Immobilized enzyme reactors in HPLC and its application in inhibitor screening:A review

This paper sets out to summarize the literatures based on immobilized enzyme bio-chromatography and its application in inhibitors screening in the last decade.In order to screen enzyme inhibitors from a mass of compounds in preliminary screening,multi-pore materials with good biocompatibility are used for the supports of immobilizing enzymes,and then the immobilized enzyme reactor applied as the immobilized enzyme stationary phase in HPLC.Therefore,a technology platform of high throughput screening is gradually established to screen the enzyme inhibitors as new anti-tumor drugs.Here,we briefly summarize the selective methods of supports,immobilization techniques,co-immobilized enzymes system and the screening model.

Microfluidic reactor with immobilized enzyme-from construction to applications:A review

Microfluidic,as the systems for using microchannel(micron-or sub-micron scale)to process or manipulate microflow,is being widely applied in enzyme biotechnology and biocatalysis.Microfluidic immobilized enzyme reactor(MIER)is a tool with great value for the study of catalytic property and optimal reaction parameter in a flourishing and highly producing manner.In view of its advantages in efficiency,economy,and addressable recognition especially,MIER occupies an important position in the investigation of life science,including molecular biology,bioanalysis and biosensing,biocatalysis etc.Immobilization of enzymes can generally improve their stability,and upon most occasions,the immobilized enzyme is endowed with recyclability.In this review,the enzyme immobilization techniques applied in MIER will be discussed,followed by summarizing the novel developments in the field of MIER for biocatalysis,bioconversion and bioanalysis.The preponderances and deficiencies of the current state-of-the-art preparation ways of MIER are peculiarly discussed.In addition,the prospects of its future study are outlined.

RAPID DETERMINATION OF L-GLUTAMIC ACID WITH AN ENZYME REACTOR OF L-GLUTAMIC DECARBOXYLASE IMMOBILIZED ON ION EXCHANGE RESIN

The preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) were studied. This work is to develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N?methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The properties of the immobilized enzyme on CM-CADB were studied by investigating the initial rate of the enzyme reaction, the effect of various parameters on the immobilized GDC activity and its stability. An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamate acid. The limit of detection is 1.0×10-5 M. The linearity response is in the range of 5×10 -2-5×10 -5 M . The equation of linear regression of the calibration curve is y= 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamate acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

A novel organic-inorganic hybrid monolith for trypsin immobilization

In proteomics, attention has focused on various immobilized enzyme reactors (IMERs) for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in a 100 μm i.d. capillary with 3-glycidoxypropyltrimethoxysilane (GLYMO) as the monomer and tetraethoxysilane (TEOS) as the crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups, which were obtained from hydrolysis of epoxy groups, and the amino groups of trypsin. Bovine serum albumin was digested thoroughly by this IMER in 47 s. After micro-reverse phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has potential for high throughput digestion.