J. Biol. Macromol., 1(2), 45-48, 2001
ArticlesMicroanalysis of GABA: An application for evaluating GABA production in yeast strains and theeffect of spice extracts on glutamate decarboxylase activity
Sachie Kurohara, Maiko Asai, Miyuki Hayashi, Kumio Yokoigawa and Hiroshi Ueno†
Department of Food Science, Nara Women’s University, Nara 630-8506, Japan
Received March 31, 2001; Accepted May 25, 2001

A high performance liquid chromatography(HPLC) system was optimized for quantitating
sub-nmol levels of γ-amino butyric acid(GABA) by using cation exchange columnchromatography and O-phthalaldehyde (OPA)
fluorescence post-column derivatization. Thissystem was used for routine analysis ofglutamate decarboxylase (GAD) activity fromyeast for the endogenously and exogenously
expressed enzymes. Although the levels ofGABA and GAD activity highly depend upon
yeast strains, significant amounts of GABA as
well as GAD activity are found in cultured yeast
cell extracts. The system is applied to evaluate(1) the GABA producing ability of yeast cellsand (2) the effectiveness of the spice extracts on
GABA-producing ability of GAD.
Key words: GABA, GAD, spice, yeast
γ-Aminobutyricacid(GABA),synthesized by glutamate decarboxylase (GAD)from L-glutamic acid,1) is an inhibitory
neurotransmitter in central nervous system(CNS).1) It also involves important biological
functions, such as inducing hypotensive anddiuretic effects.2, 3) Because of the GABA’srole, there is a great interest in raising theamount of GABA contents in food products.
Yeast can be an ideal candidate to be used forproducing GABA-containing health food, since
it has been used for major food production†including bread and alcohol beverages;
however, we have limited information on bothGABA contents and GABA production in yeast.
Our first aim of this study is to evaluate various
yeast strains for their ability in producing
GABA and GAD.
The physiological function of GAD is toproduce GABA and its role in neuronal system
is well reported; however, very little is known
in non-neuronal system. The presence ofGABA in small intestine suggests a possible
role of GAD in food digestive system.4) We
have suspected that certain food components,
such as spices, may interact directly withenzymes found in the food digestive system
like GAD, and may alter the catalytic role ofthese enzymes. Our second aim in this study
is to test this hypothesis. The effects of spices
on GAD activity were investigated by ourHPLC system.
Materials and methods
Materials - Strain of Saccharomyces
cerevisiae (pWIG32-2) which carries rat brainGAD655) was described previously.6) Spices
were gifts from Somatech Center of House
Foods Co., Japan.
Other yeast strains,Candida lipolytica (No. 1066), Debaryomyces
hansenii (No. 1104), Saccharomyces sake (No.
1460), Saccharomyces rosei (No. 1465),
Schizosaccharomyces pombe (No. 1490) werefrom the collection of this laboratory.

To whom correspondence should be addressed. E-mail: hueno@cc.nara-wu.ac.jp, Phone/Fax:+81-742-20-3493.
Abbreviations : AET, 2-aminoethyl isothiouronium bromide hydrobromide; CNS, central nervous system ; DTT,
dithiothreitol ; EDTA ; ethylenediaminetetraacetic acid; GABA, γ-amino butyric acid; GAD, glutamate
decarboxylase; HPLC, high performance liquid chromatography; OPA, O-phthalaldehyde; PCA, perchloricacid; PLP, pyridoxal 5’-phosphate

S. Kurohara et al.
O-Phthalaldehyde (OPA) was purchased from
Wako Chemical Co.
Protease inhibitor
cocktail tablets were purchased from
Boehringer Mannheim. Other reagents were
the highest grade obtained locally.
Yeast cultivation - Yeast cultivation,
except for pWIG32-2, was carried out in yeast
extract/peptone/dextrose (YPD) liquid medium7)
at 28 °C in the rotary shaker (200 rpm).
Pre-cultivation was done in 50 ml medium forovernight. Aliquots of pre-cultivation medium
were transferred to 1 l medium to start theinoculation where initial optical density wasadjusted to 0.05. Typical cultivation wascarried out for 24 h.
For pWIG32-2 strains, after thepre-cultivation, aliquots were transferred toyeast extract/peptone/acetate (YPA) medium(pH 6.0) to induce GAD production which wasunder the control of isocitrate lyase genepromoter.6) The growth was continued for 70h.
Preparation of yeast extract - Cell pellets
were collected by centrifugation at room
temperature, washed once with distilled water,and stored at -20 °C until further use.
Defrosted cell pellets were suspended in lysis
buffer containing one tablet of protease inhibitor
cocktail, 1 mM AET, 0.5 mM PLP, 0.1 mM
DTT, 1 mM EDTA in 60 mM sodium phosphatebuffer, pH 7.1. The suspension was subjected
for cell disruption either by ultrasonication(Heat Systems) or beads beater (Biospec) andcentrifuged at 4 °C. Supernatant was used for
endogenous GABA determination, GAD assay
and incubation with various spice extracts.
When the supernatant was not used immediately,
it was kept at -80 °C, under which conditions
enzyme activity was maintained.
Proteindetermination
Proteinconcentrations in yeast extracts were estimatedwith Bradford method by using a protein assay
kit from BioRad, where serum albumin wasused as a reference protein.
Enzyme assay - Crude protein extract waspassed through a Pharmacia Desalting column
to remove endogenous GABA and other low
molecular weight materials, which potentiallydisturbed GAD assay. Enzyme solution wasthen incubated with assay mixture, containing
20 mM sodium glutamate, 0.1 mM PLP in 100
mM sodium phosphate buffer, pH 7.1, at 30 °Cfor 30 or 60 min.6) At the end of incubation
period, 60% perchloric acid (PCA) was added
to stop the reaction and the mixture wascentrifuged. Supernatant was subjected forGABA analysis on HPLC.
Amount ofendogenous GABA found in the extract wasexpressed as µmol/mg protein. A unit of GAD
activity was expressed as nmol of GABA
produced/min/mg protein.
Spice extracts - Grounded powder ofrandomly selected five different spices among
popularly used kinds in Japan, turmeric, ginger,
peppermint, Japanese pepper, and sage, wasweighed and mixed individually with eitherdistilled water or 70% ethanol to make up 0.2g/ml suspension. Extraction was carried out
by gently rotating the container for over night at4 °C. Centrifugation of the mixture gave an
extract that was kept at room temperature underdark. Effect of spice extract on GAD activity
was evaluated by incubating the water orethanol extract of spice (3% v/v) with desalted
enzyme extract, obtained from the culture ofpWIG32-2 strain, and GAD assay mixture for
30 min at 30 °C. After the addition of 60%
PCA and following centrifugation, thesupernatant was applied for HPLC analysis. In
place of spice extract, just water or ethanol wasadded for taking control GAD activity that wasreferred as 100%.
HPLC conditions - High performanceliquid chromatography (HPLC) system wasconstructed with two Shimadzu model LC-6A
pumps, autoinjector (model SIL-6A), column
oven (at 30 °C), Shimadzu system controller(model SCL-6A), Gilson fluorometer (Model121) and Shimadzu LC600 pump which
delivered OPA solution (made up with 1.54 g

Improved assay method for GABA
OPA, 2 ml 2-mercaptoethanol, and 25 g boric
acid in 1 liter of water to adjust pH 10.4).
Elution buffer of 0.1 N sodium citrate, pH 3.4,
was delivered to cation exchange column(Shiseido Capcell pack SCX UG80, 4 x 250mm) at 0.7 ml/min.
OPA solution wasdelivered at 0.4 ml/min. Chromatography wasrecorded on Shimadzu Chromatopac C-R3A
where peak area corresponding to authentic
GABA was integrated and compared withstandard GABA solution (1 nmol).
To protein extract, as well as GAD assay
solution, was added 60% PCA to precipitate
protein components. After brief centrifugation,
supernatant was applied to HPLC for GABA
analysis.
Results and Discussion
Analysis of GABA contents - Fig. 1
shows a typical separation profile of the mixtureof glutamate and GABA.
Under ourexperimental conditions, glutamate and GABA
were eluted at 6.3 and 27.6 min, respectively.
There are other conditions for separating thesetwo compounds; however, they are impracticalfor our purpose: the time span that two peaks
appear have to be longer than 10 min apart toavoid the interference of glutamate. Although
an assay method employing radioactive
substrate has been extensively used for GAD
activity measurement,8) we did not use themethod as it does not allow to quantitate theendogenous GABA in the sample.
Yeast endogenous GABA expression andGAD activity - Five strains of yeast wererandomly selected from our yeast stock. As
shown in Fig. 2, Schizosaccharomyces pombe
exhibited the highest amounts of GABA in its
cell lysate whereas the highest GAD activity
was found with Candida lipolytica. Thereseems to be no direct correlation between theGAD activity and endogenous GABA level inthe cell lysate. It is of interest to point out that
two similar strains, Saccharomyces sake andRetention time (min)Fig. 1. Elution profile of glutamate andGABA. 10 µl of a mixture of glutamate (20mM) and GABA (0.1 mM) was injected.
Saccharomyces rosei, showed GAD activities atthe similar level but significantly different in
GABA contents.
Although endogenousGABA contents in yeast cell lysates have notbeen thoroughly studied yet, there was a reportof GABA production into medium by lactic acidbacterium,9) where 0.01-5.2 g of GABA wasFig. 2. Endogenous GABA level and GAD
activity in the crude extract from yeast. Datashown here are average of three experiments.

S. Kurohara et al.
extracts tend to inhibit GAD activity and water
extracts showed both inhibition and activation.
It is of great interest to ask which components
of water or alcohol extracts are responsible for
affecting GAD activity.
The difference
between 1st day and 4th day extracts suggeststhat some of the effective components may besensitive to oxygen in air.
In conclusion, our HPLC method is highly
useful for searching high GABA-producing
yeast strains and evaluating the effect of spices
on GAD activity.

Fig. 3. Effects of spices on GAD activity.
Ratio was calculated as per cent inhibition oractivation in the presence of spice extract vs.
control. Positive ratio indicates inhibitionand negative ration indicates activation.
found in one liter of culture medium. There islittle information available regarding to yeast
originated GAD. Present study suggests thatyeast produces GABA and its synthetic enzyme,
GAD. It is not clear from our results themetabolic role of GABA in yeast system.
Further characterization of GAD is warranted inorder to classify the yeast enzyme, whether it isa mammalian type or bacterial type.5)
Effect of spice on GAD activity – The
five randomly selected spices were evaluatedfor their potential in affecting GAD activity. A
system expressing rat brain GAD655) in S.
cerevisiae was constructed previously.6) For
this experiment, partially purified GAD wasused. Fig. 3 summarizes our results in which
the effects of both water and ethanol extracts on
GAD activity were indicated. The inhibitory
effects were shown to the right direction andactivation effects to the left, where both effects
were expressed as % of control. The spices
affect GAD activity significantly and thepatterns appear quite complex.
Alcohol

Acknowledgement
We thank Shiseido Co. for the use ofCapcell pack column and House Food Co.,
Somatic Center for providing us with spicesamples.
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Communicated by Takii Yukio