Eiichi Nakano

A full length cDNA clone for the alkaline protease from Aspergillus oryzae: Structural analysis and expression in Saccharomyces cerevisiae

SummaryWe have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae. According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids. A consensus sequence of a signal peptide consiting of 21 amino acids is found at the N-terminus of the prepro region. The primary structure of the mature region shares extensive homology (29%–44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN′) composing the active site are preserved. The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.

Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease

SummaryThe neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.

Molecular cloning and expression in Escherichia coli of a cDNA clone encoding luciferase of a firefly, Luciola lateralis

We have cloned a cDNA encoding Luciola lateralis (a common firefly in Japan) luciferase from a cDNA library of lantern poly(A)+ RNA, using a cDNA of L. cruciata (another common firefly in Japan) luciferase as a probe. The primary structure of L. lateralis luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids with a molecular weight of 60,132. Sequence comparison indicates that L. lateralis luciferase has significant sequence identity (94%) to L. cruciata luciferase, and that it has less sequence similarity (67%) to Photinus pyralis (a North American firefly) luciferase. The isolated cDNA clone, when introduced into Escherichia coli, directed the synthesis of enzymatically active luciferase under the control of the lacZ promoter.

Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells

The gene for cyclomaltodextrinase (CDase; EC 3.2.1.54) from Bacillus sphaericus E-244 was cloned in the recombinant plasmid pCD629. Sequencing a portion of pCD629 revealed a unique open reading frame of 1,773 nucleotides coding for a 591-amino-acid polypeptide. The deduced polypeptide sequence showed about 50% homology with that of a neopullulanase, and was slightly homologous to those of the cyclodextrin glucanotransferases and the α-amylases. The optimum pH, specific activity and Km value for β-cyclodextrin of the CDase that has been produced in Escherichia coli cells were 8.0, 16.4 units/mg protein, and 0.41 mm, respectively. These values were almost identical to those from B. sphaericus E-244.

The interaction of DNA with pancreatic ribonuclease A

Abstract 1. 1. The complex formation of calf thymus DNA with pancreatic ribonuclease A (ribonucleate pyrimidinenucleotido-2′-transferase (cycling), EC 2.7.7.16) was studied through ultracentrifugation, competitive inhibition of ribonuclease activity and gel filtration on Sephadex G-200. Ultracentrifugation data indicated that under low ionic strength ribonuclease formed a soluble complex with DNA at least in the range of pH 7.5 to pH 8.5 and this ability to form the complex was strongly affected by the presence of 0.05 M NaCl. 2. 2. The activity of ribonuclease was competitively inhibited by DNA when assayed in 0.01 M phosphate buffer (pH 7.5). The denatured DNA was much more effective than native DNA in inhibiting ribonuclease activity. 3. 3. Through gel filtration on Sephadex G-200, ribonuclease was found to form a complex with DNA in a definite combining ratio in 0.01 M phosphate buffer (pH 7.5). The combining ratios were calculated to be about one ribonuclease molecule per 500 000–1000 000 daltons of native DNA and 8000–10 000 daltons of denatured DNA. In the presence of 0.1 M NaCl the complex dissociated into free enzyme and DNA. The combining ratio of ribonuclease to native DNA was about 100 times smaller than that of denatured DNA and the discrepancy is discussed in connection with specific sites on DNA. The thermal denaturation profile of DNA was not affected by ribonuclease in the amount of this combining ratio.

Cloning of the sucrose phosphorylase gene from Leuconostoc mesenteroides and its overexpression using a sleeper bacteriophage vector

Abstract The sucrose phosphorylase gene was cloned from Leuconostoc mesenteroides ATCC 12291 and was overexpressed in Escherichia coli using a ‘Sleeper’ bacteriophage vector. A recombinant phage slp- spl -1, which had four copies of the sucrose phosphorylase gene, was lysogenized into E. coli 1100. After heat induction, this lysogen produced 55.7 units per ml culture of sucrose phosphorylase, i.e. 80-times higher than that in L. mesenteroides . As the amount of this recombinant enzyme was over 30% of the soluble protein in the cell, E. coli 1100 (slp- spl -1) succeeded in overcoming problems such as, inhibited enzymes, in the case of L. mesenteroides . Thus it is possible to achieve an industrial-scale production of sucrose phosphorylase. The complete nucleotide sequence showed that it coded for a 490-amino acid protein of M T 55,749. Homology between the deduced amino acid sequences for the L. mesenteroides sucrose phosphorylase gene and Streptococcus mutans gtfA , sucrose phosphorylase gene, was 68%.

Purification and characterization of luciferases from fireflies, Luciola cruciata and Luciola lateralis

Luciferases of Luciola cruciata and Luciola lateralis, LcL and LlL, were purified to homogeneity by ammonium sulfate precipitation, gel-filtration column chromatography, and hydroxyapatite HPLC. The molecular masses of the enzymes determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were both 62 kDa, almost identical to that of Photinus pyralis (PpL). LcL was found to be similar to PpL in thermal stability, pH stability, and the wavelength of maximum light intensity. LlL was superior to LcL and PpL in thermal and pH stability, and the reaction catalyzed by LlL emits green light with a peak intensity at 552 nm, which is 10 nm shorter in wavelength than those of PpL and LcL.

Elucidation of the thermal stability of the neutral proteinase II from Aspergillus oryzae

The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds. NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis. To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system. The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation. Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis.

Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12

The phosphotransacetylase gene (pta) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A. and Nakano, A. (1990) Appl. Microbiol. Biotechnol. 33, 680-682). Overexpression in E. coli confirmed the presence of the pta gene within the cloned fragment. DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length. The carboxyterminal region of the E. coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical). Several short regions of high sequence identity may be structurally or functionally important for enzymic activity.

Cloning of a gene coding for phosphotransacetylase fromEscherichia coli

SummaryA phosphotransacetylase gene (pta) has been cloned from a genomic DNA library ofEscherichia coli 1100, a derivative of strain K-12. The phosphotransacetylase activities ofpta+ plasmid-containing strains were amplified about 150-fold under control of thelac promoter. The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thepta gene was found to be downstream ofackA by a combination of restriction analysis and plasmid subcloning. It is located about 13 kb upstream of thepurF-folC-hisT region of the chromosome.