Inoculum for fermentation trials may be grown on yeast extract peptone dextrose (YPD) in either liquid medium or onplates. In either case, the inoculum should be fresh (24-48 h). For fermentation trials, Yeast Nitrogen Base (YNB, Difco) without ammonium sulfate or amino acids should be formulated as per manufacturer's instructions. Sugars may be autoclaved in salt and phosphate-free aqueous solution. Urea and peptone can be autoclaved in 20X concentrate.[1] YNB should be prepared in 20X concentrate and sterilized by filtration. Nutritional supplements (biotin, thiamine pyrophosphate, amino acids) should be prepared in 100 to 1000X concentrates and sterilized by filtration. Use 8% of each carbon source.[2]
For fully aerobic growth, cell density should not exceed 1.0 mg/ml (OD ~ 4.0 at 600 nm[3]). Employ 35 ml of medium in a 125 ml baffled flask, shaken at 200 rpm. For oxygen limited growth, the initial cell density should be approximately 20 to 30 OD units (5 to 7.5 mg dry wt of cells/ml). Employ 50 ml of medium in a 125 ml Erlenmeyer flask shaken at 100 rpm. Fermentation is optimal at 25deg.C; growth is faster at 30deg.C. We will use 25deg. for these experiments.
Cells can be harvested at any stage of growth, but for most consistent results, mid log phase or mid growth-phase cells are preferred. Under low aeration cells begin to shift from mid log phase to linear growth around OD 2.0 (100 rpm), and under high aeration, around OD 4.0. Oxygen-limited cells always grow at a linear rate but should be harvested before one full doubling occurs. In the case of a OD 20 inoculum shaken at 100 rpm, one doubling will occur within 24 to 48 h.
100 mM MOPS, pH 6.8-7.0; 1 mM [[beta]]-mercaptoethanol[4]
Other buffers:
Witterveen et al.[5]: 20 mM-Bis-Tris pH 6.5; 5 mM MgCl2; 1 mM [[beta]]-mercaptoethanol
Wong et al.[6]: 20 mM phosphate, pH 7.0; 10 mM 2-mercaptoethanol
For some assays, it may be necessary to remove low molecular weight metabolites by passing the centrifuged homogenate over a 15 ml column of Sephadex G25 that has been equilibrated with the breaking buffer. This will reduce background activity.
Note: Enzyme assays - and especially for kinases and reactions dependent upon NADH oxidation - require controls with and without substrate.
NAD = 665.4 g/mole; disodium salt FW = 709.4; NADP = 743.4 g/mole
The extinction coefficient for NAD(P)H at 340 nm is 6220
NOTE: A quartz cuvette is necessary because glass and plastic do not transmit in the UV; glass cuvettes CANNOT be used; some disposible plastic cuvettes might be suitable but will give lower sensitivity.
The optical density of a 1 uM solution of NAD(P)H = 6.22
As NAD(P)H is oxidized to NAD(P), the optical density decreases.
[[Delta]] OD units / 6.22 = umoles of NAD(P)(H) oxidized or reduced
Report all enzyme activities as specific activities; i.e. IU/mg protein
The mg of protein used = mg/ml as determined by Bradford assay * volume; i.e 50 ul of homogenate = 0.05 ml
IU/mg protein = [[[Delta]]A340 min-1 / 6.22] / [mg protein ml-1 * vol. of homog. used]
Substrate: 100 mM xylitol, L-arabitol, D-arabitol or ribitol
Cofactor: 0.4 mM NAD(P)+
Assay is performed in a final volume of 1.0 ml
Prepare 4 mM NAD(P)+ stock solution (10X)
Dilute homogenate in assay buffer (if needed)
Place 700 ul of buffer plus 100 ul of homogenate in cuvette and allow to equilibrate at assay temperature (25deg.C).
Add 100 ul of NAD(P)+
Measure baseline rate of NAD(P)+ reduction at 340 nm in a quartz cuvette
Add 100 ul of 10x substrate[8] and mix cuvette to start reaction.
Measure rate of NAD(P)+ reduction at 340 nm and calculate activity as umol/min.
Substrate: 100 mM D-xylose, L-arabinose, D-arabinose or D-ribose
Cofactor: 0.2 mM NADPH or 0.2 mM NADH
Assay is performed in a final volume of 1.0 ml
Prepare 2 mM NAD(P)H stock solution (10X)
Dilute homogenate in assay buffer (if needed)
Place 700 ul of buffer plus 100 ul of homogenate in cuvette and allow to equilibrate at assay temperature (25deg.C).
Add 100 ul of NAD(P)H
Measure baseline rate of NAD(P)H oxidation at 340 nm in a quartz cuvette
Add 100 ul of 10x substrate and mix cuvette to start reaction.
Measure rate of NAD(P)H oxidation at 340 nm and calculate activity as umol/min.
Assay as per pentose reductases using 5 mM final concentration of each substrate.
The pentulokinase (PUK) reaction is followed by coupling the reduction of pyruvate to lactate by lactate dehydrogenase (LDH); pentulokinase provides rate-limiting amounts of ADP to allow pyruvate kinase (PK) to convert phosphoenolpyruvate (PEP) into lactate:
Substrate: 5 mM D-xylulose, D-ribulose, or L-ribulose
Cofactors: 0.2 mM NADH, 1.5 mM PEP, 1 mM ATP
Coenzymes: 1 unit pyruvate kinase; 3 units lactate dehydrogenase
Assay is performed in a final volume of 1.0 ml
Prepare [2] mM NADH stock solution (10X)
prepare 10X mixture of PK and LDH
Prepare [15] mM PEP (10X) (Prepare just before use; keep on ice)
Prepare [10] mM ATP (10X) (Prepare just before use; keep on ice)
Dilute homogenate in assay buffer (if needed)
Place 400 ul of buffer plus 100 ul of homogenate in cuvette and allow to equilibrate at assay temperature (25deg.C).
Add [100] ul of NADH and 100 ul of LDH, PK mixture
Measure baseline rate of NADH oxidation at 340 nm in a quartz cuvette
Add [100] ul of PEP and 100 ul of LDH; equilibrate 15 sec.
Add 100 ul of 10X substrate and mix cuvette to start reaction.
Measure rate of NADH oxidation at 340 nm and calculate activity as umol/min.
Substrate: 10 mM xylitol, arabitol, arabitol or ribitol
Cofactors: 0.34 mM NAD or NADP
Assay is performed in a final volume of 1.0 ml
Prepare 4 mM NAD(P)+ stock solution (10X)
Dilute homogenate in assay buffer (if needed)
Place 700 ul of buffer plus 100 ul of homogenate in cuvette and allow to equilibrate at assay temperature (25deg.C).
Add 100 ul of NAD(P)+
Measure baseline rate of NAD(P)+ reduction at 340 nm in a quartz cuvette
Add 100 ul of 10x substrate and mix cuvette to start reaction.
Measure rate of NAD(P)+ reduction at 340 nm and calculate activity as umol/min.
For comments or further information write to Tom Jeffries: twjeffri@facstaff.wisc.edu