5426 J . Org. Chem., Vol. 62, No. 16, 1997
Gavagan et al.
cerevisiae catalase T plus endogenous H. polymorpha catalase),
and catalase T activities present in extracts of H. polymorpha
double transformants ranged from 95 to 160 DCIP IU/g of wet
cells for glycolate oxidase, 165000-315000 IU/g of wet cells
for total catalase, and 90000-140000 IU/g of wet cells for
catalase T.
8.5. The reaction vessel was sealed, the reaction mixture was
cooled to 5 °C and then the vessel was flushed with oxygen by
pressurizing to 70 psig and venting to atmospheric pressure
five times with stirring. The vessel was then pressurized to
70 psig of oxygen and the mixture stirred at 5 °C. Aliquots
(0.10 mL) were removed by syringe through a sampling port
(without loss of pressure in the vessel) at regular intervals
for analysis by HPLC to monitor the progress of the reaction.
After 6 h, the HPLC yields of glyoxylate, formate, and oxalate
were 92.3, 4.36, and 5.5%, respectively, and no glycolate
remained. The remaining activity of glycolate oxidase and
catalase were 87 and 88%, respectively, of their initial values.
Oxid a tion of Glycolic Acid /AMP A Mixtu r es Usin g Co-
Im m obilized Glycola te Oxid a se a n d Ca ta la se. In a typi-
cal procedure, a 300 mL stirred autoclave was charged with
100 mL of a solution containing glycolic acid (0.50 M),
(aminomethyl)phosphonic acid (0.375 M), isobutyric acid (0.10
M, HPLC internal standard), and flavin mononucleotide (FMN,
0.01 mM), and the solution was cooled to 5 °C. The pH of the
solution was adjusted to 8.5 with 50% sodium hydroxide, and
then 80 IU of spinach glycolate oxidase and 240 000 IU of A.
niger catalase co-immobilized4 on 24 g of oxirane acrylic beads
(Eupergit C, Accurate Chemical and Scientific Corp.) was
added (0.80 IU/mL glycolate oxidase, 2400 IU/mL catalase).
The resulting mixture was stirred at 400 rpm and 5 °C under
70 psig of oxygen, while oxygen was sparged through the
mixture at 50 mL/min. The reaction was monitored by
removing a 0.40 mL aliquot of the reaction mixture at regular
intervals, filtering the aliquot using a Millipore Ultrafree-MC
10000-MW-cutoff filter unit (to remove the enzyme catalyst),
and analyzing by HPLC. After 3.5 h, the yields of glyoxylic
acid, oxalic acid, and formic acid were 87.8, 2.5, and 3.3%,
respectively, with 9.2% glycolic acid remaining. The final
activities of glycolate oxidase and catalase were 78 and 94%
of their initial values, respectively. The immobilized enzyme
catalyst was recycled by filtering the product mixture through
a 2.5 cm i.d. × 20 cm glass column equipped with a 20 µm
polyethylene bed support. The remaining liquid adsorbed on
the catalyst was removed by briefly passing a stream of
nitrogen through the column, then the catalyst was resus-
pended in 100 mL of a fresh 5 °C solution containing glycolic
acid (0.50 M), (aminomethyl)phosphonic acid (0.375 M), isobu-
tyric acid (0.10 M, HPLC internal standard), and flavin
mononucleotide (0.01 mM) at pH 8.5. The 300 mL autoclave
reactor was again charged with this reaction mixture and the
reaction repeated.
Oxid a tion of Glycolic Acid /AMP A Mixtu r es Usin g
P er m ea bilized Micr obia l Tr a n sfor m a n t Ca ta lysts. In a
typical procedure, a 300 mL stirred autoclave reactor was
charged with 100 mL of a solution containing glycolic acid
(0.500 M), AMPA (0.375 M), isobutyric acid (0.100 M, HPLC
internal standard), and FMN (0.01 mM) at pH 8.3 (adjusted
with 50% NaOH), and the solution was cooled to 5 °C. To the
reactor were then added 5.0 g of permeabilized H. polymorpha
double transformant 13.168 (357 IU glycolate oxidase and
600 000 IU total catalase (50% S. cerevisiae catalase T, 50%
H. polymorpha endogenous catalase)). The pH of the resulting
mixture was readjusted to 8.3 with 5% NaOH. This mixture
was stirred at 750 rpm, which sparged oxygen through the
mixture via the action of the turbine impeller, and at 5 °C
under 40 psig of oxygen. The reaction was monitored by taking
a 0.20 mL aliquot of the reaction mixture at regular intervals,
filtering the aliquot using a Millipore Ultrafree-MC 10000
NMWL filter unit, and analyzing the filtrate by HPLC. After
1.5 h, the yields of glyoxylic acid, oxalic acid, and formic acid
were 90.0, 3.1, and 1.8%, respectively, with 5.0% recovery of
glycolic acid. The final activities of permeabilized-cell glycolate
oxidase and total catalase were 146 and 113%, respectively,
of their initial values after permeabilization.
Wet cell weights of permeabilized or whole cell microbial
transformants used as catalysts or in assays were determined
by blotting a known weight (ca. 0.250 g) of cell paste (obtained
by centrifugation of fermentation broth or from cell suspen-
sions in buffer) on filter paper to remove excess moisture, then
reweighing the blotted cell paste to determine wet cell weight;
this procedure provided a reproducible method for determining
the fraction of water in fresh or frozen cell paste. G.O.
activities are reported in DCIP units unless otherwise noted.
P er m ea biliza tion of P . pa stor is a n d H. polym or ph a
Micr obia l Tr a n sfor m a n t s. A suspension of 10 wt % wet
cells in 50 mM phosphate buffer (pH 7.0) containing 0.1% (w/
v) of either benzalkonium chloride or Barquat MB-50 (Lonza)
was mixed for 60 min at 25 °C.3,6 The mixture was then
centrifuged, the supernatant was decanted, and the cells were
washed three times (10% w/v) in 50 mM phosphate buffer (pH
7.0) at 5 °C. The resulting cell paste could be used directly in
oxidation reactions, or frozen at -20 or -80 °C and stored at
these temperatures until needed. The amount of permeabi-
lized cell catalyst added to a reaction mixture was chosen so
as to provide the desired concentrations of glycolate oxidase
and catalase activities. Recoveries of permeabilized cell
catalyst from reactions with glycolate oxidase and catalase
activities of greater than 100% of their initial values were due
to increased permeabilization of the whole-cell catalyst during
the course of the reaction.
Glycola te Oxid a se a n d Ca ta la se Assa ys of Wh ole Cell
Tr a n sfor m a n ts. Whole cell transformant catalysts were
assayed for glycolate oxidase activity by first blotting ca. 0.25
g of permeabilized or unpermeabilized wet cells on filter paper
to remove excess water and then accurately weighing ca. 5-10
mg of the blotted wet cells into a 3 mL quartz cuvette
containing a magnetic stirring bar and 2.0 mL of a solution
which contained 2,6-dichlorophenol-indophenol (DCIP, 0.12
mM) and TRIS buffer (80 mM, pH 8.3). The cuvette was
capped with a rubber septum and the solution deoxygenated
by bubbling with nitrogen for 5 min. To the cuvette was then
added by syringe 0.040 mL of an aqueous solution of glycolic
acid (1.0 M) and TRIS buffer (1.0 M, pH 8.3), and the mixture
was stirred while the change in absorption with time at 606
nm was measured (ꢀ ) 22 000 L mol-1 cm-1).24
Catalase activity of permeabilized microbial single trans-
formants, and total catalase activity (S. cerevisiae catalase T
plus endogenous catalase) of permeabilized microbial double
transformants, was assayed by accurately weighing ca. 2-5
mg of the blotted wet cells into a 3 mL quartz cuvette
containing a magnetic stirring bar and 2.0 mL of 16.7 mM
phosphate buffer (pH 7.0), then adding 1.0 mL of 59 mM
hydrogen peroxide in 16.7 mM phosphate buffer (pH 7.0), and
measuring the change in absorption with time at 240 nm (ꢀ )
39.4 L mol-1 cm-1).25 The catalase T and endogenous catalase
activities of microbial double transformants were separately
determined by preparing extracts of the double transformants
and assaying the extracts as described above at pH 7.0 and at
pH 4.0; at pH 4.0, the endogenous H. polymorpha catalase
retains 7% of its activity at pH 7.0, while the S. cerevisiae
catalase T retained 60% of its activity at pH 7.0.
Oxid a tion of Glycolic Acid /AMP A Mixtu r es Usin g
Solu ble Glycola te Oxid a se a n d Ca ta la se. Into a 3 oz.
Fischer-Porter glass aerosol reaction vessel were placed a
magnetic stirring bar and 10 mL of an aqueous solution
containing glycolic acid (0.25 M), (aminomethyl)phosphonic
acid (AMPA, 0.20 M), FMN (0.01 mM), butyric acid (HPLC
internal standard, 0.10 M), glycolate oxidase (from spinach;
1.0 IU/mL), and catalase (from A. niger, 14 000 IU/mL) at pH
The microbial cell catalyst was recovered from the reaction
mixture described above by centrifugation. Without further
treatment the cell pellet was mixed with 100 mL of fresh
reaction mixture and the reaction repeated. After 1.5 h, the
yields of glyoxylic acid, oxalic acid, and formic acid were 84.3,
4.9, and 9.9%, respectively, with 1.7% recovery of glycolic acid.
The final activities of permeabilized-cell glycolate oxidase and
(24) Armstrong, J . McD. Biochim. Biophys. Acta 1964, 86, 194-197.
(25) Aebi, H. E. In Methods of Enzymatic Analysis; Bergmeyer, H.
U., Ed.; Verlag Chemie: Deerfield Beach, FL 1983; 3rd ed.; Vol. III,
pp 273-286.