A Procedure for Precise Determination of Glutathione Produced by Saccharomyces cerevisiae
一种精确测定酿酒酵母产生的谷胱甘肽的方法
发布: 2018年06月20日第8卷第12期 DOI: 10.21769/BioProtoc.2887 浏览次数: 7461
评审: Valentine V TrotterPushpendra SinghAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Oct 2013
Abstract
In bioproduction, yields of products must be calculated precisely for accurate evaluation of various fermentation conditions. To evaluate productivity of microorganisms, product amounts per unit of medium volume (e.g., mg-product/L-broth), and/or product amounts per unit of a microorganism amount (e.g., mg-product/mg-dry cell weight) are often used. Nonetheless, detailed procedures for calculation of these production yields are often omitted in research articles, whereas methods for product quantification are described well. Here, we describe a detailed calculation procedure from our previous studies on glutathione production by Saccharomyces cerevisiae. This procedure can be applied to various other products and microorganisms, and therefore, may prove to be useful in various other bioproduction studies.
Keywords: Saccharomyces cerevisiae (酿酒酵母)
Glutathione (谷胱甘肽)
GSH (GSH)
GSSG (GSSG)
Aggregated microorganisms (聚集的微生物)
Background
Glutathione is the most abundant thiol-containing tripeptide in all living organisms and functions as a bioactive substance with varied roles in cells, e.g., as redox and antidotal agents. Therefore, glutathione is widely used in the medical, food, and cosmetic industries nowadays, and the demand has increased in recent years. Glutathione is industrially produced mainly by fermentation using Saccharomyces cerevisiae, which originally contains a high concentration of glutathione and has served as a safe, food-producing microorganism. Studies of microbial glutathione production in various microorganisms will become more important in the future. To evaluate the productivity in terms of glutathione via fermentation by various microorganisms, here we describe our detailed procedures of sample preparation, quantification of reduced and oxidized glutathione by high performance chromatography (HPLC), and calculations of two types of yield (Hara et al., 2012; Hara et al., 2015; Kiriyama et al., 2013; Kobayashi et al., 2017).
Materials and Reagents
- 200 μl pipette tips (FCR&Bio, catalog number: AG-200-FP-Y )
- 1,000 μl pipette tips (FCR&Bio, catalog number: AG-1000B )
- 1.5 ml microcentrifuge tubes (FUKAEKASEI and WATSON, catalog number: 131-815C )
- 50 ml centrifuge tubes (Corning, catalog number: 430291 )
- Filter unit with 0.22 μm pore size (Shimadzu, catalog number: GLCTD-MCE1322 )
- Saccharomyces cerevisiae
- Yeast cell culture medium
- Ultrapure water (Milli-Q)
- Ice
- Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate (NACALAI TESQUE, catalog number: 15111-45 )
- Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
- Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 163-04265 )
- 1-Heptanesulfonate (NACALAI TESQUE, catalog number: 31528-92 )
- Phosphoric acid (Wako Pure Chemical Industries, catalog number: 162-20492 )
- Methanol (NACALAI TESQUE, catalog number: 21929-23 )
- Yeast extract dried (NACALAI TESQUE, catalog number: 15838-45 )
- Hipolypepton (NIHON PHARMACEUTICAL, catalog number: 392-02115 )
- D-(+)-Glucose (NACALAI TESQUE, catalog number: 16805-35 )
- Aureobasidin A (Takara Bio, catalog number: 630499 )
- 99.5% Ethanol (NACALAI TESQUE, catalog number: 14713-95 )
- Agar powder (NACALAI TESQUE, catalog number: 01028-85 )
- Standard Buffer Solution (pH 4.01) (NACALAI TESQUE, catalog number: 37219-75 )
- Standard Buffer Solution (pH 6.86) (NACALAI TESQUE, catalog number: 37220-35 )
- Standard Buffer Solution (pH 9.18) (NACALAI TESQUE, catalog number: 37221-25 )
- YPD medium (see Recipes)
- Aureobasidin A stock solution (see Recipes)
- 500 mM EDTA (see Recipes)
- 100 mM EDTA (see Recipes)
- HPLC mobile phase (see Recipes)
Equipment
- Micropipetter (volume range 20-200 μl) (Nichiryo, model: NPX-200 )
- Micropipetter (volume range 100-1,000 μl) (Nichiryo, model: NPX-1000 )
- Block heater (ASTEC, model: BI-516C )
- Aluminum microtube rack (Bio Medical Science, model: BMS-D081 )
- Centrifuge (KUBOTA, model: 3740 )
- Centrifuge rotor for 1.5 ml microcentrifuge tubes (maximum capacity is 21,880 x g) (KUBOTA, model: AF-2236 )
- Centrifuge rotor for 50 ml centrifuge tubes (maximum capacity is 22,140 x g) (KUBOTA, model: AF-5004CH )
- Glass test tube (NICHIDEN RIKA GLASS, model: 101015 )
- Baffled Erlenmeyer flask (AGC TECHNO GLASS, model: 4551FK200R )
- Silicone sponge tapered plug closure (Shin-Etsu Polymer, model: T-19 )
- Silicone sponge closure (Shin-Etsu Polymer, model: C-40 )
- Spectrophotometer (Shimadzu, model: UVmini-1240 )
- Spectrophotometer cell (Hellma, model: 104-10-40 )
- High-performance liquid chromatography system (Shimadzu, model: Prominence)
- Octa decyl silyl (ODS) column (YMC, model: YMC-Pack ODS-A )
- Electronic scale (Mettler-Toledo International, model: XS105DU )
- Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
- Ice-making machine (HOSHIZAKI, model: CM-100K )
- Deep freezer (temperature range: from -20 to -30 °C) (Panasonic, model: MDF-U539 )
- Deep freezer (temperature range: from -50 to -85 °C) (PHC, model: MDF-U33V-PJ )
- pH meter (Horiba, model: F-52 )
- Forced air flow oven (Tokyo Rikakikai, model: WFO-1210 )
- Disposable loop (AS ONE, model: 6-488-02 )
- Incubator for solid preculture (Sanyo, model: MIR-153 )
- Incubator for liquid preculture (TAITEC, model: BR-43FL・MR )
- Incubator for glutathione production (TAITEC, model: G・BR-200 )
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kobayashi, J., Sasaki, D. and Kondo, A. (2018). A Procedure for Precise Determination of Glutathione Produced by Saccharomyces cerevisiae. Bio-protocol 8(12): e2887. DOI: 10.21769/BioProtoc.2887.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 定量
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