O.O. Babich, S.A. Suchyh, L.S. Soldatova, A.Y. Prosekov

L-phenylalanine-ammonia-lyase (PAL, КФ 4.3.1.5) catalyzes reaction of reversible desamination of amino acid L-phenylalanine to trans-cinnamic acid and ammonia. Enzyme is of interest as therapeutic means for treatment phenylketonuria. Production of various recombinant proteins is always complicated by their limited physical and/or chemical stability. To increase stability of recombinant proteins it is possible, having transformed their firm form by liophilization. The purpose of this work is optimization of parameters of liophilization of recombinant L-phenylalanine-ammonia-lyase. As PAL it is stablest in alkaline area рН, experiments of frost and liophilization spent at рН 8,5. Unitary frost - defrosting has shown that losses of activity of enzyme in these experimental conditions make 12 %. In the subsequent experiments in the course of a dialysis of loss of activity of enzyme didn't occur. The dialysis preparation had the specific activity equal of 2,99 E/mg. Concentration of protein made 8,51 mg/ml. It is established that the best stabilizer of activity PAL at liophilization are 0,5 % solution D-tregalozy. It is known that concentration of protein influences on cryoscopic temperature of enzymes. In this connection investigated dependence of cryoscopic temperature of enzyme from concentration of protein in preparation. Results have shown that at change of concentration of protein in preparation from 3,5 to 7,5 mg/ml cryoscopic temperature changes on 0,3-0,6°С; at change of concentration of protein from 7,5 to 11,5 mg/ml cryoscopic temperature changes on 0,6-1,3°С. Quantity of frozen water  is quantity of ice at the given temperature of the freezing, carried to a total quantity of water and the ice, containing in object at the same temperature. It is shown that at temperature the minus 50-60°С in preparations is frozen to 70-80 % of moisture. It is obvious that economic efficiency of drying of enzymatic preparations in such conditions is the lowest. According to the existing theory process of sublimatic drying of biological objects is divided into three periods: system pumping out, sublimation, warming up. At temperatures of stabilization of the dried up layer 20-40°С the mass fraction of a moisture doesn't exceed 5,0 %. At temperatures less 20°С the dried up enzymatic preparations have the raised value of a mass fraction of a moisture (from 6,0 to 10,5 %). At research of influence of level of stabilization of temperature of the dried up layer on duration liophilization and properties of enzymatic preparations the microstructure of a dry preparation has been studied. It is established that the dried up enzymatic preparation PAL developed in the sublimatic way, contains about identical particles on the size with a firm and smooth surface.