Library · Biology & lab methods
What Enzyme Activity & Kinetic Assays got wrong, from 20 theses
Summary written by a model from the records below: Experimental and theoretical investigations of enzyme activity and reaction kinetics frequently fail due to matrix interferences, invalid steady-state assumptions, and flawed kinetic model formulations. Furthermore, transient and coupled assays often suffer from secondary rate limitations, poor reproducibility, and mass-transport constraints that obscure underlying catalytic rates. These records come from graduate theses, 2021 to 2026, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Plus 8 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Simplified or empirical kinetic model formulations fail to capture complex reaction dynamics
Initial-rate fitting, non-cooperative models, and empirical power laws failed to predict complete reaction yields, temperature trends, or non-monotonic inhibition profiles. In other cases, parameter values drifted systematically with observation time or chemical Langevin approximations failed to replicate steady-state distributions and noise.
Tried and failed
heuristic stochastic quasi-steady state chemical Langevin approximation applied to enzyme kinetics steady-state distributions. Reason: failed to capture exact steady-state distributions and overestimated noise across system sizes
Data-driven stochastic modelling of gene regulation · Imperial · PhD
Tried and failed
initial-rate kinetic parameter fitting for reaction modeling applied to enzymatic synthesis yield prediction. Outcome: did not generalise. Reason: initial-rate kinetic parameters failed to accurately capture full time-course reaction yields
Continuous enzymatic reactive crystallization of beta-lactam antibiotics · Georgia Tech · PhD
Tried and failed
empirical power-law model with inhibition terms applied to reaction kinetics modeling across temperatures. Outcome: did not generalise. Reason: lacked physical microkinetic grounding and failed to track conversion trends across varying temperatures
A Mathematical Model for Methanol Synthesis in a Water-Selective Membrane Reactor · Johns Hopkins · Master's
Tried and failed
omitting cooperativity hill coefficient from kinetic model applied to competitive receptor clearance modeling. Outcome: worse than baseline. Reason: non-cooperative kinetics could not reproduce the in vitro inhibitory response despite parameter tuning
Experiments and Simulations of Autonomous Microscale Robotics · MIT · PhD
Tried and failed
Hill inhibition kinetic modeling applied to bacterial inhibition kinetics under salinity stress. Outcome: did not generalise. Reason: low concentrations caused stimulation rather than monotonically decreasing inhibition
Denitrification Under Salinity Stress in Biofilm Reactors · University of Trento · PhD
Considered and rejected
Considered and rejected: Rejected four-parameter logistic end-point fitting of SWCNT traces because critical parameters (x0, h) varied systematically with probe time rather than reflecting true invariant enzymatic kinetics.
Optical probes and measurement systems for enzyme activity and fermentation process monitoring · Iowa State · PhD
Standard Michaelis-Menten kinetic assumptions break down under non-ideal experimental regimes
Classical Michaelis-Menten models were invalidated when substrate concentrations were comparable to enzyme levels or when initial rates remained linear without reaching saturation. Practitioners also rejected these formulations when high substrate concentrations caused optical inner filter effects and insolubility or when time-series data was inadequate to constrain non-linear parameters.
Considered and rejected
Considered and rejected: Rejected using non-linear Michaelis-Menten kinetics in initial SOC model iterations due to insufficient temporal 14C time-series to constrain parameters.
Modelling the carbon cycle in the soils and rivers of Switzerland with radiocarbon · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected using pure Michaelis-Menten kinetics, adopting quadratic master equations with explicit nucleotide exchange to handle substoichiometric DnaJ saturation.
Why do we need so many proteins? A physical insight into the collaboration of Hsp70 and DnaJ · EPFL · PhD
Considered and rejected
Considered and rejected: Rejected nonlinear Michaelis-Menten regression for CKM R132C kinetics because initial rate remained linear without plateauing at tested ATP concentrations, using a linear fit instead
Impact of interindividual differences on muscle-type creatine kinase: implications for tenofovir-based PrEP · Johns Hopkins · PhD
Considered and rejected
Considered and rejected: Rejected traditional Michaelis-Menten initial-rate kinetics to determine 3CLpro catalytic efficiency because 10x KM required millimolar substrate concentrations blocked by the inner filter effect and peptide insolubility
Exploring and Exploiting Ribonuclease 1: from Protein Biochemistry to Protein Engineering · MIT · PhD
Optical and matrix interferences corrupt spectrophotometric and colorimetric readouts
Absorbance-based assays experienced signal failure when matrix turbidity, aromatic compounds, buffer precipitation, or tissue volume changes dominated optical density measurements instead of catalytic activity. Colorimetric methods also exhibited non-linear calibrations, overestimation, or an inability to distinguish product pools in mixed-substrate reactions.
Tried and failed
spectrophotometric absorbance detection in turbid matrices applied to enzymatic screening in complex wastewater. Outcome: no signal. Reason: optical turbidity and background aromatic compounds interfered directly with absorbance-based measurement
Applications of Systems-Driven Protein Engineering Toward In Situ Water Safety Technologies · Cornell · PhD
Tried and failed
enzymatic assays and HPLC applied to organic acid quantification in fruit juice. Outcome: unstable. Reason: lack of correlation upon dilution and buffer precipitation during analysis
BROADENING THE BASE OF APPLE GENETICS: A STUDY OF TRAITS USING MALUS PRUNIFOLIA · Cornell · PhD
Lost to a baseline
Salicylate colorimetric method exhibited non-linear calibration kinetics (Pearson's r < phenol) and ~10% overestimation compared to the phenol (indophenol blue) method.
Real-time ammonia quantification in electrochemical nitrogen reduction · Imperial · PhD
Considered and rejected
Considered and rejected: Phosphomolybdate spectrophotometric method rejected for mixed-substrate kinetics due to inability to track substrate-specific Pi
REACTIVITY AND STRUCTURAL DYNAMICS OF EXTRACELLULAR PHOSPHATASES: IMPLICATIONS FOR PHOSPHORUS RECYCLING · Cornell · PhD
Considered and rejected
Considered and rejected: Abandoned measuring ACE enzymatic activity in maternal kidney and placental lysates because optical density changes were driven solely by absorbance of increasing tissue volume rather than catalytic activity.
Defining the impact of paternal diet on maternal cardiometabolic ill-health in late gestation · Nottingham · PhD
Coupled and progress-curve assay designs suffer from lag phases and temporal resolution limits
Coupled and real-time enzyme assays failed because pronounced lag phases at low substrate concentrations or in high salt distorted pseudo-first-order rate estimates. Additionally, instruments could not resolve basal reaction rates below detection thresholds, and high kinetic variance in stochastic motor enzymes obscured state distinctions.
Tried and failed
spectrophotometric coupled-enzyme activity assay applied to enzyme acting as own substrate. Outcome: no signal. Reason: Low signal-to-noise ratio, high required protein concentration, and inability to determine Michaelis-Menten kinetic parameters.
A Chemical toolbox to Study Histone Glycation · Cornell · PhD
Considered and rejected
Considered and rejected: FucA (fuculose 1-phosphate aldolase) coupled assay was rejected for steady-state kinetics of MtnA because of a significant lag phase at low substrate concentrations.
Mechanistic Investigation of the Reaction Catalyzed by 5-Methylthioribose-1-phosphate Isomerase in the Methionine Salvage Pathway · University at Buffalo · PhD
Tried and failed
measuring dimerization dissociation constant via enzymatic progress curves applied to homodimeric viral protease kinetics. Reason: concentration-dependent lag phase in high salt overestimated pseudo-first-order rate constants
Exploring and Exploiting Ribonuclease 1: from Protein Biochemistry to Protein Engineering · MIT · PhD
Considered and rejected
Considered and rejected: Single-parameter dwell-time/current-blockade analysis rejected because stochastic motor enzyme kinetics caused high variance and could not cleanly distinguish bound from unbound states.
Multiplexed detection of microRNAs, proteins and neurotransmitter using barcoded molecular probes and nanopore sequencing · Imperial · PhD
Screening protocols and dilution steps undermine assay signal and reproducibility
Single-run screening approaches produced weak activation and inhibition signals that could not be replicated in subsequent trials. Activity was also lost through unmonitored dilution protocols or was completely absent due to missing partner proteins, cofactors, or incompatible stereochemistry.
Lost to a baseline
Fixed 1:40 dilution of Batch 2 solubilizate without IPC suffered an over 50% decrease in enzymatic activity compared to Batch 1.
Quality by Design strategies in Inclusion Body processing · TU Wien · PhD
Tried and failed
enzymatic S-adenosylmethionine-dependent methyltransferase assay applied to synthetic substrate analogue. Outcome: no signal. Reason: the methyltransferase required additional cofactors, partner proteins, or a different substrate stereochemistry to act
Biosynthetic studies of thiosugar natural products ; Mechanistic studies of hyoscyamine 6 [beta]-hydroxylase · UT Austin · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Test the STAR-based comparator using light-inducible promoters in Chi.bio turbidostats to measure dynamic RNA-RNA unbinding and sequestration kinetics. Blocker: Requires a wet lab, Chi.bio turbidostat hardware, engineered E. coli strains, and physical reagents
Towards engineering microbial consortia using RNA-based genetic controllers · Imperial · PhD
Left open
Determine the enzyme activity recovery rate after cross-linking compared to the crude extract under industrial parameters. Blocker: Requires wet lab equipment, sludge samples, and physical enzyme extraction and activity assay apparatus
Enzyme recovery from biological wastewater treatment · Imperial · PhD
Left open
Determine and compare the erythritol synthesis capacity and enzyme kinetics across human ADH1 genetic variants. Blocker: Requires wet-lab protein expression, purification, and spectrophotometric kinetic assays.
Understanding Reductive Enzymes in the Erythritol Biosynthesis Pathway: “The Secrets of Sweet Blood” · Cornell
Left open
Measure soil enzyme kinetics (Km) across diverse soil samples to evaluate plot-level versus treatment-level variation requirements. Blocker: Requires wet lab facilities, soil sample collection, and fluorometric enzyme assays
Potential enzyme activities in soils as affected by perennial cropping and nitrogen rates in central Iowa · Iowa State · PhD
Left open
Measure enzyme kinetics data for Vibrio natriegens to parameterize resource balance analysis and proteome-constrained models. Blocker: Requires wet-lab biochemistry and experimental enzyme assays for V. natriegens proteins
V. natriegens genome-scale metabolic model development and its application for the production of polyhydroxybutyrate · Imperial · PhD
Left open
Quantify the calibration factor gamma relating RFU to molar concentration of mCherry protein for arsenic biosensor kinetics. Blocker: Requires wet lab experiments with purified mCherry protein and fluorometry apparatus.
Effects of carbon substrate quality and iron on microbial arsenic metabolisms: implications for arsenic speciation and fate in rice paddy soils · Cornell · PhD
Left open
Test SBP-catalyzed enzymatic removal of acetaminophen and parabens at low concentrations representative of wastewater treatment plant effluents. Blocker: Requires a wet lab, soybean peroxidase enzyme, target chemical contaminants, and analytical equipment (e.g., HPLC-UV).
Soybean Peroxidase Catalyzed Oligomerization of Acetaminophen and Selected Parabens for Wastewater Treatment · University of Windsor · PhD
Left open
Characterize individual kinetics and activity of CgtA/MBP-Δ15CgtAII and MBP-WlaN/CgtB across varied pH, temperature, and substrate concentrations. Blocker: Requires a biochemistry wet lab, recombinant enzymes, lipid bilayers, and analytical assays to measure kinetics.
MAKING MEMBRANES SWEET: DEVELOPMENT OF A BIOMIMETIC PLATFORM FOR THE CELL-FREE GLYCOSYLATION OF GLYCOLIPIDS · Cornell · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Evans Christian Wralstad · MIT · PhD · 2024
Exploring and Exploiting Ribonuclease 1: from Protein Biochemistry to Protein Engineering - Mei-Tsan Kuo · Iowa State · PhD · 2026
Optical probes and measurement systems for enzyme activity and fermentation process monitoring - Artem Khobnya · Imperial · PhD · 2025
Real-time ammonia quantification in electrochemical nitrogen reduction - Alessio D'Aquino · University of Trento · PhD · 2025
Denitrification Under Salinity Stress in Biofilm Reactors - Julian Ebner · TU Wien · PhD · 2025
Quality by Design strategies in Inclusion Body processing - Lucas Coppens · Imperial · PhD · 2024
V. natriegens genome-scale metabolic model development and its application for the production of polyhydroxybutyrate - Alexander Brunmayr · Imperial · PhD · 2024
Modelling the carbon cycle in the soils and rivers of Switzerland with radiocarbon - Dimitris Volteras · Imperial · PhD · 2024
Data-driven stochastic modelling of gene regulation - Ferra Pinnock · Cornell · PhD · 2024
MAKING MEMBRANES SWEET: DEVELOPMENT OF A BIOMIMETIC PLATFORM FOR THE CELL-FREE GLYCOSYLATION OF GLYCOLIPIDS - Caroline Koch · Imperial · PhD · 2024
Multiplexed detection of microRNAs, proteins and neurotransmitter using barcoded molecular probes and nanopore sequencing - Afsaneh Khoshkerdar · Nottingham · PhD · 2024
Defining the impact of paternal diet on maternal cardiometabolic ill-health in late gestation - Hyun Yoon · Cornell · PhD · 2023
Effects of carbon substrate quality and iron on microbial arsenic metabolisms: implications for arsenic speciation and fate in rice paddy soils
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