Processes 2018, 6,38
Differences between the current measurements and previously reported values may reflect differences
in culturing conditions or the influence of specific procedural details.
Polypropylene centrifuge tubes were used for safety during centrifugation rather than glass tubes,
but it was noted that polypropylene is not completely chemically resistant to chloroform and may cause
leaching of compounds from the polymer into chloroform. This error was accounted for by performing a
blank reaction (0.6 mL water). The mass of the blank was then subtracted from the mass of the biological
sample to obtain the mass of lipid. Additionally, removal of the lower chloroform phase can be difficult to
perform reproducibly. Glass Pasteur pipettes were used initially; however, micropipettes with 200-µL tips
provided more control over phase removal and yielded the most reproducible results.
7. Protein
7.1. Literature Review
Protein is typically the largest fraction of bacterial biomass. Many methods have been reported
for determining protein quantity, including UV absorption spectroscopy and dye-based assays such
as Bradford, Lowry, BCA, and others, for which Noble et al. [63] and Noble and Bailey [64] provided
thorough discussions. UV absorption depends on tyrosine and tryptophan residues and the molar
extinction coefficient of the protein under examination, and it requires a highly purified sample.
Dye-based assays are influenced by different amino acid distributions and are subject to different
interfering compounds as well as variability between proteins. Bovine serum albumin is a commonly
used protein standard, but its amino acid sequence may not be representative of the total cellular
protein. Amino acid analysis, or hydrolysis of cellular protein followed by derivatization and
identification of individual amino acids via HPLC, is an alternative to these methods and is often
considered the gold standard for protein analysis [63,64].
Amino acid analysis was selected for the current study due to improved accuracy and less
bias as opposed to UV absorbance or dye-based methods. Some amino acids, such as cysteine and
tryptophan, degrade during hydrolysis; special hydrolysis conditions may be used to retain them [65],
or their proportions may be estimated based on genome codon distribution. Amino acid analysis
provides experimental amino acid distribution in addition to total protein quantification, which serve
as important parameters for metabolic modeling.
7.2. Procedure (After Henderson et al., 2000)
7.2.1. Reagents
•
Cell pellet (1–3 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
6 M HCl and 6 M NaOH.
•
0.1 M HCl, 0.22 µm filtered.
•
Borate buffer: 0.4 N borate, titrate to pH 10.2 with NaOH, 0.22 µm filtered.
•
OPA and FMOC derivatizing reagents (Agilent 5061–3335 and 5061–3337, Santa Clara, CA, USA).
•
Amino acid standard: 1 nmol/mL (Agilent 5061–3330).
•
Solvent A: 40 mM sodium phosphate buffer Na 2 HPO 4 , titrate to pH 7.8 with 10 M NaOH, 0.22 µm
filtered. (A ratio of different sodium salts can be used to prepare a 40 mM phosphate solution
with an initial pH closer to 7.8, e.g., 1:1 molar ratio of NaH 2 PO 4 and Na 2 HPO 4 .)
•
Solvent B: 45:45:10 acetonitrile:methanol:water (v/v/v), 0.22 µm filtered (nylon filter recommended
for organic solvents).
7.2.2. Assay
(1) Transfer cell pellet to borosilicate HPLC vial with PTFE/silicone cap.
(2) Add 50 µL 6 M HCl per mg biomass.
(3) Tightly cap the vial and hydrolyze at 105 ◦ C for 24 h (VWR analog heat block).
165
Differences between the current measurements and previously reported values may reflect differences
in culturing conditions or the influence of specific procedural details.
Polypropylene centrifuge tubes were used for safety during centrifugation rather than glass tubes,
but it was noted that polypropylene is not completely chemically resistant to chloroform and may cause
leaching of compounds from the polymer into chloroform. This error was accounted for by performing a
blank reaction (0.6 mL water). The mass of the blank was then subtracted from the mass of the biological
sample to obtain the mass of lipid. Additionally, removal of the lower chloroform phase can be difficult to
perform reproducibly. Glass Pasteur pipettes were used initially; however, micropipettes with 200-µL tips
provided more control over phase removal and yielded the most reproducible results.
7. Protein
7.1. Literature Review
Protein is typically the largest fraction of bacterial biomass. Many methods have been reported
for determining protein quantity, including UV absorption spectroscopy and dye-based assays such
as Bradford, Lowry, BCA, and others, for which Noble et al. [63] and Noble and Bailey [64] provided
thorough discussions. UV absorption depends on tyrosine and tryptophan residues and the molar
extinction coefficient of the protein under examination, and it requires a highly purified sample.
Dye-based assays are influenced by different amino acid distributions and are subject to different
interfering compounds as well as variability between proteins. Bovine serum albumin is a commonly
used protein standard, but its amino acid sequence may not be representative of the total cellular
protein. Amino acid analysis, or hydrolysis of cellular protein followed by derivatization and
identification of individual amino acids via HPLC, is an alternative to these methods and is often
considered the gold standard for protein analysis [63,64].
Amino acid analysis was selected for the current study due to improved accuracy and less
bias as opposed to UV absorbance or dye-based methods. Some amino acids, such as cysteine and
tryptophan, degrade during hydrolysis; special hydrolysis conditions may be used to retain them [65],
or their proportions may be estimated based on genome codon distribution. Amino acid analysis
provides experimental amino acid distribution in addition to total protein quantification, which serve
as important parameters for metabolic modeling.
7.2. Procedure (After Henderson et al., 2000)
7.2.1. Reagents
•
Cell pellet (1–3 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
6 M HCl and 6 M NaOH.
•
0.1 M HCl, 0.22 µm filtered.
•
Borate buffer: 0.4 N borate, titrate to pH 10.2 with NaOH, 0.22 µm filtered.
•
OPA and FMOC derivatizing reagents (Agilent 5061–3335 and 5061–3337, Santa Clara, CA, USA).
•
Amino acid standard: 1 nmol/mL (Agilent 5061–3330).
•
Solvent A: 40 mM sodium phosphate buffer Na 2 HPO 4 , titrate to pH 7.8 with 10 M NaOH, 0.22 µm
filtered. (A ratio of different sodium salts can be used to prepare a 40 mM phosphate solution
with an initial pH closer to 7.8, e.g., 1:1 molar ratio of NaH 2 PO 4 and Na 2 HPO 4 .)
•
Solvent B: 45:45:10 acetonitrile:methanol:water (v/v/v), 0.22 µm filtered (nylon filter recommended
for organic solvents).
7.2.2. Assay
(1) Transfer cell pellet to borosilicate HPLC vial with PTFE/silicone cap.
(2) Add 50 µL 6 M HCl per mg biomass.
(3) Tightly cap the vial and hydrolyze at 105 ◦ C for 24 h (VWR analog heat block).
165