Introduction
L-phenylacetylcarbinol is a compound used in the synthesis of ephedrine, itself used as a precursor to amphetamines. Industrially, it is synthesized by the aerobic fermentation of benzaldehyde by baker's yeast (S. cerevisiae/SC). While the process is quite simple, it can require a bit of equipment and knowledge, especially when talking about maintaining sterile cultures and proper growth conditions. I will therefore explain two methods of different sophistication for the production of L-PAC through biotransformation. The growth medium should be sterilized by autoclaving before use.
First method:
One might describe this technique as "sloppy but easy". It requires less equipment, is easier, but is also less effective and will result in lower yields.
Procedure:
In 500ml of sterile 0.1M pH 6 citrate buffer add 44g of yeast and 55g glucose. Place in a water bath heated at 30°C. Incubate for 40/50 minutes.
Add benzaldehyde (25 mmol) in 5 mL ethanol and acetaldehyde (25 mmol). Incubate for 60 minutes.
Filter through Celite.
Extract with 3*100 ml ethyl acetate. Break emulsion using saturated Sodium Chloride solution, dry over anhydrous sodium sulphate, filter, evaporate solvent. This is your L-PAC.
Explanations:
Citrate buffer: this is used to maintain a stable pH, as the growth of the cell will lower it overtime (not good). To prepare this buffer, follow the procedures given by this calculator: https://www.aatbio.com/resources/buffer-preparations-and-recipes/citrate-buffer-ph-3-to-6-2. Don't forget to change the pH to 6.
Temperature: allows higher metabolic activity of the yeast.
Ethanol: used to dissolve the benzaldehyde and acetaldehyde and increases their bioavailability.
Acetaldehyde: used as hydrogen acceptor instead of benzaldehyde, thus limiting the production of benzyl alcohol.
This is an easy procedure that almost anyone can follow after a bit of reading on standard sterile techniques, but gives lower yields than the other one (between 20 to 40%). It can be improved either by having better aeration using either orbital shaking at 150 rpm or by pumping sterile air into the medium, with 1L of air per liter of medium per minute, or by immobilizing the cells in alginate beads. You can look this up on youtube, it's very easy to do, and can increase yields significantly.
Second method:
This protocol is more advanced, but can increase yields up to 60 or 70% if done correctly. It will however require more material. Even though it isn't perfect, it is way better.
This method was designed for the yeast Candida utilis instead of S. cerevisiae. You can use SC instead but you will see a decrease in yields. You can buy C. utilis here: https://www.biomall.in/product/candida-utilis-atcc-9950-0779p-0779p
Procedure
The growth medium is composed of 10g/L urea (brings nitrogen), 10g/L MgSO4 (brings magnesium and sulfur) and between 50 to 70g/L of sugar using molasses. Dissolve in adequate volume of 0.1M pH 6 citrate buffer.
The day before, inoculate a starting culture of yeast and incubate at 30°C overnight.
Read optical density (absorbance) at 595nm. Inoculate your fermentation reactor with 15% v/v of your starting culture adjusted to 0.4 OD. Example:to inoculate a 1L container, if OD was measured at 0.2, add 300mL to 700mL. If OD is already at 0.4, add 150mL to 850mL etc... (240x106 cells/ml in the inoculum, see explanations)
Incubate at 30°C with either orbital shaking at 150 rpm or pumping sterile air at 1 v/v/min rate. If pumping air, you might want to add some slow stirring in there.
Incubate until OD reaches between 0.3 and 0.4. This should take between 4 to 6 hours, can be less or more. Check OD every 30 minutes until you reach the desired value.
The addition of benzaldehyde is done in multiple doses. For a 50mL, the volumes are 68, 62, 56, 50, 43, 37, 31, 25. Add an equivalent volume of acetaldehyde at the same time. Wait one hour between each addition.
Extract using 1:2 toluene/medium v/v, evaporate.
Explanation:
Optical density: it is measured using a spectrophotometer. Here we read absorbance at 595nm, which is proportional to the number of cells in solution. As a rule of thumb, remember 0.1 OD=60x10^6 cells/mL
General notes:
For aeration, on a small scale orbital shaking at 150 rpm is better. On a larger scale, you will need to bubble air passed through a filter.
In the second protocol, the addition of benzaldehyde is done in multiple doses to increase yields. This can be further improved by continuously injecting benzaldehyde in decreasing rate, but this complex to do in an amateur setting.
Substituted benzaldehydes can be used and will yield the corresponding L-PAC analog.
Finally, you can borrow some element of method 2 and incorporate them in method 1 in order to customize the experimental design to your liking. Be aware that other strains and species can be used, greatly increasing yields. See "production of L-phenyl Acetyl Carbinol (L-PAC) by different novel strains of yeasts in molasses and sugar cane juice as production medium" by Ravi et al for more info.
L-phenylacetylcarbinol is a compound used in the synthesis of ephedrine, itself used as a precursor to amphetamines. Industrially, it is synthesized by the aerobic fermentation of benzaldehyde by baker's yeast (S. cerevisiae/SC). While the process is quite simple, it can require a bit of equipment and knowledge, especially when talking about maintaining sterile cultures and proper growth conditions. I will therefore explain two methods of different sophistication for the production of L-PAC through biotransformation. The growth medium should be sterilized by autoclaving before use.
First method:
One might describe this technique as "sloppy but easy". It requires less equipment, is easier, but is also less effective and will result in lower yields.
Procedure:
In 500ml of sterile 0.1M pH 6 citrate buffer add 44g of yeast and 55g glucose. Place in a water bath heated at 30°C. Incubate for 40/50 minutes.
Add benzaldehyde (25 mmol) in 5 mL ethanol and acetaldehyde (25 mmol). Incubate for 60 minutes.
Filter through Celite.
Extract with 3*100 ml ethyl acetate. Break emulsion using saturated Sodium Chloride solution, dry over anhydrous sodium sulphate, filter, evaporate solvent. This is your L-PAC.
Explanations:
Citrate buffer: this is used to maintain a stable pH, as the growth of the cell will lower it overtime (not good). To prepare this buffer, follow the procedures given by this calculator: https://www.aatbio.com/resources/buffer-preparations-and-recipes/citrate-buffer-ph-3-to-6-2. Don't forget to change the pH to 6.
Temperature: allows higher metabolic activity of the yeast.
Ethanol: used to dissolve the benzaldehyde and acetaldehyde and increases their bioavailability.
Acetaldehyde: used as hydrogen acceptor instead of benzaldehyde, thus limiting the production of benzyl alcohol.
This is an easy procedure that almost anyone can follow after a bit of reading on standard sterile techniques, but gives lower yields than the other one (between 20 to 40%). It can be improved either by having better aeration using either orbital shaking at 150 rpm or by pumping sterile air into the medium, with 1L of air per liter of medium per minute, or by immobilizing the cells in alginate beads. You can look this up on youtube, it's very easy to do, and can increase yields significantly.
Second method:
This protocol is more advanced, but can increase yields up to 60 or 70% if done correctly. It will however require more material. Even though it isn't perfect, it is way better.
This method was designed for the yeast Candida utilis instead of S. cerevisiae. You can use SC instead but you will see a decrease in yields. You can buy C. utilis here: https://www.biomall.in/product/candida-utilis-atcc-9950-0779p-0779p
Procedure
The growth medium is composed of 10g/L urea (brings nitrogen), 10g/L MgSO4 (brings magnesium and sulfur) and between 50 to 70g/L of sugar using molasses. Dissolve in adequate volume of 0.1M pH 6 citrate buffer.
The day before, inoculate a starting culture of yeast and incubate at 30°C overnight.
Read optical density (absorbance) at 595nm. Inoculate your fermentation reactor with 15% v/v of your starting culture adjusted to 0.4 OD. Example:to inoculate a 1L container, if OD was measured at 0.2, add 300mL to 700mL. If OD is already at 0.4, add 150mL to 850mL etc... (240x106 cells/ml in the inoculum, see explanations)
Incubate at 30°C with either orbital shaking at 150 rpm or pumping sterile air at 1 v/v/min rate. If pumping air, you might want to add some slow stirring in there.
Incubate until OD reaches between 0.3 and 0.4. This should take between 4 to 6 hours, can be less or more. Check OD every 30 minutes until you reach the desired value.
The addition of benzaldehyde is done in multiple doses. For a 50mL, the volumes are 68, 62, 56, 50, 43, 37, 31, 25. Add an equivalent volume of acetaldehyde at the same time. Wait one hour between each addition.
Extract using 1:2 toluene/medium v/v, evaporate.
Explanation:
Optical density: it is measured using a spectrophotometer. Here we read absorbance at 595nm, which is proportional to the number of cells in solution. As a rule of thumb, remember 0.1 OD=60x10^6 cells/mL
General notes:
For aeration, on a small scale orbital shaking at 150 rpm is better. On a larger scale, you will need to bubble air passed through a filter.
In the second protocol, the addition of benzaldehyde is done in multiple doses to increase yields. This can be further improved by continuously injecting benzaldehyde in decreasing rate, but this complex to do in an amateur setting.
Substituted benzaldehydes can be used and will yield the corresponding L-PAC analog.
Finally, you can borrow some element of method 2 and incorporate them in method 1 in order to customize the experimental design to your liking. Be aware that other strains and species can be used, greatly increasing yields. See "production of L-phenyl Acetyl Carbinol (L-PAC) by different novel strains of yeasts in molasses and sugar cane juice as production medium" by Ravi et al for more info.
