In this procedure, enzyme mixtures for converting lignocellulose biomass to free sugars are optimized using an automated system known as A-G-L-B-R-C enzyme platform or gen plat. First, a mixture model is designed in which four to 18 distinct enzymes are combined in up to 96 different ratios using design of experiment software. The biomass and enzymes are then pipetted into 96 deep well plates using a liquid handling robotic system.
The plates are sealed and incubated for 48 hours at 50 degrees Celsius. Following the incubation, the wells are sampled using an automated liquid handling system, and the free glucose and xylose are measured using a color of metric enzyme coupled assay. The design of experiment software is then used to determine which combinations of enzymes yield the maximum free glucose and silos.
The overall goal of this procedure is to simultaneously test the efficiency of various enzyme cocktails for converting lignocellulose biomass materials into fermentable sugars for ethanol with the overall aim of lowering biofuel production costs. Although we use this method mainly for analyzing enzyme cocktails on alkaline pretreated corn stove, it is adaptable to any pretreatment in any feedstock such as acid treated poplar switch grass and dried distillers grains. This technique can be used to discover novel enzymes that are important for biomass deconstruction and to find better examples of the known enzymes.
Although the enzymes used for gem plats may come from various sources, the enzymes used in this demonstration are produced by expressing PPIC or PPIC alpha in Pia Pastis gro cells in 300 milliliter batches and baffled 500 milliliter flasks for three days. Then for the next four days, perform methanol induction every 24 hours after the last day of methanol induction. Pour the samples into a tangential flow filtration system with a 10 kilodalton cutoff membrane to concentrate and desalt the culture.
Filtrates then perform a buffer exchange with five volumes of 25 millimolar sodium acetate pH five, and concentrate the sample to bring the volume to between 10 and 25 milliliters. Measure protein using the BCA method with BSA as the standard, the final concentration of the enzyme stock solutions should be 0.5 to 10 milligrams per milliliter. Store the enzymes in small aliquots in 20%glycerol at minus 80 degrees Celsius to create simplex lattice designs.
Begin by opening the design expert program on a pc. Input the number of enzymes to be tested and the upper and lower proportions. For example, a lower proportion of 5%For core enzymes then indicate whether the experimental design is quadratic or cubic.
Quadratic is necessary for mixtures with more than eight components in Excel. Calculate the amount in micrograms of each enzyme to be added to load a total of 15 milligrams per gram of glucan per sample. Then calculate the amount of each enzyme in microliters to add to each reaction based on the protein concentrations of the stocks.
Generate additional Excel worksheets, specifying source LabWare source wells, destination LabWare, destination wells, and transfer volumes for dispensing of water and enzymes according to the experimental design. Finally, import the worksheet into Biome FX software using the transfer from file function to generate a pretreated corn stove biomass slurry. Pour the dried biomass into a Wiley mill and grind it to pass a 0.5 millimeter screen.
Collect the ground biomass in a beaker. The glucan content is measured by standard methods as described in the JoVE particle comprehensive compositional analysis of plant cell walls, part one lignin. Once the glucan content has been determined, mix the biomass with sodium citrate buffer so that the final glucan concentration is 0.5%Turn on the paddle reservoir and pour the biomass slurry into it.
Now though the enzymes on ice, the amount of each enzyme needed is dependent on the size of the experiment to dispense biomass. Slurries generate wide boar tips by using a razor blade to cut the last 0.5 centimeter off the span. Eight 100 microliter pipette tips.
The trimmed span eight tips can be reused. Once trimmed, place the tips in a pipette box so that they can be automatically picked up by the SPAN eight. This instrument is equipped with a SPAN eight pod with a disposable tip function that can dispense individual solutions into individual wells and a fixed AP 96 pod, which can transfer 96 identical solutions at one time into a 96 well plate next arrange the deck of the liquid handling system.
This consists of 1 96 deep well reaction plate, a tip box lid containing water, an enzyme rack containing the enzymes in 1.5 milliliter eend orph tubes, and several full tip boxes in the deck positions as programmed in the instrument set up. Once the deck is ready, check the program for any errors. Start the Beckman fx.
The SPAN eight arm will first pipette 200 microliters of the biomass slurry into each well of the 96 well deep well reaction plates. The SPAN eight pod will then dispense enzymes into each of the wells, according to the spreadsheet imported into the Biome FX software. Once the pipetting is complete, remove the 96 deep well plate from the deck.
Seal the plate with a pi or cap mat. Then invert the plate and tap it once or twice to break the surface tension. Place the plate in the hybridization oven for 48 hours following the incubation centrifuge.
The 96 steep well plate for three minutes at 1, 500 times G in a swinging bucket centrifuge while the plate is spinning. Program the Beckman FX to use the AP 96 pod to pipette 100 microliters from each well into a regular 96 well plate. Then after the spin, place the plate in the instrument and start the pipetting.
When the pipetting has finished. Incubate the plates at 100 degrees Celsius for 10 minutes on a plate heater to inactivate the enzymes centrifuge the plate at 1, 500 times G for 30 seconds. For GLC measurements, first, add 192 microliters of the glucose oxidase peroxidase reagent, using a program written for the AP 96 pod of the Beckman fx.
Then use the AP 96 pod to transfer 12 microliters of the sate into a regular 96. Well plate incubate the plates of 50 degrees Celsius for 20 minutes and measure the absorbance of 510 nanometers in a microplate reader. For xlo measurements, add the assay reagents according to the kit instructions.
Then use the AP 96 pod to transfer four microliters of each well into 384 well plates and measure the absorbance at 340 nanometers. Perform data analysis as described in the accompanying document using design expert software. Five substrates and three pre-treatments were compared under similar conditions of grinding 0.5 millimeter particle size enzyme loading 15 milligrams per gram of glucan and hydrolysis conditions.
48 hours at 50 degrees Celsius as can be seen here. There are striking differences between feedstocks and between pre-treatments. Clearly, the enzyme cocktail accelerates 1000 alone, cannot deal with all of the variability seen in pre-treatments and feedstocks.
This experiment compares A FEX pretreated corn stove referred to as A-F-E-X-C-S, alkaline peroxide, pretreated corn stove, A-H-P-C-S and A FEX pretreated dried distillers grains, A-F-E-X-D-D-G. The numbers along the top of the bars are the glucose yields as a percentage of the total glucose in the biomass sample. Note that the composition of cocktails differ significantly depending on the pretreatment and the biomass substrate.
Although the main use of gem plat is to make synthetic mixtures of pure enzymes, it can also be used to produce optimized mixtures of commercial enzyme preparations. Panel A shows a ary diagram of four enzymes optimized for release of glucose from A-F-E-X-D-D-G panel B shows the resulting yields using this optimized mixture compared to three other enzyme preparations. We have just demonstrated the use of gen plat to assist biomass breakdown using individual pure enzymes.
Each enzyme has a specific catalytic activity that contributes to the full breakdown of plant cell walls into individual sugars. However, it is not necessary to know the precise catalytic activity of an enzyme in order to evaluate its importance in biomass deconstruction. Using Gen pla, Knowing which enzymes are important and in what proportions is critical for guiding bioprospecting.
For new enzymes for protein engineering and for development of consolidated bioprocessing organisms, Sometimes the results are surprising. For example, we found that for release of glucose from Conover, a combination of two unrelated Xin ais is superior to either Xin alone. We also found that better manase is important for release of glucose from dry distiller grain.
Even though this material has a low betterman and content, This method will pave the way for researchers to develop even better enzyme cocktails for a variety of conditions. You don't even have to know what a particular protein does enzymatically in order to test its contribution to lignocellulose deconstruction.
