Hiroshi Motai

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.

Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae

SummaryA chemostat culture system was investigated in order to produce protease by Aspergillus species effectively in the presence of 10% NaCl which was added to avid bacterial contamination. A salt tolerant fungus Aspegillus oryzae NISL 1913 produced protease even in the presence of 10% NaCl. The protease production by this strain was accelerated by proteins. Isolated soy protein or defatted soybean fluor (DSF) was used as a nitrogen source and an inducer of protease production, and starch was used as a carbon source. Continuous protease production was performed in a carbon-limited chemostat culture (dilution rate = 0.02). The maximum activity reached 2200 protease units (PU)/ml of the culture broth (130 PU/mg dry weight) with DSF as a nitrogen source. The culture could be continued for more than 50 days without any bacterial contamination.

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

SummaryThe effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h−1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)−1·h−1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as α-amylase, indicating that the protease production was selectively stimulated by the oils.

Continuous production of soy sauce by a bioreactor system

Abstract Continuous fermentation of soy sauce with immobilized glutaminase and immobilized cells of Pediococcus halophilus, Zygosaccharomyces rouxii , and Candida versatilis was investigated. Glutamic acid production and both lactic acid and alcohol fermentation continued constantly for over 100 days without any problems. The total time required for the production of soy sauce by the bioreactor system was much less than of the conventional method (about 1/10). The soy sauce made by this system was similar to a conventional one in regard to chemical components and the quality of flavour.

Continuous fermentation of soy sauce by immobilized cells of Zygosaccharomyces rouxii in an airlift reactor

SummaryThe optimum conditions for continuous alcohol fermentation of soy sauce with immobilized Zygosaccharomyces rouxii cells were investigated using an airlift reactor. The optimum pH and temperature of the fermentation were 4.5–5.5 and 25°–27.5° C, respectively. Ethanol content in the fermented liquid was increased with increasing height to diameter ratio of the reactor and the ratio of air to nitrogen in the supplied gas (total supplied gas: 0.08 vvm). A notable decrease in ethanol content was observed when only nitrogen gas was supplied. The products fermented by supplying air (0.02 vvm) had a higher conent of aroma components than that by supplying only nitrogen gas, and the aroma of the former products was similar to that of conventional soy sauce. This alcohol fermentation using an airlift reactor was continued for about 50 days without problems even if conditions such as residence time and aeration were altered.

Detection of γ-Polyglutamic Acid (γ-PGA) by SDS-Page

γ-Polyglutamic acid (γ-PGA) produced by Bacillus suhtilis (natto) was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and basic dye staining. Using this method, the molecular weight of γ-PGA was estimated at 275,000. This value was almost the same in all bacterial strains tested. As other applications of this SDS-PAGE method, degradation of γ-PGA by acid and heat treatment and a cross-linking reaction with carbodiimide and ethylenediamine were made visible in acrylamide gel. In the growth curve of the bacteria, γ-PGA production was detected in early stationary phase by SDS-PAGE.

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.

Purification and Properties of γ-Glutamyltranspeptidase from Bacillus subtilis (natta)

To understand the mechanism by which γ-poly glutamic acid (γ-PGA) in the sticky material of natto was synthesized, we purified the γ-glutamyltranspeptidase (γ-GTP) (EC 2.3.2.2) from the culture broth of Bacillus subtilis (natto) to homogeneity. γ-GTP was composed of two subunits with molecular weight of 45,000 and 22,000. The N-terminal amino acid sequence of light subunit was homologous with that of γ-GTP from Escherichia coli. The optimum pH and temperature of activity were 8.5 and 60°C. The enzyme was inactivated by incubation for 15 min at pH 8.0 and 55°C, but little loss of the activity was detected at 40°C. γ-GTP used glutamine as a γ-glutamyl donor and acceptor for γ-PGA synthesis. Dipeptides were better γ-glutamyl acceptors than free amino acids.

Continuous production of 4-ethylguaiacol by immobilized cells of salt-tolerant Candida versatilis in an airlift reactor

Abstract The effects of pH, temperature, aeration, and residence time on the continuous production of 4-ethyl-guaiacol (4-EG), which is one of the characteristic aroma components in soy sauce, by immobilized cells of the salt-tolerant yeast Candida versatilis were investigated using an airlift reactor. The optimum pH and temperature were about 4.0 and 30–33°C, respectively. The amount of 4-EG in the liquid was constant even during alterations of nitrogen/air ratio in the supplied gas. A large amount of 4-EG (over 20 ppm) was produced at a residence time from 5 to 28 h and 1–3 ppm of 4-EG, which was the optimum concentration in conventional soy souce, was produced at a shorter residence time of 0.5 h. The 4-EG production by immobilized C. versatilis cells using the airlift reactor was stable for 40 d. It was found that the immobilized cell method was effective for the production of 4-EG by C. versatilis cells.

Contributions of immobilized and free cells of salt-tolerantZygosaccharomyces rouxii andCandida versatilis to the production of ethanol and 4-ethylguaiacol

SummaryThe contribution of immobilized cells and free cells released from gel beads to ethanol production by the salt-tolerant yeastsZygosaccharomyces rouxii andCandida versatilis, and 4-ethylguaiacol (4-EG) production byC. versatilis were investigated using an airlift reactor. The amounts of ethanol produced by free cells were about 65% and about 90% of total ethanol in the reactor forZ. rouxii andC. versatilis, respectively. It was found that immobilized cells gave a much lower specific productivity of ethanol (ethanol production per hour per cell) than free cells of both yeasts, especially ofC. versatilis. 4-EG was produced mainly by immobilized cells ofC. versatilis; the amount of 4-EG produced by free cells was about 20% of the total 4-EG, in contrast to the results of ethanol production. However, the specific productivity of 4-EG (4-EG production per hour per cell) by immobilized and free cells was fairly similar.