stable in the presence of organic solvents, but it also exhibited a higher activity than in the absence of organic solvent, except for pyridine which inhibited the protease activity. The enzyme retained 100, 99 and 80% of its initial activity, after the heat treatment for 30 min at 50, 55, and 60 degrees C, respectively. Strain Rand has been found to be able to secrete extra-cellular thermostable organic solvent-tolerant protease into the culture medium. The protease exhibited a remarkable stability towards temperature and organic solvent. This unique property makes it attractive and useful to be used in industrial applications.
    Microbial Cell Factories 05/2009; 8(1):20. DOI:10.1186/1475-2859-8-20 · 4.22 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: First Page of the Article
    1974 Ultrasonics Symposium; 02/1974
Show more