Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

Table 1

Summarization of flow-based method for enzyme study.

Flow-based technique	Enzyme	Methodology	Comments	Reference No.
Continuous flow	Tyrosine phosphatase	Use 3 syringe pumps for delivering solutions with constant combined flow rate of 100 μL/min for reaction with kinetic time scale of a minute or more	General application of flow system based on laminar flow, possible to be applied to faster reactions by reducing the diameter of tubing	[7]
	Alkaline phosphatase	Randomly and site directed immobilized his-tag alkaline phosphatase on beads were studied using FIA-chemiluminescence system	Site directed enzyme had 𝑉max (activity) higher than randomly immobilized enzyme	[19]
	Acetylcholinesterase	Online dialysis of product before mixing with chromogenic reagent	Online pretreatment	[27]
	Acetylcholinesterase and angiotensin-converting enzyme	SI-LOV system with microreservoir and fiber optic/stirrer	Kinetic parameters obtained agree well with literature values	[41]
	Urease	Thermal inactivation, flow colorimetry, and model equation	Can conclude about reversibility and irreversibility of denature and native forms	[58]
	α-amylase	Basic FIA system for measuring enzyme activity at different pHs	Very stable at neutral pH 5–8, but loss of activity out of this pH range	[59]
	Glucanase	Fluorescence probe flow injection	Confirm the use of theoretical equation to predict kinetic parameters	[60]
	Alkaline phosphatase	Immobilized enzyme on Sepharose beads and packed in the reactors	Study effect of orthophosphate inhibitor	[61]
	α-amylase	Immobilized with glutaraldehyde on polyurethane foam and study activity under magnetic field	Show oscillatory behavior of enzyme reaction	[62]
	Alkaline phosphatase	Open-closed flow injection, theophylline as inhibitor	Km and inhibitor constant were determined	[63]
Stopped flow	Fructose bisphosphatase	Label free, mid IR detection	Alternative detection method	[57]
	Glutathione transferase	Potentiometric detection for mechanism of different isoenzymes	Alternative detection method	[18]
	Elastase	Conventional stopped flow-spectrometric system for slow binding kinetic approach	Conclude that inhibition is 2 step mechanism and gain insight understanding of the effect of heparin on the inhibition mechanism	[65]
	5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthase	Fluorescence measurement at equilibrium	Evaluation of substrate and inhibitor binding	[66]
	Tannase	Immobilized enzyme on glass beads, packed in conductometric flow cell	Check activity of immobilized tannase which is commonly repetitively used in industry	[67]
	Total and prostatic acid phosphatase	Double injection flow analysis	Increase injection concentration 2 fold to compensate dilution	[68]
	Protein kinase	Phosphorylation of peptide substrate, quench with acetic acid	This is the first chemical observation and characterization of phosphoryl transfer at the active site of protein kinase	[69]
Quench flow	3-Deoxy-D-manno-2-octulosonate-8-phosphate synthase	Anion exchange HPLC for detection of radiolabel	Gain conclusion about reaction intermediate	[70]
	5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthase	Radioactively label the enol moiety and then separate and quantitate products with HPLC after acid quench	Observe tetrahedral intermediate	[71]
Zone trapping/Bypass trapped flow	Hexokinase	Coupled to glucose-6-phosphate dehydrogenase, and monitored production of reduced NADPH fluorometrically	Comparable Km and Ki value to the published data obtained from manual techniques	[74]
Air segmented flow	Peroxidase	LAV with microreservoir with air segments	Mimic manual operation and eliminate dilution/dispersion effect	[80]