[show abstract][hide abstract] ABSTRACT: cis-6-Hexadecenoic acid is a major component of human sebaceous lipids that is involved in skin self-sterilization and atopic dermatitis amelioration. It can be prepared by hydrolysis of isopropyl cis-6-hexadecenoate produced by resting cells of Rhodococcus sp. strain KSM-MT66. To devise an economical industrial-scale process for the production of this rare fatty acid, we optimized the conditions for growing rhodococcal cells. Mg(2+) and Fe(2+) ions are essential for the efficient production of isopropyl cis-6-hexadecenoate. To further increase the production of isopropyl cis-6-hexadecenoate, we created a mutant strain (T64) with reduced esterase activity by random mutagenesis using UV irradiation of MT66. Under an optimized condition, the mutant T64 produced more than 60 g l(-1) isopropyl cis-6-hexadecenoate in a 4-d cultivation, corresponding to about 52 g l(-1)cis-6-hexadecenoate.
Journal of Bioscience and Bioengineering 11/2007; 104(4):300-3. · 1.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: alpha-Amylase (LAMY) from alkaliphilic Bacillus sp. strain KSM-1378 is a novel semi-alkaline enzyme which has 5-fold higher specific activity than that of a Bacillus licheniformis enzyme. The Arg124 in LAMY was replaced with proline by site-directed mutagenesis to increase thermostability of the enzyme. The wild-type and engineered LAMYs were very similar with respect to specific activity, kinetic values, pH-activity curve, and degree of inhibition by chelating reagents. Thermostability and structure stiffness of LAMYs as measured by fluorescence were increased by the proline substitution. The change of Arg124 to proline is assumed to stabilize the loop region involving amino acid residues from 122 to 134. This is the first report that thermostability of an alpha-amylase is improved by proline substitution.
Bioscience Biotechnology and Biochemistry 10/1999; 63(9):1535-40. · 1.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).
Bioscience Biotechnology and Biochemistry 10/1998; 62(9):1720-5. · 1.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: alpha-Amylase from alkaliphilic Bacillus KSM-1378 (LAMY) is a novel semi-alkaline enzyme which has a high specific activity, a value 5-fold higher than that of a Bacillus licheniformis enzyme at alkaline pH. Thermostability of this enzyme could be improved by deletion of the Arg181-Gly182 residue by means of site-directed mutagenesis. The wild-type and engineered LAMYs were very similar with respect to specific activity, pH-activity curve, temperature-activity curve, susceptibility to inhibitors, and pattern of hydrolysis products from soluble starch and maltooligosaccharides. However, the engineered enzyme also acquired increased pH stability and resistance to sodium dodecyl sulfate and especially chelating reagents, such as ethylenediaminetetraacetate and ethyleneglycol-bis (beta-aminoethylether)tetraacetate. This is the first report that thermostability of alpha-amylase is improved by enhanced calcium binding to the enzyme molecule.
Biochemical and Biophysical Research Communications 08/1998; 248(2):372-7. · 2.41 Impact Factor