DEVELOPMENTS OF ENZYME SYSTEMS TOLERANT TO ARTIFICIAL CONDITIONS AND ANALYSIS OF THEIR FUNCTIONS

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Developments of enzyme systems which can be used in rather hard artificial conditions such as high temperature, high salt conditions, high contents of organic solvents, and high pressure, are necessary to the wider applications of enzymatic processes into chemoenzymatic applications.In this project two extreme artificial conditions, namely solvent-tolerance and pressure-tolerance, are chosen as the targets and in order to obtain microbilas which produce these tolerant enzymes convenient screening procedures are to be investigated. In addition to this, chemical modification methods and amino-acid replacements are also applied to produce the aimed enzyme systems.Namely, direct observations of microbilas through micrographic methods under high pressure conditions were made possible by investing pressure micrgoraph cell and a very convenient method of pressure-tolerance screening was proposed by use of a commercially available high-pressure pressing apparatus.For the chemical modifications, high pressure-induced partial refolding of enzyme proteins was utilized to introduce the midifier reagent into the depth of the protein molecules, which is original to the investigator's laboratory.The functional analysis of the obtained enzymes under artificial conditions were further performed by use of various spectrophotometric methods, such as 4th derivative absorbance, FTIR, fluorescence, and high pressure electrophoresis, both in situ and ex situ manners.