Abstract
A Beta-galactosidase-producing bacterium, strain KNOUC114 isolated from a hot spring was identified, and its gene of Beta-galactosidase and properties of the enzyme were studied. The strain KNOUC114 showed typical properties of genus Thermus with phenotypic characteristics of rod-shape (0.2x3.5 um), Gram negative, non-motile, endospore not observed, forming yellow-pigmented colonies, growing aerobically and optimally at 68-70 oC. The strain could grow at the temperature above 80 oC, which is a typical characteristic of Thermus thermophilus. The main cellular fatty acids of KNOUC114 were isobranched-C17:00 and C15:00 fatty acids that are the predomonant acyl chains of the strains of genus Thermus. In pylogenetic analysis based on 16S rDNA sequence, the strain KNOUC114 was finally identified as Thermus thermophilus species, and named as Thermus thermophilus KNOUC114. The Beta-galactosidase gene of KNOUC114 (KNOUC114 Beta-gal) was cloned and expressed in Eschericia coli. KNOUC114 Beta-gal was composed of 1938bp encoding 645 a.a with deduced MW. of 72,784 dalton. The purified recombinant Beta-galactosidase of KNOUC114 (KNOUC114 Beta-gal) reacted optimally at pH 5.7 and 85 oC, possessed good activity at the pH of raw milk and the temperature of HTST for raw milk, and was stable at the temperature of HTST and at the pH of raw milk, meaning that KNOUC114 Beta-gal is suitable to be used for hydrolyzation of lactose in raw milk during HTST pasteurization of raw milk.
Highlights
Thermophilic bacteria can be used in industrial and biotechnological process owing to the production of thermozymes which are active and stable at high temperature
Some thermophilic β-galactosidases have been isolated from thermophilic bacteria of Saccharopolyspora rectivirgula (Nakao et al 1994), Thermoanaerobater ethanolicus(Foknia and Velikodvorskaya (1997), Thermus sp.(Koyama et al 1990; Ohtsu et al 1998; Kang et al 2005), Thermotoga maritima(Kim et al 2004), Alicyclobacillus acidocaldarius (Gul-Guven et al 2007) and Geobacillus stearothermophilus (Soliman 2008), they have not been used in industry yet, and all of commercially available β-galactosidases are optimally active at temperature lower than pasteurization temperature
The majority of Thermus strains have a maximum growth temperature slightly below 80°C (Mania and da Costa 1991), Degryse et al (1978) suggested that strains of Thermus thermophilus could be distinguished from all other species of genus Thermus by their ability to grow at above 80°C, and Thermus thermophilus HB8 was capable of growth at above 80°C (Manania et al 1994)
Summary
Thermophilic bacteria can be used in industrial and biotechnological process owing to the production of thermozymes which are active and stable at high temperature. Thermus (Otsu et al 1998), and Geobacillus (Soliman 2008) were widely investigated for the thermostable and thermophilic enzymes useful in industry, and archaea such as Pyrococcus (Wanarska et al 2005) and Sulfolobus (Pisani et al 1990) were studied. An extremely thermostable β-galactosidase produced by a hyperthermophilic archaea of Pyrococcus woesei active up to 110°C and optimally at 93°C was reported (Dabrowski et al 1998). As an effort to find a suitable β-galactosidase fit for operation at pasteurization temperature in dairy industry, we isolated a thermophilic bacterium, strain KNOUC114 growing well at 70°C and showing excellent β-galactosidase activity (Nam et al 2004).
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