Seiji Negoro

Solubility of artificial proteins with random sequences

A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The soluble random proteins RP3–42 and RP3–45 and insoluble RP3–70 were purified. The solubility of the purified form is the same as that in the cell lysate.

Thermostable Peroxidase from Bacillus stearothermophilus

A peroxidase from Bacillus stearothermophilus was purified to homogeneity. The enzyme (Mr 175,000) was composed of two subunits of equal size, and showed a Soret band at 406 nm. On reduction with sodium dithionite, absorption at 434 nm and 558 nm was observed. The spectrum of reduced pyridine haemochrome showed peaks at 418, 526 and 557 nm; the reduced minus oxidized spectrum of pyridine haemochrome showed peaks of 418, 524 and 556 nm with a trough at 452 nm. These results indicate that the enzyme contained protohaem IX as a prosthetic group. The optimum pH was about 6 and the apparent optimum temperature was 70 degrees C. The enzyme was relatively stable up to 70 degrees C; at 30 degrees C it was stable for a month. The enzyme had peroxidase activity toward a mixture of 2,4-dichlorophenol and 4-aminoantipyrine with a Km for H2O2 of 1.3 mM. It also acted as a catalase with a Km for H2O2 of 7.5 mM.

Sequence and properties of β‐xylosidase from Bacillus pumilus IPO

The nucleotide sequence of the beta-xylosidase (xynB) gene from Bacillus pumilus has been reported previously [Moriyama, H., Fukusaki, E., Crespo, J.C., Shinmyo, A. & Okada, H. (1987) Eur. J. Biochem. 166, 539-545]. However, the sequence identified in the present study is quite different from the previously reported one. The total length of the PstI--EcoRI fragment of a plasmid pOXN295 containing the xynB gene is 2201 bp from our sequencing, while the length of the fragment in the previous data was 2466 bp. The sequences are similar in the N-terminal (500 bp) and C-terminal (260 bp) regions, but those in the central region are completely different. From the following observations, the previous sequence seems to have no reliable experimental basis. First, the restriction sites observed for pOXN295 are quite different from the sites deduced from the sequence. Second, the amino acid composition deduced from the sequence and the composition identified by amino acid analysis of the purified beta-xylosidase are very different. It is confirmed, on the other hand, that our new sequence agrees well with these experimental data. The enzyme was purified to homogeneity from Bacillus pumilus and Escherichia coli harboring a hybrid plasmid which highly expresses the xynB gene. The molecular mass of the enzyme was estimated to be 190 kDa by high performance gel filtration chromatography using TSK-G3000SW and 56 kDa by SDS/polyacrylamide gel electrophoresis. The pH optimum was 7.0, and the optimum temperature was 40 degrees C. The Vm value was estimated to be 1.23 +/- 0.14 mukat/mg (or p-nitrophenyl beta-D-xyloside) and 0.14 +/- 0.011 mukat/mg (for xylobiose), while Km was estimated to be 3.9 +/- 0.59 mM (for p-nitrophenyl beta-D-xyloside) and 8.9 +/- 1.19 mM (for xylobiose).

Purification and characterization of a new glucose dehydrogenase from vegetative cells of Bacillus megaterium

Abstract A new type of glucose dehydrogenase was purified from vegetative cells of Bacillus megaterium IAM1030. The characteristics of the vegetative-cell enzyme were investigated and compared with the enzyme from sporulating cells of B. megaterium IWG3. They are very similar in the following points: molecular size (Mr 120,000), subunit composition (homo tetramer), pH-activity profile with an optimum pH at around 8, pH-stability profile with a stable pH range of 6.0–7.5 (at 25°C, for 30 min), substrate specificity (specific for d -glucose and 2-deoxy- d -glucose), and the affinity for glucose (a Km value of 11–12 mM at pH 8.0, 2.5 mM NAD). They are a little different in the following points: slower mobility for the vegetative-cell enzyme in polyacrylamide-gel electrophoresis at pH 8, immunological determinants (some of them are common), and higher heat resistance for the vegetative-cell enzyme at pH 6.5. They are quite different in their affinity for NAD and NADP. The Km values for NAD are 0.1 mM for the vegetative-cell enzyme and 1.0 mM for the spore enzyme, while the values for NADP are 7.1 mM for the vegetative-cell enzyme and 0.09 mM for the spore enzyme, at pH 8.0, 0.1 M d -glucose. These results suggest that B. megaterium has at least two types of glucose dehydrogenase.

Structure of Isozyme Genes of Glucose Dehydrogenase from Bacillus megaterium IAM1030

Abstract Two isozyme genes of glucose dehydrogenase, gdhI and gdhII , were isolated from Bacillus megaterium IAM1030, and their nucleotide sequences were identified. Each gene has an open reading frame of 783 base pairs available to encode a peptide of 261 amino acids. There are two more open reading frames, orf1 and orf2 , upstream from gdhI ; orf1 consists of 684 base pairs available to encode a peptide of 228 amino acids, and orf2 consists of 858 base pairs available to encode a peptide of 286 amino acids. The nucleotide sequence of orf2 seems to be similar to orfX in the gdh operon of B. subtilis . Upstream of orf2 , as well as that of orfX , there is a probable promoter sequence very similar to the consensus promoter sequence recognized by B. subtilis RNA polymerase containing the sigma factor σ G , a new class of sporulation-specific promoters. Downstream from the coding region of orf2 , no transcriptional termination signal is found, and soon after orf2 , there is a ribosomal-binding sequence followed by the coding region of gdhI . Therefore, orf2 and gdhI seem to form an operon similar to the orfX-gdh operon of B. subtilis , and be transcribed during sporulation as a polycistronic message. In the upstream regions of gdhII and orfI , on the other hand, there are probable promoter sequences homologous to the postulated consensus promoter sequence recognized by B. subtilis RNA polymerase containing the sigma factor σ A . Both gdhI and gdhII are expressed in Escherichia coli cells. The phylogenic relationships among glucose dehydrogenase genes including gdhI and gdhII are also discussed.

Characterization of endo-type 6-aminohexanoate-oligomer hydrolase from Flavobacterium sp.

Abstract Cyclic oligomers of 6-aminohexanoate were fractionated from waste materials obtained from a nylon-6 factory by Sephadex LH-20 gel filtration column chromatography, and two major fractions, a cyclic tetramer and cyclic dimer, were obtained. The 6-aminohexanoate-oligomer hydrolase (EIII) from Flavobacterium sp. K172 was active toward the cyclic tetramer, but not toward the cyclic dimer. The Mr of the EIII enzyme was estimated to be 93,000 by high performance gel filtration chromatography (TSK-G3000SW), while that of the polypeptide as deduced from the nucleotide sequence was calculated to be 36,902, suggesting that the EIII enzyme has a dimeric or trimeric structure. The enzyme was stable between the pH 6.5 and 11 with an optimum pH of 7.0. The enzyme activity was stable up to 45°C with an optimal temperature of 40°C.

Alteration of catalytic function of 6-aminohexanoate-dimer hydrolase by site-directed mutagenesis

Abstract Alteration of Asp181 in a nylon oligomer-degrading enzyme, 6-aminohexanoate-dimer hydrolase (EII) of Flavobacterium sp. KI72, to Asn and to Glu by site-directed mutagenesis increased Km values toward 6-aminohexanoate-dimer 4 times and 11 times, respectively. Replacement to His or to Lys caused complete loss of the activity (less than 0.02% of the activity of the EII enzyme). Thus, a single amino acid alteration at position 181 of the enzyme drastically affects the catalytic function.

Construction and characterization of N-terminally truncated DNA polymerase from Thermus thermophilus

Abstract Various plasmids harboring the truncated DNA polymerase gene ( polA ) from Thermus thermophilus HB8 ( Tth polymerase) were constructed. The most thermostable Tth Δ NF 2 polymerase [the gene product of polA Δ NF 2, which lacked a 751-bp region (region flanked by initiation codon and Fsp I site in the polA gene)] was selected, and purified from the recombinant Escherichia coli . SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the Tth Δ NF 2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the Tth Δ NF 2 polymerase was 63% of that of the Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the Tth Δ NF 2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the Tth Δ NF 2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.

Properties of Artificial Proteins with Random Sequencesa

Abstract: A library of artificial proteins of 141 amino acid residues, of which 95 are random and which include 20 kinds of amino acids, was prepared. As the properties of the artificial random proteins are free from the evolutionary constraint, they can be used as a standard to discriminate the specialized properties of natural proteins. Out of the 25 identified random proteins, 5 are soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. Therefore, as natural soluble or insoluble proteins can arise from the line of soluble or insoluble ancestry, respectively, solubility does not seem a specialized property of natural proteins. The soluble random proteins RP3‐42 and RP3–45 were purified and their properties were investigated.

Characterization of hybrid enzymes between 6-aminohexanoate-Dimer hydrolase and its analogous protein

Abstract 6-Aminohexanoate-dimer hydrolase (EII) and its analogous protein (EII′), of Flavobacterium sp. K172 are composed of 392 amino acids, in which 47 are different. The enzyme activity of EII′ toward 6-aminohexanoate dimer is approximately 0.5% of that of EII. We have constructed various hybrids of the two genes by exchanging fragments flanked by conserved restriction sites such as Pvu II, Bgl II, Sal I, and Bam HI (respectively 74, 483, 771, and 1,141 bp downstream of the initiation codon), and purified their gene products to homogeneity. Hyb-12 protein, which was obtained by the replacement of the Bgl II- Sal I region of the EII′ with the corresponding region of EII, had 12 times higher specific activity towards the 6-aminohexanoate dimer and its related substrates than EII′ protein. Hyb-10, which was composed of the N-terminal - Bgl II regions of EII′ and the Bgl II-C terminal region of EII, had activity toward these substrates nearly equal to the activity of the EII enzyme. Comparisons of the activity toward 6-aminohexanoate dimer and its analogues has demonstrated that EII, EII′, and their hybrid enzymes are highly active only toward the substrates that contain 6-aminohexanoate as the N-terminal residue, while the recognition of the C-terminal residue in the substrate was not stringent. The substrate specificity, pH-activity profile, and heat stability of these enzymes varied slightly.