Immobilization Of Enzyme Molecules Research Articles

In situ Synthesis and Immobilization of Enzyme Molecules on Microreactor Array Chips

The improved catalytic activity of enzymes is required in various fields. Enzymes have conventionally been improved by the screening of bacteria possessing mutant enzymes. However, the screening conditions are limited since screening requires the growth of bacteria. Here, we report the development of a protein microarray for the analysis of enzymatic activity. A his-tagged enzyme is synthesized in situ and immobilized on the microarray, which is composed of microreactors with a diameter and depth of 4 μm and a density of 1.0 x 106 reactors/cm2. β-glucosidase, synthesized in situ using a cell-free synthesis system, was immobilized on the microreactor array chip and its catalytic activity was observed. This enzyme-immobilized microarray is expected to enable the rapid and quantitative screening of enzymes.

Ultrasound-assisted generation of ACE-inhibitory peptides from casein hydrolyzed with nanoencapsulated protease

Bioactive peptides generated from milk proteins are eminent ingredients for functional foods and nutraceuticals. Amongst several approaches to release these peptides, hydrolysis of milk proteins with proteolytic enzymes is a promising choice. It is, however, required to inactivate the enzyme after a predetermined time, which leads to impurity of the final product. Immobilization of enzyme molecules can overcome this problem as it simplifies enzyme separation from the reaction mixture. A fungal protease from Aspergillus oryzea was encapsulated within nanoparticles yielded via silicification of polyamidoamine dendrimer template generation 0. It was used to hydrolyze the dominant milk protein (casein) in the absence or presence of sonication. The production of angiotensin converting enzyme (ACE)-inhibitory peptides was monitored during hydrolysis. Sonication did not affect maximum ACE-inhibitory activity but shortened the process sixfold. Ultrafiltration permeate of the centrifugal supernatant of casein solution hydrolyzed during sonication inhibited ACE activity as efficiently as the supernatant obtained from it. The protease from Aspergillus oryzea encapsulated within nanospheres is suitable for generation of ACE-inhibitory peptides from casein. The nanoncapsulation procedure is simple, rapid and efficient. This may enable the industrial production of functional products from milk.

Pulsed-laser metal contacting of biosensors on the basis of crystalline enzyme-protein layer composites

Crystalline bacterial cell surface layers (S-layers) composed of monomolecular arrays of protein subunits are accessible to a wide variety of possible proteinchemical reactions. This enables the attachment and immobilization of enzyme molecules in a tightest packing, which has not been achieved with other immobilization matrices. When immobilized to an S-layer lattice, the enzyme entities are surrounded by nanometer pores. Thus, they can react electrochemically with the analyte liquid streaming through these pores. The control over this process has to take place by way of an inert electrical contact in a distance of less than 1 nm. The relatively voluminous, but specially shaped sensor enzyme molecules have to be connected with an optimum metallic contact, which must not disturb the protein structure. Previously, platinum films were applied on enzyme layers immobilized on S-layer protein by argon sputtering. This conventional technique, however, exhibits substantial limitations. One, for instance, is the volume change of the S-layer/enzyme composite system when it is introduced into a conventional vacuum coating apparatus. This coating problem can be circumvented by a completely new deposition method, i.e. the pulse-laser-deposition (PLD) on protein crystal composite films with optimized laser parameters and reaction atmospheres. Enzyme activities of 70–80% were achieved, thus demonstrating that composite systems consisting of the 2D-protein-layer/enzyme/metal sequence can successfully serve as highly efficient sensor systems.

A New Type Micro-Glucose Sensor Based on Amperometric Detection

A variety of biosensors have been developed for thedetermination of substance of clinical importance, since Clark-type oxygen electrode-based enzyme sensors were demonstrated. For the practical applicationof biosensors, microfabrication of enzyme electrodes is very important because biosensors with the easy handling and disposable usage can be attained easily by miniaturization techniques. Here glucose oxidase has been taken as the enzyme to be immobilized, and the enzyme product, hydrogen peroxide, is oxidized by the electrode matrix. Therefore, the amperometric signal of the micro-enzyme electrode is related to the concentration of glucose. The 100% response of the micro-enzymese nsoris no longer than 3sec, and the determinable range for glucose is from 5x 10-7 to 10-2mol/1. The time stability of the sensors are satisfactory: The sensor response to 1mM glucose is within ±2%for several weeks. We have developed a unique electrochemical fabrication technique, simultaneous platinization and immobilization of enzyme molecules. In this technique enzyme is incorporated into electrolyte solution and is embodied on the growing surface of platinized platinum during electrolysis .