Cloning and Expression of Subtilisin Amylosacchariticus Gene1

Abstract

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.

Complex between the subtilisin from a mesophilic bacterium and the Leech inhibitor eglin-C

“…The amino-acid sequences of several Bacilli subtilisins have been determined to date including subtilisin NOVO or BPN' (the same protein: SBPN) (Markland & Smith, 1967), SBCARL (Smith, De Lange, Evans, Landon & Markland, 1968), SBAS (Kurihara, Markland & Smith, 1972;Yoshimoto, Oyama, Honda, Tone, Takeshita, Kamiyama & Tsuru, 1988), SBDY (Nedkov, Oberthiir & Braunitzer, 1983) and SBI168 (Stahl & Ferrari, 1984). In addition several three-dimensional structures have been determined by X-ray diffraction.…”

“…They are numbered according to that of SBPN as is conventional, and are given in full to clear up some confusions from the published sequence data and the coordinates in the Brookhaven Protein Data Bank. The sequences given for BI168 (Stahl & Ferrari, 1984), SBAS (Yoshimoto et al, 1988), SBPN (Wells, Ferrari, Henner, Estell &Chen, 1983;Vasantha, Thompson, Rhodes, Banner, Nagle & Filpula, 1984) and SBCARL (Jacobs, Eliasson, Uhlen & Flock, 1985) are those for the corresponding genes. These differ somewhat from those reported for direct sequencing of the protein for SBAS (Kurihara et al, 1972), SBPN (Markland & Smith, 1967) and SBCARL (Smith et al, 1968); the direct sequence has not been published for SBI168.…”

“…These differ somewhat from those reported for direct sequencing of the protein for SBAS (Kurihara et al, 1972), SBPN (Markland & Smith, 1967) and SBCARL (Smith et al, 1968); the direct sequence has not been published for SBI168. [The situation is further confused by the extra four errors introduced in quoting the original protein sequence of SBAS by Yoshimoto et al (1988)]. The gene sequences are generally believed to be more accurate, and these are used in Fig.…”

Genetically Engineered Poly-γ-glutamate Producer fromBacillus subtilisISW1214

“…9,10) Intricate regulation of PGA production in B. subtilis 11,12) causes a difficulty in the interpretation of this issue. In this study, to simplify the regulation of PGA production and construct a more convenient PGA producer, genetic alterations were preformed to B. subtilis ISW1214, which is a tetracycline-susceptible derivative from the 1012 strain of B. subtilis R 13,14) and also the leucine/methionine auxotroph. 13) Similarly to B. subtilis 168, 11) B. subtilis ISW1214 is a domestic strain very useful in gene engineering, 13) which harbors no plasmid DNA.…”

“…In this study, to simplify the regulation of PGA production and construct a more convenient PGA producer, genetic alterations were preformed to B. subtilis ISW1214, which is a tetracycline-susceptible derivative from the 1012 strain of B. subtilis R 13,14) and also the leucine/methionine auxotroph. 13) Similarly to B. subtilis 168, 11) B. subtilis ISW1214 is a domestic strain very useful in gene engineering, 13) which harbors no plasmid DNA. 14) It, however, has remained obscure about whether the ISW1214 strain cannot produce PGA as well as the 168 strain.…”