Ammonium sulfate

Oolong tea leaves (Utonrousousuisen) were purchased from the Banboo Cakan Chinese tea ceremony (Kochi, Japan). d(−)-Mannitol was purchased in its β crystalline form from Merck Ltd (Tokyo, Japan). Low-substituted hydroxypropyl cellulose (L-HPC) (mean particle size, 45 µm; hydroxyepoxy group, NBD-020) was supplied by Shin-etsu Chemical Co., Ltd (Tokyo, Japan). Polyvinylpyrrolidone (PVP) (Kollidon® 25) was obtained from BASF Japan (Tokyo, Japan). Gum arabic (Gum arabic HP powder), carrageenan (Sheepy gum® FA), guar gum (Guar pack® PF-20), tamarind gum (Glyroid® 6C), and pectin (H&F pectin classic AF701) were purchased from DSP Gokyo Food and Chemical (Osaka, Japan). Yeast nitrogen base without amino acids was purchased from Nippon Becton Dickinson Co., Ltd (Tokyo, Japan). Glucose, ammonium sulfate, polypeptone S, and monopotassium phosphate were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Brain heart infusion was purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). All other reagents used were of the highest grade available from commercial sources.

Urea (CH₄N₂O, 99%), ammonium sulfate (AS, (NH₄)₂SO₄, 99.5%), triethanolamine (TEOA, C₆H₁₅O₃N, 99.5%), ethanol (EtOH, 99.5%), hydrochloric acid (HCl, 35–37%), sodium hydroxide (NaOH, 95%) and chloroplatinic acid (H₂PtCl₆·6H₂O, 98.5%) were obtained from Wako Pure Chemical Co., Ltd. (Osaka, Japan). All the chemicals were used without further purification. Ultrapure water (UPW) from a Direct-Q Millipore system was used in all experiments.

The strains corresponding to the dominant ASVs at 20 °C were isolated. The source of isolation was the muscle tissues of the Japanese horse mackerel stored at 20 °C for 48 h, as described in section 2.1. As the medium conditions, Marine Broth 2216 (BD) supplemented either of defibrinated sheep blood (Cosmo Bio Co., Ltd., Tokyo, Japan) (50 mL/L), ammonium sulfate (Fujifilm Wako Pure Chemical Corporation) (10 g/L), yeast extract (BD) (1 g/L), or fish extract (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) (20 ml/L) was used. The atmosphere conditions were modified to aerobic, microaerophilic (5%–12% O_2, 5%–8% CO₂; AnaeroPack-MicroAero; MITSUBISHI GAS CHEMICAL CO., INC., Tokyo, Japan), or anaerobic (<0.1% O₂, >16% CO₂; AnaeroPack-Anaero; MITSUBISHI GAS CHEMICAL). These medium and atmosphere conditions were combined, then total 17 culture conditions were attempted for the isolation. The sample suspension was two-fold serially diluted, used to inoculate the medium, and incubated at 20 °C for 7 days. The most diluted bacterial culture among the cultures that showed bacterial growth was subjected to 16S rRNA sequencing analysis using the Sanger method (Monciardini et al., 2002 (link)). The sequence was then aligned to the sequence of the dominant ASVs using Clustal W (Thompson et al., 1994 (link)).

In the comparative analysis of normal and high-EC yeast strains, Mahoroba-Hana was used as the normal yeast strain, and Mahoroba-Gin was used as the high-EC yeast strain [18 ,19 ]. These yeasts were prepared under two conditions, slant and stationary phases, because the stationary phase contains yeast in a relatively homogeneous phase and may have relatively high EC content. To induce the stationary phase in each yeast strain, a static culture was used, with all yeast extracted in the same lot. The timing of observation at the stationary phase was determined a priori based on the growth character of each yeast strain: two days post extraction for Mahoroba-Hana and three days post extraction for Mahoroba-Gin. The trends of growth curve of each strain grown in synthetic YNB medium (prepared using nitrogen-based medium without glucose or ammonium sulfate (Difco, United States), glucose (Nacalai Tesque, Japan), and ammonium sulfate (Wako, Japan),) were also confirmed. Growth curves were obtained from OD 660 measurement using UV spectrometer UV-1800 (Shimadzu, Japan).

E. coli strain DH5α [F⁻, ϕ80dlacZΔM15, Δ (lacZYA-argF) U169, endA1, hsdR17 (r_k⁻, m_k⁺), supE44, thi-1, λ⁻, recA1, gyrA96, relA1, deoR] was used as the host for recombinant DNA manipulation and grown in Luria–Bertani medium [1% (w/v) tryptone (Becton, Dickinson and Company, MI, USA), 0.5% (w/v) yeast extract (BD), and 1% (w/v) sodium chloride] containing 100 μg/mL ampicillin (Meiji Seika Pharma, Tokyo, Japan).
Saccharomyces cerevisiae strain BY4741/sed1Δ (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, YDR077w::KanMX4), obtained from EUROSCARF (Frankfurt, Germany), was used for the cell surface display of glutaminase (YbaS) from E. coli because sed1Δ resulted in an increase of the quantity of protein on the yeast cell surface (Kuroda et al. 2009 (link)). Yeast host cells were grown and transformed in synthetic complete without uracil (SC-Ura) medium [0.15% (w/v) yeast nitrogen base without amino acids and ammonium sulfate (BD), 0.5% (w/v) ammonium sulfate (Wako Pure Chemical Industries, Osaka, Japan), 0.19% (w/v) yeast synthetic drop-out medium supplements (Sigma-Aldrich, MO, USA), 2% (w/v) glucose]. For the production of ammonia by YbaS-displaying yeast, the transformants were cultured in SC-Ura buffered at pH 5.5 with 200 mM 2-morpholinoethanesulfonic acid (MES) (Nacalai Tesque, Kyoto, Japan).