enzyme based analytical chemistry - nitrate and the u.s. epa

Recombinant Enzymes for Analytical Chemistry

Enzyme-Based Analytical Chemistry Nitrate and the U.S. EPAEllen Campbell, Ceo & vpneci superior enzymes

BIOTECHNOLOGY CHEMISTS CAN USEEnzymes are catalysts made of proteinChemical reactions in biological systems are made possible by enzymesEnzymes have been used for medical analysis for decadesState of the art in biotechnology enables reliable enzyme productionEnzymes for Green Analytical Chemistry

ANALYTICAL ADVANTAGES OF ENZYMESSELECTIVITYFind target in complex mixturesSENSITIVITYLow detection limits in complex mixturesSPECIFICITYFalse negatives and false positives are rareSAFETYFor shipping, storage, handling, and disposalREAGENT GRADE ENZYMES ARE ACCURATE, RELIABLE, AND ENVIRONMENTALLY BENIGN

NITRATE REDUCTASE

TIGHT QC ENSURES ENZYME QUALITY

Highest purity media componentsOptimized SOPs Computer controlled fermentation

RECOMBINANT ENZYME PRODUCTION WORKFLOW

EARLY NITRATE REDUCTASE PUBLICATIONSFeb 1986: Plant Physiology: Regulation of Corn Leaf Nitrate ReductaseAug 1990: Trends in Biochemical Sciences: Functional domains of assimilatory nitrate reductases and nitrite reductasesFeb 1996: Plant Physiology: Nitrate Reductase Biochemistry Comes of AgeMar 1998: Analytical Chemistry: Construction and Characterization of Nitrate Reductase-Based Amperometric Electrode and Nitrate Assay of Fertilizers and Drinking WaterJun 1999: Annual Review of Plant Physiology and Plant Molecular Biology: Nitrate Reductase Structure, Function and Regulation: Bridging the Gap between Biochemistry and PhysiologyJun 2000: Plant Physiology: Recombinant Expression of Molybdenum Reductase Fragments of Plant Nitrate Reductase at High Levels in Pichia pastorisJan 2002: Environmental Science & Technology: Corn Leaf Nitrate Reductase A Nontoxic Alternative to Cadmium for Photometric Nitrate Determinations in Water Samples by Air-Segmented Continuous-Flow Analysis

*The above publications are only a selected few of the comprehensive list published by Dr. Bill Campbell on Nitrate Reductase

CAMPBELL, W. H. PhD NECi President, MTU Professor Emeritus

NO3NO2H+H2ONADH+++NAD++Nitrate ReductaseNO2+Sulfanilic Acid+Diazonium SaltDiazonium SaltNEDPink dye for measuring A 540 20nmSamples are measured against a standard nitrate curve to determine results in ppm Nitrate-N NITRATE REDUCTASE REPLACES CADMIUM

NITRATE ANALYSIS USING NITRATE REDUCTASENitrate Reductase (NaR) catalyzes the reduction of nitrate to nitriteNADH is the electron donor for driving the reactionResulting Nitrite reacts with Griess color reagents to produce an AZO dyeSamples are analyzed spectrophotometrically at 540 20 nmNaR simply replaces Cadmium in conventional methods. The rest of the chemistry is the same.

CADMIUM VS. NITRATE REDUCTASE

0.010.101.005.000.010.101.005.00

Line of equal correlation

Nitrate + Nitrite Concentration in mg-N/L (NaR)Nitrate + Nitrite Concentration in mg-N/L (CdR)Source: USGS Validation Study

NITRATE REDUCTASE ASSAY FOR NITRATE ANALYSISSmall sample size in relation to total assay volumeLittle sample preparation requiredHigh specificity reduces false positives and false negativesHighly purified and freeze-dried protein glass$0.21-$0.25 per sample for any Discrete analyzerCompetitive cost per sample for most FIA & SFA systems

Semi-Automated Batch Reduction System for Enzymatic Nitrate DeterminationsStoryboards courtesy of Charles Patton, USGS, 2003

RECOMBINANT ENZYME PRODUCTIONGuaranteed lot-to-lot reproducibilityIncreased production capacityReasonable production costsEngineered for Improved StabilityStable during analytical runsShipping at AmbientStorage at controlled RT

GUARANTEED ENZYME PURITYOne unit will reduce 1.0 mol nitrate to nitrite per minute in NADH system at pH 7.5 and 30CStable indefinitely at -20C; Min 2 years at 4C (freeze-dried)Expected Purity: 20 40 units/mg proteinAll additives are reagent grade, no animal-derived or hazardous materials

NITRATE REDUCTASE METHOD VALIDATIONSValidated in 2011 for DACollaborative study with NWQLStatistically equivalent to Cd reduction methodValidated in 2014Replaces nitrate methods D1254 and D992Can be used in place of D3867 (Cd Reduction)Validation study submitted 2013ATP Letter received April 2014No negative comments to address Methods Update Rule for CWA Reporting in 40 CFR Part 136.6

SAMPLES TESTED FOR VALIDATION STUDIES

Source: U.S. EPA Validation Study for CWA 40 CFR Part 136

Acceptance Standard is 90% or greater reduction efficiency2nd Source = Nitrate Standard in mg N/L Nitrate Standard = mg N/LENZYMATIC REDUCTION EFFICIENCY SUMMARY


TABLE 5 FROM EPA VALIDATION REPORT: INITIAL PERFORMANCE AND RECOVERY (IPR) SUMMARY


TABLE 7: METHOD DETECTION LIMIT (MDL) SUMMARYDA = Discrete AnalyzerNA = Not AnalyzedOne replicate discarded due to issue with blank


NITRATE + NITRITE BY DA: ENZYME VS. CADMIUM


NITRATE METHOD VALIDATIONSAS OF JULY 2016Inclusion in 40 CFR Part 136 of Clean Water Act Methods pending

Validation Study for U.S. EPA Safe Drinking Water Act pending publication in the Methods Update Rule

Standard Methods for the Examination of Water & Wastewater (standardmethods.org) in progress

EPA Drinking Water Study

Source: U.S. EPA Validation Study for SDWA

EPA Drinking Water StudyStandard Deviationmg Nitrate-N/LFinalmg Nitrate-N/LCompared to ERALab 1 - NaRR0.033755.3989105.0195%Lab 1 - CdR0.079985.093799.0992%Lab 2 - NaRR0.075524.979596.8783%Lab 2 - CdR0.021325.064098.5214%Lab 3 - NaRR0.0051005.125899.7218%Lab 3 - CdR0.018165.1800100.7782%


SAME METHODDIFFERENT FORMATSLABORATORY TEST KITS

SIMPLIFIED TEST KITS

NECI HANDHELD PHOTOMETER

NECI HANDHELD PHOTOMETEROther devices use round vials or outdate chemistry methods which are inaccurate or toxicNSF Grant in collaboration with Dr. Pearce at MTU resulted in PLOS ONE Publication in August 2015 for open-source technologyOpen source technology makes science more accessible to a wide audienceMakes simplified test kits more accurate for field measurement

NECI HANDHELD PHOTOMETERSame data and technologies in the field as in the laboratoryEnzyme assay standard curve programmed inLEDs for specific wavelengths Blank function for calibrationUses standard square cuvettes to eliminate optic distortion

NECI HANDHELD PHOTOMETERGPS and Sample Time RecordedData Collection on Android DevicesExport of CSV for Sharing & AnalysisPhosphate & Nitrate Analysis

RECENT RESEARCH & DEVELOPMENTPLOS ONEEnzyme-Catalyzed Oxygen Removal System for Electrochemical Analysis under Ambient Air: Application in an Amperometric Nitrate BiosensorANALYTICAL CHEMISTRYOpen-Source Photometric System for Enzymatic Nitrate QuantificationELSEVIER METHODS XDetermination of Phosphate in Soil Extracts in the Field: A Green Chemistry Enzymatic Method

201220152015

PNP FOR PHOSPHATE MEASUREMENT

Graphical Abstract: Determination of Phosphate in Soil Extracts in the Field: A Green Chemistry Enzymatic Method

PNP ASSAY FOR MEAURING PHOSPHATEReaction mixture is prepared: Buffer (200 mM HEPES, pH 7.6, 20 mM MgCl2) + MESG (80 nmol) + Recombinant PNP Enzyme (1 unit)Sample is added to reaction mixture and mixedReaction goes to completion within 20 minutesAbsorbance at 360 nm is measured after blanking spectrophotometer with HEPES bufferAbsorbance of sample is compared with standard curve (prepared in advance with certified1000 ppm KH2PO4 standard diluted in deionized water to the desired range)

PNP ASSAY CHEMISTRYH2PO4-2-amino-6-mercapto-7-methylpurine ribonucleoside+2-amino-6-mercapto-7-methylpurinePurine Nucleoside PhosphorylaseMESGAfter background absorbance of reagent blank is subtracted, the linear standard curve, ppm phosphate is easily determined

Ribose-1-phosphate

+

The purine product yields an increase in absorbance at 360 nm in equal molar ratio to the inorganic phosphate in the reaction mixture

TOTAL NITROGEN AND TOTAL PHOSPHORUSWere validating methods for analyzing Total N and Total P

Involves one sample preparation method prior to analysis using enzyme based test kits for nitrate and phosphate

Application Notes coming soon Ask us about priority notification

ENZYME FOR MEASURING GLYCEROLHigh glycerol content in biodiesel may cause problems during storage, in the fuel system, injector fouling, and formation of deposits.Important to monitor glycerol content during biofuel productionPure Glycerol QA/QC Thanks to USDA funding, we have successfully made a recombinant enzyme and are researching properties

ENZYME FOR MEASURING ETHANOLAlcoholic beverage QA/QCGrowing biofuels demand & marketEthanol content analysis in conventional fuelsConcerns of Vehicle Compatibility: Meeting E10 requirements?

NITRATE BIOSENSORPublication in 2012 on oxygen removal system for electrochemical analysis under ambient airUSDA grant application submitted for the development of a Nitrate BiosensorThis technology will further develop accessibility of science to the general publicMore samples analyzed, more data collected, more environmental analysis means better resource management and protection

ENZYMATIC OXYGEN REMOVAL SYSTEM

United States Patent No. US 9,187,779Oxygen 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.

WE APPRECIATE SUPPORT FROMSmall Business Innovation Research Programs of the USDA, NIH, and NSF. Michigan MEDC SBIR matching funds and the UofM FCP.USEPA CWA and SDWA Offices, ASTM, USGS, TNI, Standard Methods, Equipment ManufacturersCharles Patton, William Lipps, Lemuel Walker, Lynn Egan, J Kevin Roberts, and many othersVisit www.nitrate.com to learn more

Lab 1Lab 2Lab 3*

NaR-RCd-RNaR-RCd-RNaR-RCd-R

CatalyticReductionEfficiency (NO3/NO2)Percent93.2551

93.927998.0040

105.9603104.7569

103.0221

102.5518100.2798

96.713997.008698.572799.850993.3010

94.3464101.8339

96.7978104.0904