Isobutanol Production by Using a Non-Cell-Based Enzymatic Method
with HiTrap FF-, HiPrep 26/10 Desalting- and HiTrap Q-Sepharose
FF-columns (GE Healthcare). Cell lysates were prepared with
a Basic-Z Cell Disruptor (Constant Systems, Northants, UK), cell
debris was removed by centrifugation at 35,000ꢃg and 48C for
duced to NADH. Assay mixture contained 50mm HEPES (pH 7),
+
[23]
2.5mm MgCl , 4mm NAD and 5mm d,l-glyceraldehyde.
2
ALS activity: ALS activity was determined by following pyruvate
consumption at 508C. Reaction mixtures contained 25 mm HEPES
3
0 min (Sorvall RC6+, SS-34 rotor, Thermo Scientific). For lyophili-
(pH 7), 0.1 mm thiamine pyrophosphate, 2.5mm MgCl , 15mm
2
zation an Alpha 2–4 LD Plus freeze dryer (Martin Christ GmbH, Os-
terode am Harz, Germany) was used. GDH and DHAD were purified
by heat denaturation (30 min at 708C, respectively). GDH was sub-
sequently freeze-dried (SpeedVac Plus, Thermo Scientific), DHAD
concentrated using a stirred Amicon cell (Milipore, Darmstadt, Ger-
many) and either stored at ꢀ808C or directly applied to experi-
ments. KDGA, AlDH and KDC were purified as previously de-
sodium pyruvate. Pyruvate concentration in the samples was deter-
mined via lactate dehydrogenase as described elsewhere.
KARI activity: KARI activity was assayed by following the NADH
consumption connected to the conversion of acetolactate to 2,3-
dihydroxyisovalerate at 508C. The assay mixture contained 5mm
[21]
acetolactate, 0.3mm NADH, 10mm MgCl and 50mm HEPES, pH 7.
2
KDC activity: KDC activity was assayed by following the decarboxy-
lation of 2-ketoisovalerate to isobutyraldehyde at 508C and
340 nm. Assay mixture contained 50mm HEPES (pH 7), 0.1mm thia-
[21,23,31]
scribed
and stored as lyophilisates. ALS and KARI were puri-
fied via IMAC using 25 or 50 mm HEPES, pH 7. Elution was ach-
ieved with 500 mm imidazol. Enzymes were desalted and stored as
a liquid stock (ALS) or lyophilisate (KARI).
mine pyrophosphate, 2.5mm MgCl and 60mm 2-ketoisovalerate.
2
Decarboxylation rate was calculated using the molar extinction co-
ꢀ
1
ꢀ1 [27]
efficient of 2-ketoisovalerate (e=0.017 Lmmol cm ).
ADH activity: ADH activity was determined by following the
NADH-dependent reduction of isobutyraldehyde to isobutanol at
Protein determination
5
08C. Assay mixture contained 10mm HEPES (pH 7.2), 5mm isobu-
Protein concentration was determined with the Roti-Nanoquant re-
agent (Carl Roth) according to the manufacturer’s recommenda-
tions using bovine serum albumin as a standard.
tyraldehyde and 0.3mm NADH.
Glucose analysis: Glucose oxidase was used for the quantification
of glucose. Assay mixture contained 20mm potassium phosphate
(
(
pH 6), 0.75mm 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid
ABTS), 2 U glucose oxidase and 0.1 U peroxidase. After the addi-
tion of samples the reaction mixture was incubated for 30 min at
08C and the extinction at 418 and 480 nm measured. Assay cali-
bration was performed using defined glucose standard solu-
SDS-PAGE
3
[40]
Protein samples were analyzed as described by Laemmli using
a Mini-PROTEAN system from Bio-Rad (Munich, Germany).
[42]
tions.
GC–FID analysis
Enzyme assays
Isobutyraldehyde and isobutanol or acetaldehyde and ethanol
were quantified by GC–FID using a Thermo Scientific Trace GC
Ultra, equipped with a flame ionization detector (FID) and a Head-
space Tri Plus autosampler. Alcohol and aldehyde compounds
were separated by a StabilWax column (30 m, 0.25 mm internal di-
ameter, 0.25 mm film thickness; Restek, Bellefonte, USA), whereby
All photometrical enzyme assays were performed in microtiter
plate format using a Thermo Scientific Multiskan or Varioskan pho-
tometer. When necessary, reaction mixtures were incubated in
a waterbath (Julabo, Seelbach, Germany) for accurate temperature
[41]
control. Buffers were prepared according to Stoll, adjusting the
+
pH to the corresponding temperature. Reactions using NAD or
ꢀ
1
helium (0.8 or 1.2 mLmin ) was used as the carrier gas. The oven
NADH as coenzymes were followed at 340 nm (molar extinction
temperature was programmed to be held at 508C for 2 min, raised
ꢀ1
ꢀ1
coefficient NADH=6.22 Lmmol cm ) and the glucose concentra-
ꢀ1
with a gradient 108Cmin to 1508C and held for 1 min. Injector
and detector were kept at 2008C. Samples were incubated prior to
injection at 408C for 15 min. Injection was done in the split mode
[27]
tions were measured at 418 nm and 480 nm as indicated. One
unit of enzyme activity is defined as the amount of enzyme neces-
sary to convert 1 mmol substrate per minute. In addition to the
standard reaction conditions described below, enzyme activity was
tested under reaction conditions (100mm HEPES, pH 7, 2.5 mm
ꢀ1
with a flow of 10 mLmin , injecting 700 mL using headspace
mode.
MgCl , 0.1 mm thiamine pyrophosphate) prior to alcohol synthesis
2
experiments.
HPLC analysis
GDH activity: GDH activity was assayed at 508C by oxidizing d-glu-
cose to gluconate, whereby the coenzyme NAD is reduced to
NADH. Assay mixture contained 50 mm HEPES (pH 7), 2mm NAD
and 50mm d-glucose.
DHAD activity: DHAD activity was measured by an indirect assay.
The assay mixture containing DHAD, 20mm substrate and 100mm
HEPES (pH 7) was incubated at 508C. Afterwards the conversion of
glycerate to pyruvate, gluconate to 2-keto-3-desoxygluconate or
+
Gluconate, 2-keto-3-desoxygluconate, pyruvate, glycerate, 2,3-dihy-
droxyisovalerate and 2-ketoisovalerate were separated and quanti-
fied by HPLC, using an Ultimate-3000 HPLC system (Dionex, Idstein,
Germany), equipped with autosampler and a diode-array detector.
Chromatographic separation of gluconate, 2-keto-3-desoxygluco-
nate, pyruvate and glycerate was achieved on a Metrosep A
Supp10–250/40 column (250 mm, particle size 4.6 mm; Metrohm,
Filderstadt, Germany) at 658C by isocratic elution with 12mm am-
monium bicarbonate (pH 10), followed by a washing step with
30mm sodium carbonate (pH 10.4). Mobile phase flow was adjust-
+
[20]
2
,3-dihydroxyisovalerate to 2-ketoisovalerate, respectively, was de-
termined via HPLC as described below.
KDGA activity: KDGA activity was followed in cleavage direction at
ꢀ1
5
08C. Reaction mixture contained 50mm HEPES (pH 7), 0.1 mm thi-
ed to 0.2 mLmin . 2,3-dihydroxyisovalerate and 2-ketoisovalerate
were separated using a Nucleogel Sugar 810H column (300 mm,
7.8 mm internal diameter; Macherey–Nagel, Dꢀren, Germany) at
608C by isocratic elution with 3mm H SO (pH 2.2). Mobile phase
amine pyrophosphate, 2.5mm MgCl , 20 U PDC and 10mm KDG.
KDG cleavage was followed by HPLC as described below.
AlDH activity: AlDH activity was assayed at 508C by oxidizing d-
glyceraldeyde to glycerate, whereby the coenzyme NAD is re-
2
2
4
+
ꢀ1
flow was adjusted to 0.6 mLmin . Sample volume was 10 mL in
ChemSusChem 2012, 5, 2165 – 2172
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2171