Name: GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization (Video) | JoVE Journal
Uploaded: 2011-10-24T13:00:00.000+00:00
Duration: 11 min 38 s
Description: Discover automated enzyme cocktail optimization for biomass degradation using GENPLAT. This method enables efficient conversion of lignocellulose to fermentable sugars for biofuel production.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was funded in part by the U.S. Department of Energy Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) and grant DE-FG02-91ER200021 from the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences. We thank John Scott-Craig and Melissa Borrusch for their material and conceptual contributions.

1. Enzyme Production
<ol>
<li>Produce proteins in Pichia pastoris by cloning of the corresponding genes into vectors pPICZ or pPICZα and transformation into P. pastoris. Grow cells in 300-ml batches in baffled 500-ml flasks with methanol induction every 24 hr (Banerjee et al., 2010a).</li>
<li>Concentrate and desalt the culture filtrates by tangential flow filtration.</li>
<li>Store the enzymes in small aliquots in 20% glycerol at -80°C. The concentration range is 1-10 mg/ml.</li>
</ol>
2. Design of Experiment
<ol>
<li>Input the number of enzymes to be tested, their upper and lower proportions (e.g. a lower proportion of 5% for core enzymes), and the type of experimental design (e.g., quadratic or cubic) into the Design-Expertsoftware. </li>
<li>Calculate the amount in μg of each enzyme to be added to make a total loading of 15 mg/g glucan, and then calculate the amount of each enzyme in μl to add to each reaction. </li>
<li>Generate 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 and import it into the Biomek FXP software using the transfer-from-file function.</li>
</ol>
3. Enzymatic Hydrolysis
<ol>
<li>Suspend the biomass slurry in 50 mM sodium citrate, pH 4.8, containing 5 μg/ml tetracycline and 5 μg/ml cycloheximide, in the paddle reservoir. </li>
<li>Robotically pipet 200 μl of the slurry into each well of a 96-well deep-well reaction plates using Span-8 1000-μl pipet tips with the last 0.5 cm cut off. Mix the slurry once at 100 μl/sec and then aspirate the same volume from the paddle reservoir and dispense into the plates.</li>
<li>Dispense the enzymes into the same plate using the program entered into the Beckman FX. </li>
<li>Seal the plates with pierceable capmats. </li>
<li>Invert and tap the plates several times to overcome surface tension. Place the plates at 50°C for 48 hr in the hybridization incubator rotating at 10 rpm. </li>
</ol>
4. Determination of Glc and Xyl 
<ol>
<li>Centrifuge the reaction plates for 3 min at 1250 x g in a swinging bucket centrifuge. </li>
<li>Transfer 100 μl of the supernatant into regular 96-well plates using the AP96 pod of the Biomek FX. </li>
<li>Incubate the plates at 90-95°C for 10 min in order to inactivate the enzymes, and then centrifuge at 1250 x g for 30 sec. </li>
<li>For Glc measurements, transfer 12 μl of the supernatants into regular 96-well plates. Add 192 μl of the glucose oxidase/peroxidase (GOPOD) reagent. Incubate the plates at 50°C for 20 min and read the absorbances at 510 nm in a microplate reader. Blank is reaction mixture with no enzyme.</li>
<li>For Xyl measurements, transfer 4 μl into 384-well plates. Add the assay reagents and read the plates at 340 nm. Blank is the reaction mixture with no enzyme.</li>
</ol>
5. Data Analysis
<ol>
<li>Convert the absorbance values to their % Glc and Xyl yields based on the known Glc and Xyl content of the original biomass. Import these percentages as the responses into the Design-Expert software. Check the statistical paramenters to be sure that the criteria for a robust model are fulfilled.</li>
<li>Confirm the best model prediction experimentally.</li>
</ol>
6. Representative Results with GENPLAT: 
(1) Creation of optimized mixtures of individual pure proteins. The results indicate which enzymes are important, and in what proportions, and also which enzymes are not important. Some proteins that are abundant in the T. reesei secretome (Nagendran et al., 2009), such as Cip1 and Cip2, appear to play no role in Glc release from corn stover pretreated by ammonia fiber expansion (AFEX) or alkaline hydrogen peroxide (AHP) (Banerjee et al., 2010b). On the other hand, some proteins are unexpectedly important. For example, endo-xylanases of Glycosyl Hydrolase families 10 and 11 are both important for Glc release; one xylanase cannot substitute for the other (Banerjee et al., 2010b,c). Cel61A, a minor protein in the T. reesei secretome, is very important for Glc but not Xyl release. Some enzymes are important for release of both Glc and Xyl, whereas others are necessary only for one or the other (Banerjee et al., 2010c). 
A synthetic mixture containing 16 components, each at a concentration of 0.94 mg/ml (i.e., a non-optimized mixture), released 38% of the available Glc from AFEX-pretreated DDG. Under identical hydrolysis conditions, an optimized mixture, in which the concentrations of the 16 components ranged from 0% to 32%, released 52% of the Glc (Fig. 2). This experiment illustrates the utility of constructing defined mixtures.
(2) Creation of optimized cocktails for different pretreatment/substrate combinations. Current commercial cellulase preparations are "one-size-fits-all". In reality, the best cocktail depends on both the substrate and the pretreatment. A single product such as Accellerase 1000 gives different yields from different substrates pretreated in the same way, and from the same substrate exposed to different pretreatments (Fig. 1). We have used GENPLAT to optimize mixtures of pure enzymes for multiple pretreatments and mutiple feedstocks (Banerjee et al., 2010c). Figure 2 shows how the optimal enzyme proportions of 16 pure enzymes differs for AFEX-pretreated corn stover, AHP-pretreated corn stover, and AFEX-pretreated DDG. Only 11 enzymes are shown in Fig. 2 because the optimal proportions of the other five (Cel61B, AbfB, Cip1, Cip2, and Cel12A) were found to be 0% (Banerjee et al., 2010c).
(3) Novel enzyme discovery. The construction of optimized mixtures of pure proteins is an ongoing process. As new proteins become available in pure form (from commercial sources, by heterologous expression, or by purification), they can be systematically tested by adding them to the current optimized set. Novel proteins that contribute to Glc or Xyl release then become part of a new, optimized set. In this way, GENPLAT becomes a platform for the identification of new enzymes for biomass deconstruction. We have made extracts of fungi other than T. reesei and A. niger grown on different substrates. One particular extract boosted Glc yield when added to our core set. The responsible protein was purified and shown to be a novel glycosyl hydrolase not present in any commercial enzyme preparation (Banerjee and Walton, unpublished results).
(4) Discovery of better enzymes. Having shown with GENPLAT which enzymes are most important for degradation of a particular biomass, we have a better understanding of which enzymes should be the focus for improvement. Better examples of the critical enzymes could be found by searching in nature or made by protein engineering. (An enzyme could be "better" than its T. reesei homolog in several ways, depending on the desired property, e.g., higher specific activity, greater synergy, decreased proteolytic sensitivity, or enhanced thermal stability). GENPLAT provides a meaningful platform for assaying potentially better enzymes, because it utilizes a realistically complex cocktail (important because no biomass enzyme works in isolation) and a realistic substrate (important because results with pure cellulose or other artificial substrates may not be relevant to natural lignocellulosic materials).
(5) Commercial enzyme optimization. Sufficiently large quantities of pure enzymes do not yet exist in the real world. Until they do, ethanol producers must rely on commercial products such as Accellerase 1000 and MultifectXylanase. However, mixtures of commercial enzymes generally perform better than any individual one, and GENPLAT can be used to design optimized cocktails of multiple commercial enzymes. Figure 3 shows the use of GENPLAT to optimize a mixture of four commercial enzyme preparations. The optimal proportions for release of Glc from AFEX-pretreated DDG were found to be 47% Accellerase1000, 27% Multifect Pectinase, 22% Novozyme 188, and 4% Multifect Xylanase (Banerjee et al., 2010c). Figure 3B shows the differences in Glc yield from AFEX-DDG with four enzyme preparations; the optimized commercial mixture gives higher Glc yield than Accellerase1000 alone, SpezymeCP alone, or a 16-component synthetic mixture. This experiment also indicates that the 16-component synthetic mixture is missing one or more enzymes needed for optimal Glc release, which are present in one or more of the commercial enzymes. GENPLAT can be used to identify such components.
<img src="/files/ftp_upload/3314/3314fig1.jpg" alt="Glucose yield comparison bar charts for different feedstocks and pretreatments in biofuel research." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. No single commercial enzyme preparation is optimal for all substrates and all pretreatments. Five substrates and three pretreatments were compared under similar conditions of grinding (0.5 mm particle size), enzyme loading (15 mg/g glucan), and hydrolysis conditions (48 hr, 50°C). A. Digestion of different AFEX-pretreated substrates with Accellerase 1000. B. Digestion of corn stover pretreated by three different methods with Accellerase 1000 (see Banerjee et al., 2010c).
<img src="/files/ftp_upload/3314/3314fig2.jpg" alt="Enzyme proportion bar chart; pretreatment/feedstock comparison: AFEX CS, AHP CS, AFEX DDG." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Optimal proportions of 11 enzymes for release of Glc from AFEX-pretreated corn stover (AFEX-CS), alkaline hydrogen peroxide-pretreated corn stover (AHP-CS), or AFEX-pretreated dried distillers’ grains (AFEX-DDG). Data are from Banerjee et al. (2010c). Numbers along the top of the bars are the Glc yields as a percent of the total Glc in the biomass sample.
<img src="/files/ftp_upload/3314/3314fig3.jpg" alt="Glucose yield: (A) Ternary diagram; (B) Bar chart comparing enzyme treatments, analytical results." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. A. The use of GENPLAT to produce an optimized mixture of four commercial enzyme preparations for release of Glc from AFEX-pretreated DDG. Multifect Xylanase made the smallest contribution (4%) and was therefore omitted from this ternary diagram. B. Glc yields from AFEX-DDG with different enzyme treatments under identical hydrolysis conditions (15 mg/g glucan enzyme, 48 h, 50°C). "Four-component commercial" has the proportions determined from the experiment shown in Fig. 3A .

<ol><li>Anderson, M. J., Whitcomb, P. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAnderson%2C+MJ+%22DOE+Simplified%3A+Practical+Tools+for+Effective+Experimentation%22">DOE Simplified: Practical Tools for Effective Experimentation</a>. , 2nd Edition, Productivity Press. New York. (2007).</li>
<li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Aslam, N., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+enzyme+mixtures+for+biomass+deconstruction%3A+production+and+optimization+of+a+core+set%5BTitle%5D)+AND+%22Biotechnol.+Bioengineer%22%5BJournal%5D)">Synthetic enzyme mixtures for biomass deconstruction: production and optimization of a core set.</a> Biotechnol. Bioengineer. 106, 707-720 (2010).</li>
<li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Bongers, M., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+multi-component+enzyme+mixtures+for+deconstruction+of+lignocellulosic+biomass%5BTitle%5D)+AND+%22Bioresour.+Technol%22%5BJournal%5D)">Synthetic multi-component enzyme mixtures for deconstruction of lignocellulosic biomass.</a> Bioresour. Technol. 101, 9097-9105 (2010).</li>
<li>Banerjee, G., Car, S., Scott-Craig, J. S., Borrusch, M. S., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+optimization+of+enzyme+mixtures+for+deconstruction+of+diverse+pretreatment%2Fbiomass+feedstock+combinations%5BTitle%5D)+AND+%22Biotechnol.+Biofuels%22%5BJournal%5D)">Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations.</a> Biotechnol. Biofuels. 3, 22-22 (2010).</li>
<li>Berlin, A., Maximenko, V., Gilkes, N., Saddler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+enzyme+complexes+for+lignocellulose+hydrolysis%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">Optimization of enzyme complexes for lignocellulose hydrolysis.</a> Biotechnol. Bioeng. 97, 287-296 (2007).</li>
<li>Decker, S. R., Brunecky, R., Tucker, M. P., Himmel, M. E., Selig, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+screening+techniques+for+biomass+conversion%5BTitle%5D)+AND+%22Bioenerg.+Res%22%5BJournal%5D)">High throughput screening techniques for biomass conversion.</a> Bioenerg. Res. 2, 179-192 (2009).</li>
<li>Gao, D., Chundawat, S. P., Krishnan, C., Balan, V., Dale, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mixture+optimization+of+six+core+glycosyl+hydrolases+for+maximizing+saccharification+of+ammonia+fiber+expansion+%28AFEX%29+pretreated+corn+stover%5BTitle%5D)+AND+%22Bioresour.+Technol%22%5BJournal%5D)">Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover.</a> Bioresour. Technol. 101, 2770-2781 (2010).</li>
<li>Harris, P. V., Welner, D., McFarland, K. C., Re, E., Poulsen, J. C. N., Brown, K., Salbo, R., Ding, H., Vlasenko, E., Merino, S., Xu, F., Cherry, J., Larsen, S. Y., LoLeggio, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulation+of+lignocellulosic+biomass+hydrolysis+by+proteins+of+glycoside+hydrolase+family+61%3A+Structure+and+function+of+a+large%2C+enigmatic+family%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: Structure and function of a large, enigmatic family.</a> Biochemistry. 49, 3305-3316 (2010).</li>
<li>Kim, E., Irwin, D. C., Walker, L. P., Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factorial+optimization+of+a+six-cellulase+mixture%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">Factorial optimization of a six-cellulase mixture.</a> Biotechnol. Bioeng. 58, 494-501 (1998).</li>
<li>King, B. C., Donnelly, M. K., Bergstrom, G. C., Walker, L. P., Gibson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optimized+microplate+assay+system+for+quantitative+evaluation+of+plant+cell+wall-degrading+enzyme+activity+of+fungal+culture+extracts%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.</a> Biotechnol. Bioeng. 102, 1033-1044 (2009).</li>
<li>Nagendran, S., Hallen-Adams, H. E., Paper, J. M., Aslam, N., Walton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+genomic+potential+for+secreted+plant+cell-wall-degrading+enzymes+in+the+ectomycorrhizal+fungus+Amanita+bisporigera%2C+based+on+the+secretome+of+Trichoderma+reesei%5BTitle%5D)+AND+%22Fung.+Genet.+Biol%22%5BJournal%5D)">Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei.</a> Fung. Genet. Biol. 46, 427-435 (2009).</li>
<li>Rosgaard, L., Pedersen, S., Langston, J., Akerhielm, D., Cherry, J. R., Meyer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+minimal+Trichoderma+reesei+cellulase+mixtures+on+differently+pretreated+barley+straw+substrates%5BTitle%5D)+AND+%22Biotechnol.+Prog%22%5BJournal%5D)">Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated barley straw substrates.</a> Biotechnol. Prog. 23, 1270-1276 (2007).</li>
</ol>

No conflicts of interest declared.

It is widely recognized that reducing the cost of enzymes is important to the development of an economical lignocellulosic ethanol industry.
Currently available commercial enzyme cocktails are complex and poorly defined mixtures of many proteins (Nagendran et al., 2009), and they are adapted mainly for use on acid-pretreated corn stover. 
In order to accelerate the development of better enzyme cocktails, several laboratories have developed high-throughput platforms for enzyme discovery and characterization. Efforts in this area have incorporated one or more of the following properties also found in GENPLAT: robotic dispensing of enzymes and biomass slurries, statistical design of experiment, and/or automated determination of Glc and Xyl (Berlin et al., 2007; Decker et al., 2009; Kim et al., 1998; King et al., 2009). GENPLAT extends these earlier efforts, most significantly in the complexity of the enzyme mixtures that can be analyzed from at most 6 components in the earlier studies to more than 16 in our latest work (Banerjee et al., 2010c). Additional key features of GENPLAT are the use of a bead mixing chamber (paddle reservoir) that can keep stover slurries suspended during dispensing; gentle mixing during digestion by end-over-end rotation; and automated colorimetric determination of Glu and Xyl.

genplat: an automated platform for biomass enzyme discovery and cocktail optimization

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
<div id="myEventWatcherDiv">
	 </div>

GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

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.

genplat-an-automated-platform-for-biomass-enzyme-discovery-cocktail

Research

JoVE Journal

Bioengineering

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

High-throughput Saccharification Assay for Lignocellulosic Materials

In this video, we demonstrate a high throughput assay for carbohydrate-active enzyme screening using insoluble chromogenic polymer hydrogel substrates. The enzyme degrades these substrates into a soluble colored product that can be quantified by measuring the color intensity of the product.

Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates

This video demonstrates an assay to screen enzymes effective in degrading complex polysaccharides in plant biomass using insoluble chromogenic biomass or ICB. The enzymes hydrolyze ICB to release soluble dye-linked oligosaccharides, and the enzyme activity is assessed by colorimetric analysis of the reaction product.

Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

The MultiBac Protein Complex Production Platform at the EMBL

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate).

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

This protocol provides a method for the systematic global optimization of genetically encoded biosensors through automation-assisted genetic library generation and assessment. This is coupled with design-of-experiment methodologies to streamline experimentation and enable the selection of genetic components to tune biosensors to specific design outcomes.

Efficient Sampling of Genetically Encoded Biosensor Design Space Enabled with a Design of Experiments and Automation Workflow

This protocol presents a standard high-throughput screening workflow for industrial microorganisms. It involves constructing a mutant library via ARTP mutagenesis, performing positive droplet screening with automated microfluidics, and subsequently verifying the results. The workflow’s reliability was validated by screening high-yield B. coagulans strains.

High-Throughput Screening of L-Lactic Acid-Hyperproducing Bacillus coagulans Mutants Using an Automated Droplet Microfluidic Platform

Discover automated enzyme cocktail optimization for biomass degradation using GENPLAT. This method enables efficient conversion of lignocellulose to fermentable sugars for biofuel production.

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

Explore More Videos