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the reaction was stopped with 200 g l−1 K2CO3 (half of the reac-
tion volume). It was centrifuged for 10 min at 16,000 × g and the
absorption of the supernatant was measured at 405 nm.
The concentration of the hydrolyzed pNP--d-gal was deter-
mined from a pNP/K2CO3 calibration curve.
One Unit (1 U) of enzyme activity was defined as the amount
of enzyme required to liberate 1 mol pNP by the hydrolysis of
pNP--d-gal per min at 37 ◦C, pH 6.5.
C18, MZ Analysentechnik, Mainz, Germany, 150 × 3.0 mm, S: 5 m,
mobile phase: acetonitrile and water with 11.53 g l−1 phosphoric
acid, gradient: 0–5 min, 5% acetonitrile, 5–33 min 5–95% acetoni-
trile; 33–38 min 95% acetonitrile; 38–48 min 5% acetonitrile, flow:
0.4 ml min−1, temperature: 24 ◦C). Ethyl benzoate in methanol was
used as an internal standard for quantification.
2.10. Selective hydrolysis of allyl ˛-d-gal and allyl ˇ-d-gal
4 g l−1 of allyl ␣-d-gal and allyl -d-gal was incubated with
1 U ml−1 enzyme in a total volume of 625 l at 37 ◦C with shaking
for 24 h. A sample was removed at various times and the reaction
stopped by heating for 5 min at 95 ◦C. It was mixed with an internal
standard (glycerin, 50 mg ml−1) and analyzed with an HPLC fitted
with a refractive index detector (column: YMC-Pack Polyamine II,
YMC, 250 × 4.6 mm, S: 5 m, 12 nm, mobile phase: acetonitrile and
water, 85:15, flow 1 ml min−1, temperature: 24 ◦C).
2.6. Determination of molecular mass and KM of ˇ-galactosidase
The determination of the -galactosidase molecular mass was
done by gel filtration using SuperdexTM 200 (Amersham Bio-
sciences, UK). The flow rate was 1 ml min−1 and fractions of about
pH 6.5 + 0.15 M NaCl). A calibration curve was constructed using
bovine serum albumin, ovalbumin, catalase and alcohol dehydro-
genase as standards.
The KM value for the substrate pNP--d-gal was determined
using the Tate and Reynolds method [25]. The solution was
buffered at pH 6.5 with 50 mM sodium acetate buffer, contain-
ing 10 mM ␣-cyclodextrin. The reaction was followed for two min
and the increase in absorbance from the complex formation was
recorded. From these data, enzyme kinetics were calculated, and
Michaelis–Menten and Hanes plots were drawn. Determinations
were done in duplicate. The extinction coefficient of pNP in the
buffer was determined via Beer–Lambert equation (A = · c · d,
where A = absorbance, b = path length (cm) and c = concentration
(M)).
3. Results
The Xplor®2 transformation/expression platform was used for
the generation of a -galactosidase producing yeast strain because
it allows the construction of resistance marker-free transformants
[14]. The system is based on a bacterial vector backbone, with yeast
selection markers and expression modules inserted between two
25S rDNA segments. After the construction of the plasmids in E. coli,
all bacterial sequences are removed by AscI and SbfI restriction. The
choice of the restriction enzyme determined whether the rDNA
fragment flanks the expression cassette (AscI) or not (SbfI), so that
the -galactosidase gene can be integrated in the yeast genome
as a Yeast rDNA Integrative Cassette (YRC) or a Yeast Integrative
Cassette (YIC).
The -galactosidase concentration was determined using a
Coomassie stained SDS-PA gel for the calculation of kcat [26].
2.7. Protein analysis
The first step in producing a -galactosidase producing yeast
strain was to ligate one or two of the amplified LAC4-6H genes
with ATRP1m and AADE2 selection marker modules (Fig. 1).
Linearized DNA containing one or two LAC4-6H expression
modules and the ATRP1m selection marker module without
rDNA sequences (YIC102-LAC4-6H, YIC102-2LAC4-6H) or with
rDNA sequences (YRC102-LAC4-6H, YRC102-2LAC4-6H) were inte-
grated in the genome of the atrp1 auxotrophic mutant strain A.
adeninivorans G1212. In addition, the double mutant strain A.
adeninivorans MS1006 [atrp1, aade2] was co-transformed with
YRC102-2LAC4-6H and YRC104-2LAC4-6H or YIC102-2LAC4-6H
and YIC104-2LAC4-6H. Thus, recombinant yeast strains containing
one, two or four copies of the LAC4-6H expression module were
generated.
SDS-PAGE and Western analyses were performed as described
specific primary antibody produced in rabbits (Antibodies online
GmbH, Germany) and a goat anti-rabbit IgG alkaline phosphatase
conjugate (BIO-RAD, USA), and subsequently stained by incubation
with NBT/BCIP substrate (Roche Diagnostics, Switzerland).
The dye-binding method of Bradford [27] was used for protein
quantification (BIO-RAD, USA), with bovine serum albumin used as
the standard.
2.8. TLC for the analysis of lactose, glucose and galactose
The analysis of lactose, glucose and galactose in the supernatant
of different yeast strains was done by thin-layer chromatogra-
phy (Polygram SIL G/UV254 activated by 0.1 M boric acid and then
heated to 130 ◦C for 30 min) using acetic acid and n-butanol, 1:1, as
the solvent and 1% 4-methoxybenzaldehyd, 4% sulphuric acid, 95%
ethanol for development.
All cassettes were successfully transformed in both yeast strains
with around 96 transformants per cassette. After stabilization of
the transgenic yeast strains by passaging (see Materials and meth-
ods), no loss of YRCs or YICs was observed after cultivation on
non-selective medium for a period of 30 days.
2.9. Enzymatic synthesis of benzyl ˇ-d-gal by
transgalactosylation
3.2. Expression of LAC4-6H gene in A. adeninivorans
The enzymatic synthesis of benzyl -d-gal by transgalactosyla-
tion was done in a total volume of 500 l. Lactose was dissolved
in the purified enzyme preparation (0.1 U) and water. The reaction
was started by the addition of benzyl alcohol in a ratio of water:
benzyl alcohol 9:1 (v/v). It was incubated at 35 ◦C with shaking for
one day. Then the sample was centrifuged for 10 min, 16,000 × g
and enzyme activity was stopped by heating the supernatant for
5 min at 95 ◦C. Other solvents (acetone, acetonitrile, tert. butanol
or dioxane) if present, were evaporated until only the benzyl alco-
hol was left. Methanol was then added to give a total volume of
500 l. The sample was centrifuged again and the supernatant
was analyzed by HPLC with a UV-detector (column: Kromasil 100
Transgenic yeast strains carrying one LAC4-6H expression mod-
ule were grown in YMM-glucose-NaNO3 at 30 ◦C for 48 h. Since
LAC4-6H does not contain an obvious secretion sequence, the
recombinant protein should be localized intracellularly. Whole-cell
extracts were analyzed for the presence of recombinant Lac4-6hp
activity in the initial screen by testing the supernatant of whole
cells, disrupted cells and the supernatant of the disrupted cells for
-galactosidase activity. Most activity was detected in the super-
natant of the disrupted cells, suggesting the enzyme is located
intracellularly and is soluble. However, one third of the activity
was also detected in the disrupted cells (activity of the intracellular
soluble fraction excluded).