Establishment of a hyper-protein production system in submerged Aspergillus oryzae culture under tyrosinase-encoding gene (melO) promoter control.

UV-mediated mutagenesis generated a high glucoamylase-producing mutant of Aspergillus oryzae exhibiting strong melanization in solid-state culture. Expression of the glucoamylase-encoding gene (glaB), which is specifically expressed in solid-state culture, and the tyrosinase-encoding gene (melO), was analyzed using an E. coli beta-glucuronidase (GUS) reporter assay to investigate this phenomenon. Although no common regulation was found for melO and glaB expression, the former was greatly enhanced in submerged culture. Interestingly, the melO promoter was about four times stronger for GUS production than the powerful promoters amyB, glaA, and modified agdA, previously isolated for industrial heterologous gene expression in A. oryzae. These findings indicated that the melO promoter would be suitable for hyper-production of heterologous protein in Aspergillus. The glaB-type glucoamylase selected as the target protein was produced in a submerged culture of A. oryzae under the control of the melO promoter. The maximum yield was 0.8 g/l broth, and the total extracellular protein purity was 99%. Repeated batch culture, to improve productivity, gave a maximum yield of 3.3 g/l broth. The importance of this work is in the establishment of a both high-level and high-purity protein overproduction system in A. oryzae by use of the melO promoter.

Pharmacokinetics of a new parenteral oligosaccharide antibiotic, SCH27899 (Ziracin), in healthy subjects.

The pharmacokinetic properties of an everninomicin antibiotic (SCH27899; Ziracin) were studied with healthy Japanese male volunteers by single (1, 3, 6, and 9 mg/kg of body weight) and multiple 60-min intravenous infusions (3, 6, and 9 mg/kg once daily for 10 consecutive days following a 2-day interval after the initial dose). At single doses the peak serum concentration and the area under the serum concentration-time curve linearly increased with the dose. While total body clearance (CL; 31.2 to 45.6 ml/kg/h) and percent cumulative urinary recovery as unchanged drug (4.9 to 7.1%) were rather constant irrespective of doses, the terminal half-life of gamma phase (t(1/2 gamma); 14.2 to 19.6 h) were slightly prolonged at the higher two doses compared with the lower two doses. With repeated doses of SCH27899, a statistically significant decrease and increase were found in CL and t(1/2 gamma) of about 36 and 21%, respectively, although these changes may be clinically irrelevant. The most commonly reported adverse events were local reactions such as erythema, pain, and palpable venous cord of mild to moderate degree around the injection site, which could be managed by changing the injection sites.

Identification of functional elements that regulate the glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae.

Aspergillus oryzae has-two glucoamylase-encoding genes, glaA and glaB, whose expressions are distinguished by the type of culture used. The glaB gene is markedly expressed in solid-state culture but is little expressed in submerged culture. In solid-state culture, glaB expression at the transcriptional level is enhanced by low-Aw (water activity), high-temperature, and physical barriers to hyphal extension, as well as by starch. To determine the cis-acting factors in the glaB promoter, deletion analysis of the promoter was done with GUS (beta-glucuronidase) as the reporter. Deletion of the 27 bp from -350 to -324 (Region A) in 1.1 kb of the glaB promoter completely abolished starch, low-Aw, and high-temperature induction. Substitution of the 12-bp GC-rich motif from -335 to -324 (GC-box) resulted in significant loss of starch and low-Aw inductivities. These findings suggest that the GC-box is a cis-element essential for the high-level expression of glaB in solid-state culture.

Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae.

The DNA (glaB) and a cDNA-encoding glucoamylase produced in solid-state culture of Aspergillus oryzae were cloned using oligodeoxyribonucleotide probes derived from internal amino acid sequences of the enzyme. Comparison of the nucleotide sequences of a genomic DNA fragment with its cDNA showed the glaB gene carried three exons interrupted by two introns and had an open reading frame encoding 493 aa residues. The 5'-flanking region had a TATA box at nt -87 from the start codon and two putative CAAT sequences at nt -276 and -288. The glaB gene shared 57% homology at the aa level with the glaA gene which was cloned previously from A. oryzae. Interestingly, the glucoamylase encoded by the glaB gene had no C-terminal domain such as that proposed to have starch binding activity in Aspergillus glucoamylases. Introduction of cDNA of the glaB gene to Saccharomyces cerevisiae caused the secretion of active glucoamylase to culture medium and introduction of the glaB gene to A. oryzae increased glucoamylase productivity in solid-state culture. Northern blot analysis showed the glaB gene was expressed in solid-state culture, but not in submerged culture.

Cysteine Protease Inhibitors Produced by the Industrial Koji Mold, Aspergillus oryzae O-1018.

Aspergillus oryzae O-1018 (FERM P-15834) separated from industrial koji for brewing sake was found to produce five papain-inhibitory compounds in the culture supernatant. The five isolated inhibitors were named CPI-1 to CPI-5, and their structures were elucidated by spectroscopic analyses and chemical degradation. We determined the structures of CPI-2, CPI-3 and CPI-4 as 4-amino-1-[[N- [(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl] amino]butane, 5-amino-1-[[N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleucyl]amino]pentane and N (8)- [N-[(2S, 3S)-3-trans-carboxyoxiran-2-carbonyl]-L-isoleu-cyl]spermidine, respectively. We also confirmed by a degradation experiment that CPI-1 consisted of L-trans-epoxysuccinic acid, L-tyrosine and spermidine, and that CPI-5 was composed of L-trans-epoxysuccinic acid, L-phenylalanine and spermidine. Although CPI-4 was identified as kojistatin A,(1)) the other CPIs seemed to be novel compounds. All CPIs were cysteine protease-specific inhibitors with appreciable selectivity toward cathepsin B and L. The inhibition potency of CPIs against cysteine proteases was as high as or higher than that of E-64. In particular, CPI-2, -3 and -4 were ten times more effective than E-64 toward cathepsin B and L, and CPI-1 and -5 were about 100 times more inhibitory than E-64 toward cathepsin L.

Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees.

Nine peptides to inhibit angiotensin I converting enzyme (ACE) were isolated from sake and sake lees. They were short peptides with 5 or fewer amino acid residues, and many of them had a tryptophan or tyrosine residue at the C-terminus. We synthesized the peptide fragments of IYPRY and YGGY, and measured their inhibitory activity. As a result, we have concluded that hydrophobic amino acids in the sequence and amino acid at C-terminus had an important role in the inhibition. When digested with pepsin and pancreatin, YGGY lost its inhibitory activity but IYPRY maintained its activity. YGGY and IYPRY were orally administered to spontaneously hypertensive rats (SHR) at the dose of 100 mg/kg. YGGY didn't change the blood pressure of SHR, but IYPRY reduced their blood pressure. The hypotensive effect of IYPRY continued for 30 h after administration. Also, three dipeptides among the IYPRY fragments, IY, YP and RY, had hypotensive effects, and the effect of RY continued for 30 h after administration.

Antihypertensive effects of peptide in sake and its by-products on spontaneously hypertensive rats.

Systolic blood pressure (SBP) decreased significantly when the hydrolysate of sake lee (HSL) and peptide fraction of sake (PFS) were orally administrated to spontaneously hypertensive rats (SHR). SBP of "young" SHR decreased significantly after orally administering Val-Tyr, His-Tyr, Arg-Phe, Val-Trp, and Tyr-Trp that were isolated from PFS and HSL. The hypotensive effect of Val-Tyr and His-Tyr that was observed in "young" SHR disappeared as they got older, but PFS and HSL maintained their antihypertensive effect on "aged" SHR. SHR fed on a diet with HSL replacing half of the protein source for 3 weeks showed a significant decrease in SBP after 10 days of feeding.