Abstract
The genes for extracellular neutral protease (Npr) and intracellular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple beta-amylases and an alpha-amylase from a large amylase precursor. The npr gene was composed of 1,770 bp and 570 amino acids, while the isp gene was composed of 978 bp and 326 amino acids. Both proteases produced by E. coli cleaved the amylase precursor to generate beta- and alpha-amylases. Furthermore, several other proteases produced the same products from the precursor. A 130-kDa amylase precursor has two large domain structures responsible for the generation of beta- and alpha-amylases. The junction region of approximately 200 amino acids may be exposed on the surface of the molecule and susceptible to proteolytic enzymes, which results in the formation of multiple amylases.
Highlights
The genes for extracellular neutral protease (Npr) and intracelular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple,B-amylases and an a-amylase from a large amylase precursor
The major extracellular proteolytic enzymes are alkaline serine proteases, such as subtilisin produced by B. subtilis [25], and neutral proteases, such as thermolysin produced by B. thermoproteolyticus [28]
To characterize enzymes involved in the proteolytic processing of the amylase precursor into 13- and a-amylases, two protease genes were cloned from B. polymyxa and sequenced: the extracellular neutral protease gene and the intracellular serine protease gene
Summary
The genes for extracellular neutral protease (Npr) and intracelular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple ,B-amylases and an a-amylase from a large amylase precursor. To characterize enzymes involved in the proteolytic processing of the amylase precursor into 13- and a-amylases, two protease genes were cloned from B. polymyxa and sequenced: the extracellular neutral protease gene (npr) and the intracellular serine protease gene (isp). After removal of the precipitate by centrifugation (10,000 rpm, 10 min, 4°C), the dialyzed sample was applied to a column of DEAE-cellulose (2.9 by 8 cm) that had been equilibrated and washed extensively with the same buffer to elute amylases produced simultaneously by B. polymyxa.
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