Manganese (Mn2+)-dependent storage stabilization of Rhodotorula glutinis phenylalanine ammonia-lyase activity

Article abstract of DOI:10.1021/jf072614u

Phenylalanine ammonia-lyase (PAL; E C 4.3.1.5) reverse reaction has been exploited for the commercial production of optically pure L-phenylalanine from trans-cinnamic acid. Optimal conditions for the growth and PAL activity of Rhodotorula glutinis cells a

Full text of DOI:10.1021/jf072614u

894 J. Agric. Food Chem. 2008, 56, 894–902
Manganese (Mn2+)-Dependent Storage Stabilization
of Rhodotorula glutinis Phenylalanine
Ammonia-lyase Activity
MARK J. WALL,† ANDREW J. QUINN,§ AND GODWIN B. D’CUNHA*
Department of Chemistry, Cape Breton University, 1250 Grand Lake Road,
Sydney, Nova Scotia, Canada B1P 6L2
Phenylalanine ammonia-lyase (PAL; E C 4.3.1.5) reverse reaction has been exploited for the
commercial production of optically pure L-phenylalanine from trans-cinnamic acid. Optimal conditions
for the growth and PAL activity of Rhodotorula glutinis cells and an improved method for the synthesis
of L-phenylalanine have been reported. A major problem encountered during these studies was rapid
loss of PAL activity during storage of the yeast cells, which were therefore unsuitable for long-term
and repeated use. Enhancement of enzyme stability in the presence of various additives including
polyhydric compounds and metal ions is described. Whole cells retained nearly 85% of the original
enzyme activity for at least 12 weeks when a low concentration of Mn2+ (0.01%) was included in the
storage buffer medium (50 mM Tris-HCl, pH 8.8). In contrast, <3.0% activity was present in the
control within 4 weeks. Mn2+-dependent stabilization of PAL was also observed with an isolated
enzyme preparation (73% retention in activity for 12 weeks) obtained by ultrasonication of R. glutinis
whole cells. The data suggest that Mn2+ ions may be responsible for the specific stabilization of a
more active conformation of the enzyme. In addition, enzyme stability as a function of temperature
was studied, and the optimal temperature for maximal activity retention was 0–2 °C. The effects of
various additives on the induction of PAL have also been examined. These results could have direct
implications in studies on activity, inhibition, and reaction mechanism of this biotechnologically
important enzyme.
KEYWORDS: Rhodotorula glutinis yeast cells; phenylalanine ammonia-lyase; Mn2+-dependent storage
stability; enzyme induction; GC-MS determination of PAL reverse activity
INTRODUCTION
Commercially, Rhodotorula yeast has been the principal
source of PAL because of its high levels of PAL and nonfas-
tidious requirements for growth and PAL synthesis (8, 10). PAL
reaction is of great interest in commercial applications including
treatment of certain mouse neoplastic tumors (11), quantifying
serum L-Phe (12), treatment of phenylketonuria (13), and
preparation of low-phenylalanine diets (14). The reverse of the
PAL physiological reaction has been utilized for the synthesis
of L-Phe and its methyl ester (15–18).
Phenylalanine ammonia lyase (EC 4.3.1.5, PAL) is the first
enzyme of the phenyl propanoid metabolic pathway, a secondary
metabolic pathway operative in higher plants (1). The enzyme
is present in higher plants (1–3), some fungi (4, 5), yeasts (6–8),
and a single prokaryote, Streptomyces (9). However, it is absent
in true bacteria and animal tissues. The enzyme is soluble and
cytoplasmic in origin in the majority of cases. PAL catalyzes
the spontaneous nonoxidative deamination of L-phenylalanine
(L-Phe) to form trans-cinnamic acid (t-CA) and ammonia (1).
We have recently reported optimal conditions for the maximal
growth and PAL activity of R. glutinis yeast cells and an
improved method for the synthesis of L-Phe using PAL reverse
reaction (19). Although the yeast cells could be used as a rich
enzyme source, especially for effective transformation of t-CA
to L-phe (>85% conversion), PAL activity declined rapidly
during storage. The present study was undertaken to explore
the possibility of stabilizing the biocatalyst so that it could be
repeatedly used over the long term. This study also included
induction of PAL enzyme during the growth of R. glutinis yeast
cells. In this paper, we also describe the modification of a gas
* Author to whom correspondence should be addressed [e-mail
godwin_dcunha@cbu.ca; fax (902) 563-1880; telephone (902) 563-
1967].
† Present address: Department of Chemistry, Dalhousie University,
Halifax, NS, Canada B3H 4J3.
§ Present address: Undergraduate Pharmacy Program, Dalhousie
University, Halifax, NS, Canada B3H 4J3.
10.1021/jf072614u CCC: $40.75
2008 American Chemical Society
Published on Web 01/15/2008

Storage Stabilization of PAL Activity
J. Agric. Food Chem., Vol. 56, No. 3, 2008 895
an orbital shaker incubator. The reaction was allowed to proceed
overnight to obtain an appreciable amount of product, L-Phe. PAL
reverse reaction product was then identified by paper chromatography
and quantified by spectrophotometric determination at 520 nm, using
a method reported earlier (19).
Stabilization of PAL Activity during Storage. Stability studies
included analyzing the PAL forward reaction using both R. glutinis
whole cells as the enzyme source and the crude enzyme extract obtained
by ultrasonication treatment of the yeast cells.
chromatography–mass spectrometry (GC-MS) method for the
rapid and sensitive determination of PAL reverse reaction of
R. glutinis.
Enzyme stabilization is an extremely important aspect of
biocatalysis in commercial applications to reduce production
costs. The main objective of our study is focused on Mn2+
-
dependent stabilization of PAL, which has not been reported
before. In addition to further information on stability of PAL
(a tetrameric protein), these data may be significant in predicting
metal ion stabilization of other multimeric enzymes and proteins.
The influence of the presence of various compounds on the stability
of PAL activity of R. glutinis during storage at 0–2 °C was investigated.
The effect of different temperatures (-20, 0–2, 25, 30, and 37 °C) and
various concentrations of Mn2+ (0.0025-0.05%) on the stability of R.
glutinis PAL during storage was studied separately. Cells were grown
in a yeast extract medium (27 h, 30 °C, and 150 rpm), washed twice
with 0.9% NaCl, harvested, suspended in 10 mL of 50 mM Tris-HCl
buffer, pH 8.8, and stored at 0–2 °C. Each of the compounds used for
enzyme stabilization was added to the storage buffer medium prior to
adjustment of the pH to 8.8. Cells stored in storage buffer without any
additive served as a control. PAL activity of the stored cells was
monitored periodically every 2 weeks for a total of 12 weeks.
MATERIALS AND METHODS
Microorganism. The yeast strain R. glutinis RE4607095D used in
this study, procured from Oxoid Inc. (Nepean, ON, Canada), was
maintained by weekly transfers on 3.0% agar plates and slants
containing 1.0% peptone, 1.0% yeast extract, and 0.5% NaCl.
Chemicals. Yeast extract, peptone, and bacteriological agar were
obtained from Oxoid Ltd. (Basingstoke, Hampshire, U.K.). Sabouraud
dextrose agar was procured from Acumedia Manufacturers, Inc.
(Baltimore, MD). L-Phe, L-tyrosine (L-Tyr), L-isoleucine (L-Ile), L-
alanine (L-Ala), L-aspartic acid (L-Asp), L-tryptophan (L-Trp), L-serine
(L-Ser), L-glutamic acid (L-Glu), t-CA, Tris, n-butanol, trifluoroacetic
anhydride, cetyl pyridinium chloride (CPC), glycerol, sorbitol, and
sucrose were purchased from Fisher Scientific (Fair Lawn, NJ).
Polyethylene imine (PEI), polyethylene glycol (PEG), ethylene glycol,
ATP, pyridoxal phosphate, and glutaraldehyde were obtained from
Sigma-Aldrich Canada (Oakville, ON, Canada). Commercially available
inorganic chemicals including metal ion salts of the highest analytical
grade were used without any further treatment.
The influence of selected metal ion additives (Mn2+, Mg2+, Zn2+
,
Ca2+, and Li+) and temperature (from –20 to 37 °C) on the stability
of PAL isolated from R. glutinis whole cells was studied in a similar
manner. Aliquots of supernatant fluid (obtained from sonicated cell
extracts) were mixed with buffers containing the respective
additives.
Induction of PAL Activity. Various additives were incorporated
into the yeast extract growth medium to determine their effect on
induction of PAL activity. The following additives were tested: L-Ala,
L-Asp, t-CA, L-Ile, L-Phe, L-Ser, L-Trp, and L-Tyr. Cells were then
grown for 27 h at 30 °C on an orbital shaker. Cells grown in a medium
without any of the above additives served as control. The effect of
different concentrations of L-Phe on the induction of PAL activity was
studied separately.
Culture Conditions. R. glutinis cells from Sabouraud dextrose agar
plates were grown in a culture medium containing 1.0% yeast extract,
1.0% peptone, and 0.5% NaCl at 30 °C on an orbital shaker (150 rpm).
Cells were harvested in the late log phase (24–27 h) when maximal
PAL activity was observed. Cell amounts are reported on a dry weight
basis.
Gas Chromatography–Mass Spectrometric (GC-MS) Determi-
nation of PAL Reverse Reaction. Initially PAL reverse reaction
product, L-Phe, was identified and quantified by using a paper
chromatography-visible spectrophotometric method developed in our
laboratory earlier (19). Because paper chromatography is an out-dated
and time-consuming method, a modified GC-MS method based on a
protocol reported by Deng and Deng (21) was optimized for determining
L-Phe formed as a result of PAL reverse reaction.
Secretion of PAL Enzyme during Incubation. Ten milliliter
aliquots of a 27 h grown culture were centrifuged (7500g for 15.0 min).
The enzyme activities of the whole cells and supernatant fluid were
determined independently to check for secretion of PAL during the
growth incubation period.
Crude Enzyme Isolation. R. glutinis whole cells suspended in Tris-
HCl buffer (50 mM, pH 8.8) were subjected to the following treatments:
(a) intermittent ultrasonication (50 Hz/ sec) in a Branson 1510 ultrasonic
bath for an hour in ice, (b) CPC (0.001%) detergent permeabilization
for an hour, and (c) synergistic ultrasonication and CPC detergent
solubilization treatment. After the respective treatment, the cell extracts
were centrifuged at 7500g for 15.0 min using a Damon IEC HN-SII
centrifuge. PAL activity of the supernatant fluids and cell debris residue
was monitored at the end of each treatment. Aliquots of supernatant
fluid corresponding to 10.0 mg of R. glutinis cells were used for enzyme
assays.
PAL Forward Assay. The PAL forward activity of R. glutinis was
measured by a modification of a method reported earlier: spectropho-
tometric determination of t-CA formation from L-phe at an absorbance
of 290 nm (17). The reaction mixture (5.0 mL) containing 50 mM Tris-
HCl buffer, pH 8.8, 37.5 mM L-Phe, 0.005% CPC, and 10.0 mg R.
glutinis cells was incubated at 30 °C for 10.0 min. Inactivating the
enzyme with concentrated HCl and separating the cells by centrifugation
then terminated the reaction. The absorbance of the clear supernatant
fluid was measured at 290 nm. The reaction mixture in which substrate
was added after termination of the reaction served as the blank. The
enzyme activity is expressed as units; 1 unit of enzyme is defined as
the amount required to convert 1 nmol of L-Phe per minute per
milligram of dry cells at room temperature.
Samples included PAL reverse reaction control (R. glutinis cells
boiled for 15 min) and experimental and commercial L-Phe and t-CA
standards.
Derivatization. One hundred microliters of each sample was
evaporated under a stream of N2 at 40 °C, and the residue was reacted
with 100 µL of n-butanol at 150 °C for 60 min. The solvent was
evaporated to dryness under a stream of N2 at 40 °C, and the butyl
derivatives were reacted with 100 µL of a mixture of trifluoroacetic
anhydride and acetonitrile (1:1 v/v) at 100 °C for 30 min. Finally, the
derivatized samples were evaporated to dryness under a stream of N2
at 40 °C and redissolved in 100 µL of methanol.
Separation. Analytes were separated using an Agilent Technologies
Inc. HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film
thickness) inserted in a Hewlett-Packard GC-MS in 70 eV EI mode.
One microliter of derivatized standards (L-Phe and t-CA) and 3 µL of
control and experimental were injected in splitless mode.
Oven Temperature Program. An initial temperature of 50 °C for
2 min was increased to 300 at 15 °C/min and maintained at 300 °C for
10 min. Helium carrier gas at a flow rate of 1 mL/min was used, and
the detector was set at a temperature of 280 °C. The qualitative analysis
was performed under full-scan acquisition mode within 50–550 amu
range, and quantification was carried out under scan mode: m/z
50–300.
PAL Reverse Assay. The reaction mixture was made up of 5 mL
with 0.1 M Tris-HCl buffer, pH 9.0, containing 0.1 M t-CA, 4.0 M
NH4OH, 0.005% CPC, and 400.0 mg of R. glutinis cells. Samples
containing cells boiled at 100 °C for 15 min served as controls. The
reaction mixtures were incubated at 30 °C with shaking (200 rpm) on
Data Analysis. The values reported are the mean of at least three
independent determinations.

896 J. Agric. Food Chem., Vol. 56, No. 3, 2008
Wall et al.
Table 1. Optimal Parametersa for the Assay of R. glutinis Yeast PAL
this aim, an effort was made to stabilize PAL by eliminating/
retarding the factors that could be possibly contributing to
enzyme inactivation. As these inactivation mechanisms are
poorly understood, conventionally used methods of protein
stabilization were employed.
parameter
cell amount
concentration of L-Phe
time
temperature
concentration of CPC
50 mM buffer, pH 7.5
pH
range/parameter tested
optimal value/parameter
2.0–40.0 mg
10.0–75 mM
5.0–60.0 min
25, 30, 37 °C
0.001-0.05%
borate, phosphate, Tris-HCl
7.0–10.0
10.0 mg
37.5 mM
10.0 min
30 °C
0.005%
Tris-HCl
8.8
(a) PAL Stabilization by Exogenous Compounds. From the
data presented in Table 3, it is clear that among the additives
tested for their effect on the stability of PAL, only polyhydric
alcohols (25% glycerol, 5% ethylene glycol, and 1% PEG) gave
significant reduction in enzyme inactivation during storage.
Appreciable PAL activity (about 30–35%) was retained for
about 10–12 weeks in the presence of glycerol and PEG.
It is evident from Table 4, which summarizes the effect of
different metal ions on the storage stability of PAL, that both
Mn2+ and Mg2+ had influenced a remarkable stabilizing effect
on PAL and that appreciable enzyme activity was retained on
at least 12 weeks of storage at 0–2 °C. The retention of PAL
activity in the presence of Mn2+ and Mg2+ at the end of 12
weeks was 85 and 65%, respectively. Mn2+ gave very encour-
aging results; therefore, the concentration dependency of this
divalent cation for PAL stabilization was explored further. PAL
activity could be extended to about 10 weeks in the presence
of Zn2+ and Li+.
(b) Effect of Mn2+ Concentration and Temperature. The
optimal concentration of Mn2+ required for obtaining maximum
stabilization of PAL was determined. At an optimal Mn2+
concentration of 0.01%, R. glutinis cells retained nearly 85–90%
of the original PAL activity after 12 weeks of storage (Figure
1). A higher concentration (>0.01%) of Mn2+ did not make a
significant difference in enzyme stability. It can be seen from
Figure 2 that cells retained maximum PAL activity (about 85%
for 12 weeks) when stored at 0–2 °C. Cells stored at –20 °C
retained nearly 80% of the original enzyme activity after 12
weeks of storage. However, a progressive decline in enzyme
activity was observed when cells were stored at 25 °C or higher
temperatures.
a Under these optimized conditions, PAL activity of R. glutinis cells was 34.0
units. Cells grown in a yeast extract medium for 27 h at 30°C were used for
determining PAL activity. One enzyme unit is defined as nanomoles of L-Phe
transformed per minute per milligram of dry cells.
Table 2. PAL Activitya of Enzyme Isolated from R. glutinis Whole Cells
treatment
PAL activity (units)
control (untreated cells)
ultrasonication
0.001% CPC
34.0
30.3
16.0
30.5
ultrasonication + CPC
a PAL activity is reported in terms of nanomoles of L-Phe transformed per minute
per milligram of dry cells. The details of each treatment procedure for extracting
the enzyme from R. glutinis yeast whole cells are discussed under Materials and
Methods.
RESULTS
Optimal Parameters for Assay of R. gluitinis PAL. The
various parameters that were optimized to obtain maximal PAL
activity in R. glutinis whole cells are presented in Table 1. The
enzyme activity was proportional with time and the amount of
cells in the range selected for routine assays. The optimal
concentration of the substrate, L-Phe, was found to be 37.5 mM,
which is about 18 times the Km value of 2.1 mM reported for
PAL from Rhodotorula (22). The amount of cells and concen-
tration of L-Phe exceeding the optimal values (10 mg and 37.5
mM, respectively) did not further increase PAL activity of cells
significantly. The presence of a low concentration (0.005% v/v)
of detergent, CPC, in the assay mixture was necessary to
permeabilize the yeast cells to substrate and products. Omitting
the detergent from the assay mixture gave only 50% of the PAL
activity obtained in its presence. CPC concentrations of >0.1%
were found to be inhibitory to PAL.
Secretion of PAL Enzyme during Incubation. The PAL
activity of the supernatant fluid (0.068 unit) obtained after the
centrifugation of the cells from the culture was negligible compared
to that of the whole yeast cells (34.0 units). This ruled out the
secretion of the enzyme by the yeast cells into the medium during
growth. Therefore, it can be concluded that there is no rupture of
cells during incubation due to osmotic factors.
PAL Activity of Isolated Enzyme. The PAL activity of the
enzyme extracts obtained at the end of each isolation treatment is
shown in Table 2. It is evident that ultrasonication was an effective
method of PAL extraction (about 89% enzyme activity was
recovered in the supernatant fluid). Because the presence of 0.001%
CPC along with ultrasonication did not show a significant effect
in enhancing enzyme recovery, enzyme extracted by ultrasonication
treatment alone was used for stability studies. The cell debris
residue obtained on centrifugation of the sonicated cell suspension
was discarded as it had very low PAL activity (<5% activity of
the original whole cells).
(c) Effect of Metal Ions and Temperature on Stabilization of
Isolated PAL. The effect of selected metal ions on stabilization
of PAL isolated from R. glutinis whole cells is shown in Table
5. The protective effects of Mn2+ and Mg2+ on isolated PAL
were similar to their effects on PAL-containing cells. In the
presence of 0.01% of Mn2+ and Mg2+, the isolated enzyme
could retain 73 and 58% activity, respectively, for at least 12
weeks. The optimal temperature for maximal retention of activity
of isolated enzyme upon storage was also 0–2 °C. At this
temperature, the extracted PAL preparation retained 70–75%
activity for about 12 weeks.
Induction of R. glutinis PAL Activity. The effect of t-CA
and individual amino acids including L-Phe on PAL enzyme
during fermentation was assessed in shake flask studies (Table
6). L-Phe, L-Tyr, and L-Ile gave significant enhancement of PAL
activity, whereas t-CA was found to be a strong inhibitor.
Addition of 0.05% L-Phe to fermentation medium resulted in a
nearly 4-fold increase in enzyme activity (Figure 3). Higher
concentrations of L-Phe (>0.05%) gave negligible increases in
PAL activity. Although L-Tyr at a concentration of 0.05%
showed inducive effects on PAL activity of the yeast cells, a
decrease in enzyme activity was observed when the concentra-
tion was increased beyond 0.25% (data not shown). Inhibition
of PAL activity was observed with the use of t-CA at a
concentration as low as 0.025%.
Stability of R. glutinis PAL Activity during Storage. The
PAL enzyme has several applications (11–14) including the
synthesis of L-Phe (15–17) and its methyl ester (18); therefore,
a source of stable PAL is of considerable significance. With
Determination of R. glutinis PAL Reverse Reaction. An
improved method for the synthesis of L-Phe, its identification,
and quantification by paper chromatography was developed in

Storage Stabilization of PAL Activity
J. Agric. Food Chem., Vol. 56, No. 3, 2008 897
Table 3. Effect of Various Compoundsa on Storage Stability of R. glutinis PAL
PAL activity on storage (units) after
compound
2 weeks
4 weeks
6 weeks
8 weeks
10 weeks
12 weeks
control (no additive)
1.0 mMATP
0.5 mM pyridoxal phosphate
1.0% L-isoleucine
25.0% glycerol
5.0% ethylene glycol
1.0% polyethylene glycol
10.0 mM glutamic acid
1.0 M sorbitol
0.1 M sucrose
1.0% polyethyleneimine
0.5% glutaraldehyde
10.3
10.4
10.4
10.5
33.4
32.7
30.2
10.3
10.7
21.1
9.5
2.9
3.2
3.2
2.9
25.7
20.4
26.8
2.9
0.1
2.0
1.9
0.2
21.4
16.7
21.3
0.2
2.1
6.1
0.6
0.5
0.1
17.5
8.2
16.7
0.1
0.6
2.7
0.2
0.5
12.6
2.1
6.9
4.0
1.8
3.7
14.2
2.4
0.5
0.9
1.8
9.8
2.8
a Each of these compounds was added to the storage buffer medium prior to adjustment of the pH to 8.8. Original PAL activity of R. glutinis cells was 34.0 units. PAL
activity is reported in terms of units (nanomoles of L-phe transformed per minute per milligram of dry cells). Cells were stored at a refrigerated temperature (0–2 °C).
Table 4. Effect of Various Metal Ionsa on Storage Stability of R. glutinis PAL
PAL activity on storage (units) after
metal ion (0.01%)
2 weeks
4 weeks
6 weeks
8 weeks
10 weeks
12 weeks
control (no metal ion)
10.3
8.6
2.9
2.5
4.2
10.8
28.9
31.9
33.4
18.1
9.4
0.1
1.1
1.1
6.6
16.0
30.6
32.9
8.4
Na+
K+
0.4
10.3
11.7
32.4
33.5
33.6
31.7
30.9
15.7
34.0
Li+
3.6
7.1
1.5
2.1
Zn2+
Mg2+
Mn2+
Ca2+
Co2+
Cu2+
Fe2+
27.2
32.0
2.0
24.5
30.6
22.1
28.7
5.2
1.9
3.9
9.9
4.2
1.7
a Each of these metal ions was added to the storage buffer medium prior to adjustment of the pH to 8.8. Original PAL activity of R. glutinis cells was 34.0 units. PAL
activity is reported in terms of units (nanomoles of L-Phe transformed per minute per milligram of dry cells). Cells were stored at refrigerated temperature (0–2 °C).
Figure 1. Effect of Mn2+ concentration on the storage stability of R. glutinis PAL. Mn2+ was added to the storage buffer medium prior to adjustment
of the pH to 8.8. The details of the storage buffer are mentioned under Materials and Methods. Original PAL activity of R. glutinis cells was 34.0 units.
PAL activity is reported in terms of units (nanomoles of L-Phe transformed per minute per milligram of dry cells). Cells were stored at refrigerated
temperature (0–2 °C).
our laboratory earlier (19). An optimized GC-MS method for
the determination of R. glutinis PAL reverse reaction is reported
in the current study. Figure 4 shows the chromatograms of the
standards (L-Phe and t-CA) and PAL reverse reaction samples
(control and experimental); the mass spectra of the derivatized
standards are shown in Figure 5. The retention times of
derivatized L-Phe and t-CA standards were found to be 12.75
and 12.60 min, respectively. It is evident from the chromatogram
of control that the substrate, t-CA, was unreacted because the
enzyme was inactivated by boiling. The experimental gave
distinct peaks for L-Phe (formed as a result of PAL reverse
reaction) and unreacted t-CA at retention times of 12.75 and
12.60 min, respectively. These retention times coincided very
well with the retention times of L-Phe and t-CA standards.

898 J. Agric. Food Chem., Vol. 56, No. 3, 2008
Wall et al.
Figure 2. Effect of temperature on the storage stability of R. glutinis PAL. 0.01% Mn2+ was added to the storage buffer medium prior to adjustment of
the pH to 8.8. Original PAL activity of R. glutinis cells was 34.0 units. PAL activity is reported in terms of units (nanomoles of L-Phe transformed per
minute per milligram of dry cells).
Table 5. Effect of Various Metal Ionsa on Storage Stability of Isolated PAL
PAL activity on storage (units) after
metal ion (0.01%)
2 weeks
4 weeks
6 weeks
8 weeks
10 weeks
12 weeks
control (no metal ion)
Li+
5.4
9.4
1.0
7.0
3.4
27.8
24.7
8.2
Mn2+
Mg2+
Zn2+
Ca2+
29.5
29.1
25.2
20.5
29.0
27.3
17.9
15.7
26.5
22.6
2.6
24.9
21.2
22.1
17.4
5.2
1.3
a Each of these metal ions was added to the storage buffer medium prior to adjustment of the pH to 8.8. Aliquots of supernatant fluid containing isolated PAL were added
to the storage buffer medium. Original activity of isolated enzyme was 30.3 units. PAL activity is reported in terms of units (nanomoles of L-Phe transformed per minute
per milligram of dry cells). Storage buffer medium containing extracted enzyme was stored at refrigerated temperature (0 –2 °C).
Table 6. Effect of Various Compoundsa on Induction of R. glutinis PAL
Although not the highest PAL producer, the value of 34.0 units
obtained in the present study (Table 1) is higher than most
compound (0.05%)
PAL activity (units)
values reported before. No significant amount of PAL secretion
by R. glutinis has been shown to occur, although a related
fungus, Neurospora crassa, is known to secrete this enzyme
during growth (5). The absence of secretion of enzyme in the
growth medium is advantageous in preventing loss of available
enzyme. Ultrasonication treatment of R. glutinis whole cells was
very effective in enzyme isolation, and we were able to extract
about 90% of the cell PAL activity.
control (no additive)
L-alanine
L-aspartic acid
trans-cinnamic acid
L-isoleucine
L-phenylalanine
L-serine
L-tryptophan
L-tyrosine
34.0
37.3
31.6
7.18
116.5
128.5
35.4
42.5
101.9
The use of stabilizing additives is a common method of
maintaining enzyme activity (10, 25, 26). Additives are known
to stabilize enzymes by either binding to the active form, shifting
the equilibrium to that form, or forming a more stable
conformation, thereby preventing denaturation (25, 26). In the
present study (Table 3), it was found that polyhydric alcohols
(25% glycerol, 5% ethylene glycol, and 1% PEG) gave
appreciable protection to PAL and prolonged the PAL activity
of stored cells to about 10 weeks. The most pronounced
influence was observed with glycerol, and cells retained nearly
37% activity for 10 weeks. On the basis of a study involving
the interaction of glycerol with a number of purified model
proteins in aqueous solutions, Gekko and Timasheff (26) have
concluded that the conformation of protein is stabilized due to
preferential hydration of protein in the presence of glycerol.
Although mere extrapolation of the results of the data obtained
with purified proteins on complex cellular systems is not strictly
valid, the polyhydric alcohols tested here somehow maybe
a Each of these additives was added to the fermentation medium prior to
inoculation with R. glutinis seed culture. The cells were assayed for PAL activity
after 27 h of growth in the yeast extract medium. The details of the medium are
mentioned under Materials and Methods. PAL activity is reported in terms of units
(nanomoles of L-Phe transformed per minute milligram of dry cells).
DISCUSSION
The availability of a rich and stable enzyme source is a
prerequisite for a biocatalyst in commercial applications. In addition
to a number of potential applications (11–14), PAL mediates the
stereospecific formation of optically pure L-Phe (15–17) and
L-phenylalanine methyl ester (18). However, there have been very
few reports on stabilization (10) and induction (8). The present
study confirms and further extends the earlier work on Rhodotorula
PAL stabilization, induction, and determination.
A compilation of PAL activity of different Rhodotorula
species from earlier work reveals that the enzyme activity varies
in the range of 2.0–80.0 units/mg of dry cells (8, 10–12, 15).

Storage Stabilization of PAL Activity
J. Agric. Food Chem., Vol. 56, No. 3, 2008 899
Figure 3. Effect of phenylalanine concentration on the induction of R. glutinis PAL. L-Phe was added to the yeast extract fermentation medium prior to
inoculation with R. glutinis seed culture. The details of the fermentation medium are mentioned under Materials and Methods. The cells were assayed
for PAL activity after 27 h of growth in the yeast extract medium. Original PAL activity of R. glutinis cells (cells grown in the absence of an additive) was
34.0 units. PAL activity is reported in terms of units (nanomoles of L-Phe transformed per minute per milligram of dry cells).
Figure 4. Chromatograms of (a) standard L-phenylalanine, (b) trans-cinnamic acid, (c) control, and (d) experimental. Retention time of derivatized
standards was 12.75 (L-Phe) and 12.60 (t-CA) minutes, respectively. In the control, the substrate t-CA was not consumed because the enzyme was
inactivated by boiling R. glutinis cells at 100 °C for 15 min. In the experimental, peaks for L-Phe formed as a result of PAL reverse reaction and
unreacted trans-cinnamic acids were obtained at 12.75 and 12.60 min, respectively. These retention times are identical to the ones obtained with the
standards.
stabilizing the PAL active conformation inside the cells in a
similar manner. It is pertinent to mention here that L-Ile, which
has been reported to have a stabilizing influence by retarding
the decline of PAL activity during growth of cultures (15), did
not have a pronounced effect on PAL stability during storage.
The most significant and novel finding of the present study

900 J. Agric. Food Chem., Vol. 56, No. 3, 2008
Wall et al.
Figure 5. Mass spectra of derivatized standards (a) L-phenylalanine and (b) trans-cinnamic acid.
is the stabilization of PAL (contained in R. glutinis cells and in
isolated form) during storage by a low concentration of Mn2+
of Rhodotorula PAL with Mg2+ at a concentration of 2 M. It is
difficult to interpret their data in light of the present findings
because they have chosen a single and rather high (2 M)
concentration of the metal ion to test retention of PAL activity.
However, it is interesting to note that in the present study, a
higher concentration of Mn2+ (>0.01%) did not make a
significant difference in enzyme stability. It should also be
mentioned that in addition to 85% retention of enzyme activity
with 0.01% Mn2+, about 65% of the original enzyme activity
was retained for 12 weeks when 0.01% Mg2+ was present in
.
Salts containing Mn2+ are readily available and not expensive;
therefore, these additives can be effectively used in protein
stabilization procedures. Inclusion of 0.01% Mn2+ in the storage
buffer medium resulted in retention of nearly 85% of the original
enzyme activity in the case of PAL containing R. glutinis cells
(Figure 1) and 73% with isolated PAL (Table 5) for a period
of at least 12 weeks. In their study involving the bioconversion
of t-CA to L-Phe, Evans et al. (10) have reported stabilization

Storage Stabilization of PAL Activity
J. Agric. Food Chem., Vol. 56, No. 3, 2008 901
the storage buffer (Table 4). The role of Mg2+ in PAL
stabilization reported earlier (10) is not known, and we have
yet to investigate the possible biochemical mechanism of PAL
stabilization by Mn2+. It could be due to the specific stabilization
of a more active conformation of the enzyme in the presence
of these divalent metal ions. Another unique observation made
during the investigation of the influence of metal ions on enzyme
stabilization was that K+, which is known to be cofactor for a
number of enzymes, was not very effective in PAL stabilization,
and activity was almost negligible in about 6 weeks. The rapid
decline in PAL activity in the presence of Cu2+ could be
possibly brought about by the heavy metal ion binding to
essential SH group(s) on the enzyme protein, thereby distorting
its biologically active form.
PAL is an inducible enzyme (7, 8, 10, 15, 27), and the results
obtained from this study (Table 6) were in good agreement with
earlier findings. Addition of 0.05% L-Phe in the fermentation
medium resulted in a 3.8-fold enhancement of PAL activity of
the yeast cells. The PAL activity of 128.5 units with L-Phe-
induced cells is significantly higher than the values of 35, 50,
and 79 units reported by Yamada et al. (15), Evans et al. (10),
and Orndorff et al. (8), respectively. Inducible enzymes such
as PAL show an increase in activity, as the inducer concentration
is increased to a certain concentration when enzyme activity
reaches a maximum. In the present study it was found that a
higher concentration (>0.05%) of L-Phe did not show significant
increase in PAL activity of the cells (Figure 3). L-Tyr, which
is structurally similar to L-Phe, when present at a concentration
of 0.05% in the fermentation medium, gave a 3-fold increase
in PAL activity. However, a decrease in enzyme activity was
observed when the concentration of L-Tyr was increased beyond
0.25% (data not shown). Because the role of L-Tyr in PAL
induction is not known and was not investigated in this study,
more work is needed in this direction. A report by Nakamichi
et al. (27) suggested that other amino acids such as D- or
L-isoleucine, D- or L-leucine, L-methionine, L-tryptophan, and
L-tyrosine induced PAL activity in R. glutinis. Although we
obtained a 3-fold enhancement in enzyme activity with 0.05%
L-Ile, there was no significant increase in PAL activity in the
presence of L-Ala, L-Asp, L-Ser, and L-Trp. It was found that
the addition of t-CA (in a concentration as low as 0.025%) to
growth media resulted in negligible PAL activity of the yeast
cells. This is possibly because of the enzyme being inhibited
by its end product.
at studying the mechanism of stabilization of Rhodotorula PAL
by Mn2+ and Mg2+
.
ACKNOWLEDGMENT
We thank Dr. Allen Britten and Dr. Krishnat Naikwadi for
their help during GC-MS studies; Dr. Michael Tanchak and Paul
McDougall for the use of their respective laboratories and
equipment during culturing and harvesting of R. glutinis yeast
cells; and M. Jason MacDonald for assistance in preparing
figures.
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Received for review September 4, 2007. Revised manuscript received
November 16, 2007. Accepted November 30, 2007. Financial support
for this work was funded by Cape Breton University research office.
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