|Alcohol Oxidase 1||Pichia pastoris||IMAC||Ethanol/Methanol Quantification||15 Minute Rxn w/ ABTS @405 nm|
|Alcohol Oxidase 2||Aspergillus sp.||IMAC||Ethanol/Methanol Quantification||15 Minute Rxn w/ ATBS @ 405 nm|
|Catalase||Aspergillus oryzae||IMAC||Removal of Hydrogen Peroxide, NECi's Patented O2 Removal System|
|Galactose Oxidase||Fusarium graminearum||IMAC||Galactose Quantification||15 Minute Rxn w/ ABTS @ 405 nm|
|Pyranose 2-Oxidase||Phanerochaete chrysosporium||IMAC||NECi's Patented Oxygen Removal System|
When biodiesel is made - from any feedstock - approximately 10% of the product will be glycerol (also called glycerin). Glycerol has many uses, but it destroys engines because it's a sugar alcohol, and therefore must be removed from biodiesel before it can be used or sold as fuel. ASTM International has set a limit of 0.02% glycerol in biofuels (ASTM Standard D6751). ASTM's standard analytical chemistry method for glycerol detection in biodiesel is based on gas chromatography (GC). This is not a barrier for large producers with well-equipped labs and trained technicians, but nonetheless has limitations that biofuel producers must work around, such as being excessively complicated for production personnel and inaccuracy in certain important circumstances. This is assuming that producers are equipped with a GC and trained personnel to operate it, which most producers are not. This enzyme is unique; there is nothing comparable on the market anywhere. Anything that improves processing, QA, or QC will help the growing biodiesel industry flourish. Test kits for onsite use will help producers of any size improve process timings and conditions. Reagents for higher end analytical instruments will make this possible for small and medium scale biofuel producers to perform some of their own testing on-site, saving time and testing costs. This enzyme will result in faster, better, and cheaper results - especially for on-site production spot checks. This enzyme-based glycerol analysis method will benefit biodiesel producers of all sizes, from individual farm-based operations to large producers with their own QA/QC laboratories. We envision that this method will also be employed at commercial laboratories performing multiple analysis per day for contract analysis.
Producing an enzyme-based chemistry method for the biodiesel industry is in line with NECi Superior Enzymes' priorities and values. We believe in benefiting rural communities that thrive on agriculture, and we plan to do so by helping them become more self-sufficient and sustainable. Biofuels are produced using agricultural waste, used cooking oil, and animal fats can be converted into sustainable fuel that will be used for local transportation. This reduces fossil fuel consumption by providing a fuel that minimizes bio-waste, all while keeping production within the communities. For example, a small biofuel producer we've interviewed for this project takes waste from local restaurants and processes the used cooking oil to produce biodiesel which fuels the local K-12 school buses. This is a self-sustaining cycle which we hope to benefit by making biodiesel production more efficient and accessible with our enzyme-based glycerol test method.
During a USDA Phase I grant, our researchers determined that the native form of this enzyme can quantify glycerol in biodiesel samples during all steps of production. Since native enzymes aren't stable and reproducible, our scientists successfully developed and produced a recombinant form of this enzyme. Recombinant enzymes are stable and are reproducible for lot-to-lot consistency.
Our research team has identified an ideal clone of the enzyme, manufactured in Pichia pastoris, after many growth trials. This process involves examining stability, growth and purification efficiency, and specific enzyme properties. The enzyme has been successfully purified and demonstrates the superior qualities of NECi's commercially available enzymes. Assay development, stability studies, production improvements, and market research are currently underway. The photometer development team will then optimize our handheld device for compatibility with the enzyme-based glycerol test kits.
This project was prompted by feedback from the Michigan Department of Agriculture & Rural Development (MDARD). Ethanol has been added as an octane enhancer in some Michigan gasoline for some time, it increases the octane almost 3 Anti Knock Index points. If gasoline contains ethanol, there is a limit of 10% of total volume for conventional fuels.  Around 13 billion gallons of fuel ethanol were added to motor gasoline produced in the United States in 2014. Most gasoline in the U.S. contains up to 10% ethanol by volume (E10), also known as the "blend wall", which all gasoline vehicles can use. Gasoline containing 15% ethanol can only be used by light-duty vehicles with a model year 2001 or newer, and only flex-fuel vehicles can use gasoline with greater than 15% ethanol.
Another market to note when mentioning QA/QC ethanol testing is the alcoholic beverage industry. Alcoholic beverages are made by converting sugars to alcohol through a process called fermentation. Extracts from grapes, barley, and more are fermented, which converts the naturally occurring glucose to ethanol. Monitoring ethanol content is essential during the fermentation process and concentration can affect taste, microbial activity, solubility of other constituents, and is also important for federal and state labeling statues.
Most ethanol test kits for on-site analysis of gasoline use densitometry measurement, which are rudimentary disposable test tubes that measure ethanol by the difference in density between ethanol and water. This method requires careful calibration and an eyeball's guess using units printed or marked on the side of the tubes. Considering these circumstances brings a reliability and accuracy concern into question. With an increasing interest and importance in determining ethanol levels in conventional fuels and biofuels, more reliable and accurate test methods need to be employed on-site and in analytical laboratories.
Current quantification methods for ethanol in the alcoholic beverage industry include densitometry, ebulliometry, HPLC, FTIR, gas chromatography, and IR. Many of these methods described require a large investment in special equipment or rely on methods which are outdated, inaccurate, or not reliable. Many times, distilleries, breweries, and wineries do not have their own analytical laboratories, and therefore must send their samples to an external laboratory and wait for results, which can be very costly. Offering a colorimetric, on-site, easy to use, and cost-effective test kit to these beverage producers will allow them to make more timely decisions and save by doing these testing procedures internally.
An optimal clone of the enzyme has been chosen and successfully produced in our lab. We've completed numerous assays with ethanol standards, and have shown the enzyme to be effective in accurately quantitatively measuring ethanol concentration. We're testing the enzyme's compatibility with real-world samples, including various alcoholic beverage samples and plan to begin testing fuel samples soon.
The enzymatic oxygen removal system is a patented technology developed by Nicolas Plumere, Wilbur Campbell, and Ellen Campbell. The systems and methods for oxygen removal from aqueous solutions are presented in which a bi-enzymatic reaction sequence recycles and depletes oxygen to extinction, preferably using an oxidase and a catalase as biocatalysts and a carbohydrate as co-substrate. Contemplated systems and methods are particularly advantageous in conjunction with electrochemical reaction systems in which oxygen would adversely interfere with the reaction system.
The present application relates generally to oxygen removal systems for use with biosensors that operate with recombinant reductases, and particularly with nitrate reductases, chlorate reductases, perchlorate reductases, hydroxylamine reductases, bisulfite reductases, and isoquinoline reductases.
Enzyme Based Analytical Chemistry
Test kits for laboratories, agriculture, homeowners, and more
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