Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene

Nakajima, Ryoichi; Imanaka, Tadayuki; Aiba, Setsuya

doi:10.1128/jb.163.1.401-406.1985

Cited by 112 publications

(25 citation statements)

References 25 publications

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“…The library size of 7200 clones for the error-prone PCR library appears to be smaller than the size of other libraries described in literature, which typically ranges from 10,000 (Sibakov and Palva 1984;Stephens et al 1984), B. megaterium (BMA;Metz et al 1988), B. sp KSM-1376Igarashi et al 1998), B. sp #707 (S707;Tsukamoto et al 1988), B. stearothermophilus (BStA;Nakajima et al 1985), B. sp. TS-23 (TS-23; Lin et al 1998).…”

Section: Discussionmentioning

confidence: 92%

Directed evolution of a bacterial α‐amylase: Toward enhanced pH‐performance and higher specific activity

Bessler¹,

Schmitt²,

Maurer³

et al. 2003

Protein Science

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Abstract␣-Amylases, in particular, microbial ␣-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for ␣-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the ␣-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4-and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial ␣-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial ␣-amylases.

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Section: Discussionmentioning

confidence: 92%

Directed evolution of a bacterial α‐amylase: Toward enhanced pH‐performance and higher specific activity

Bessler¹,

Schmitt²,

Maurer³

et al. 2003

Protein Science

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“…It was transformed with plasmid pMK57 to produce strain MK57 or plasmid pMK79 to produce strain MK79 (8). Plasmid pMK57 (8) contains 3.0 kb of B. stearothermophilus DNA containing the α‐amylase gene (24) cloned into pUC8 (23). Plasmid pMK79 was constructed by inserting a 2.3 kb Vitreoscilla fragment containing the 0.6 kb vgb , as well as the 1.7 kb C‐terminal coding region of uvrA , into pMK57 (8, 25).…”

Section: Methodsmentioning

confidence: 99%

Nitrite Inhibition of Vitreoscilla Hemoglobin (VHb) in Recombinant E. coli: Direct Evidence that VHb Enhances Recombinant Protein Production

2000

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Bacteria engineered with the gene (vgb) encoding Vitreoscilla hemoglobin (VHb) typically produce more protein than unengineered cells, and it has generally been assumed that VHb is responsible for this effect. Here, using matched strains of E. coli that bear a recombinant R-amylase gene (MK57) or the R-amylase gene and vgb (MK79), we provide evidence supporting this assumption. Sodium nitrite (which is known to inhibit heme proteins) was tested over a range of concentrations regarding effects on growth, R-amylase production, respiration, and VHb function in MK57 and MK79. Nitrite concentrations were identified at which respiration of cell membranes was inhibited only slightly and to approximately equal degrees in both strains, while whole cell respiration was inhibited to a greater extent and about twice as much in MK79 as MK57. This suggests that these concentrations inhibit VHb while having a much smaller effect on cytochrome oxidase. Direct measurements of VHb showed, in fact, that the same nitrite concentrations greatly decreased the levels of active (ferrous) and, to a somewhat lesser extent, total (ferrous plus ferric) VHb in MK79. Finally, these same nitrite concentrations reversed the advantage regarding R-amylase production of MK79 over MK57 seen at 0 mM nitrite, linking the presence of active VHb with the increase in R-amylase production.

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“…(iii) aL-Amylase and penicillinase activities. a-Amylase was assayed by the 3,5-dinitrosalicylic acid method as described previously (5,25). Penicillinase activity was assayed as described previously (13,34).…”

Section: Methodsmentioning

confidence: 99%

Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT

1990

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The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes.

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Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene

Cited by 112 publications

References 25 publications

Directed evolution of a bacterial α‐amylase: Toward enhanced pH‐performance and higher specific activity

Directed evolution of a bacterial α‐amylase: Toward enhanced pH‐performance and higher specific activity

Nitrite Inhibition of Vitreoscilla Hemoglobin (VHb) in Recombinant E. coli: Direct Evidence that VHb Enhances Recombinant Protein Production

Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT

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