チュウトウド コウネツセイ サイキン Geobacillus stearothermophilus セイイク オンド ヘンイカブ ニ オケル タンパクシツ ハツゲン カイセキ

Optimum growth of moderately thermophile Geobacillus stearothermophilus (Bst) has been observed at 55ºC, and suppressed by the downshift to lower temperature. In order to understand the relationship between growth temperature and protein expression, we made Bst mutant strains which were adapted to lower temperature (30ºC) compared with wild type strain, and then we examined their protein expression under various growth temperatures. When they were cultured at 55ºC, different expression patterns of six proteins (P1-P6) were observed between wild type and mutant strains, whereas five proteins (A1-A5) were found only in mutant strains cultured at 37ºC. Furthermore, expression of five proteins (DS1-DS5) was increased according to the downshift of growth temperature to 37ºC. Among all 16 proteins, abundant three proteins (P1, DS3, and DS5) were electroblotted to PVDF membrane, followed by analyzed N-terminal amino acid sequences. As a result, it was suggested that P1 must be Surface-layer protein A (sbsA), whereas DS3 and DS5 were unknown proteins

アミトロール テイコウセイ ダイチョウキン カブ ニオケル タンパクシツ ハツゲン カイセキ

It was reported that the E.coli mutant strain exhibited the resistance against amitrole (AT) which had used as a pesticide and inhibits the bacterial growth. In order to elucidate the mechanism on the AT-resistance in E.coli, we prepared the E.coli mutant strain by treating with chemical mutagen, MNNG, and then selected the AT-resistant E. coli strain. The obtained mutant strain was possible for proliferating even in the M9 minimal medium containing 2 mg/ml AT. Therefore, we examined the expressed proteins in the mutant strain, which was cultured under various conditions. As a result, it was suggested the expression of 22 kDa protein (P1) was suppressed as the AT concentration was increased in M9 medium. By the amino acid sequence analysis, it was proved that P1 must be alkyl hydroperoxide reductase C22 protein (ahpC). On the other hand, the expression of P2 protein in mutant strain has increased in the M9 medium containing 2mg/ml AT, and partial amino acid sequence of P2 was consistent with zinT (yodA) protein. From these results, it is likely that the amitrole-resistance in E.coli might be regulated by soxRS regulon, not oxy R, like adaptation for the oxidative stress

Thermostabilization by the Improvement of Intertrimeric Residues in Thermus thermophilus Inorganic Pyrophosphatase.

Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase)is a thermostable homohexamer of 174 amino acids，and its intertrimer interface is formed mainly by the symmetric α-helix A between subunits. Amino acids and their interactions composing intertrimer interface are different in hexameric Family I PPases，and then it was deduced that Tth PPase showed high thermostability because of stabilizing this interface by interactions of these residues. In this study，we focused on Thr138 and Ala141 residues in intertrimer interface of Tth PPase to confirm the relationship between intertrimeric residues and thermostability， and then improved their combination to His and Asp/Glu (HD or HE variant). As results，the HD variant showed the highest thermostability of enzyme activity，fluorescence spectra, and quaternary structure in the wild type Tth PPase and all variants. Especially，about 38% of hexamer and almost 40% of enzyme activity were observed in HD variant after heating even at 85℃. Therefore，we suggested that the conversion to a set of ionic His138 and Asp141 at intertrimer interface had increased the thermostability of Tth PPase，and then suppressed its thermal aggregation