Enzyme preparations, characteristics

It should be noted that a number of different enzyme preparations can now be purchased directly from manufacturing chemists. It must be emphasised that the activity of an enzyme, whether purchased or prepared in the laboratory, may vary between rather wide limits. The activity is dependent on the source of the enzyme, the presence of poisons and also on the temperature. It appears, for example, that the quality of horseradish peroxidase depends upon the season of the year at which the root is obtained from the ground. It cannot be expected therefore that all the experiments described below will work always with the precision characteristic of an organic reaction proceeding under accurately known conditions. 

Some properties of Penicillium fellutanum pectinesterase were studied. The optimum of pectinesterase action was detected at pH 5 and 45 °C. The enzyme was stable at pH 4 — 5 and 40 °C (pH 5) for 240 min. and was specific towards lemon pectin. An enzyme preparation composed mainly of pectinesterase was partially purified by gel filtration. Pectinesterase activity was accumulated in one of the obtained fractions. Molecular weights of fraction determined were found to be 46,000 and 1,200. Disk electrophoresis in polyacrilamide gel of the purified preparation revealed two protein bonds with one active component. The partially purified enzyme had the kinetic characteristics =... 

HRP is a hemoprotein containing photohemin IX as its prosthetic group. The presence of the heme structure gives the enzyme its characteristic color and maximal absorptivity at 403 nm.The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However, at least seven isoenzymes exist for HRP (Shannon et al., 1966 Kay et al., 1967 Strickland et al., 1968), and their RZ values vary from 2.50 to 4.19. Thus, unless the RZ ratio is precisely known or determined for the particular isoenzyme of HRP utilized in the preparation of an antibody-enzyme conjugate, subsequent measurement after crosslinking would yield questionable results in the determination of the amount of HRP present in the conjugate. 

The characteristics of a support material are of great importance to the measured enzyme activity [79, 101]. Hydrophobic carriers have a low ability to attract water, thus leaving more available for the enzyme, hence Wehtje et al. [102, 103] have shown that celite is a suitable carrier for the PaHnl to yield an immobilized form of the enzyme. In contrast, controlled pore glass (CPG) and Sephadex G25 were found to be less well suited to enzyme support as, using these systems, cyanohydrin synthesis was significantly reduced (over 30%). Sephadex also promoted the spontaneous addition of HCN to benzaldehyde [102]. A series of batch experiments showed that if the solvent (diisopropyl ether) surrounding the immobilised PaHnl contained insufficient water (i. e. less than 2 %), it would be extracted from the enzyme preparation and consequently enzyme activity was lost [102]. These results were the basis for the production... 

Le Traon-Masson, M.-P. and Pellerin, P., Purification and characterization of two P-D-glucosidases from an Aspergillus niger enzyme preparation affinity and specificity toward glucosylated compounds characteristic of the processing of fruits. Enzyme Microb. Technol 22, 374, 1998. 

Zvyagintsev, V. I., Krasheninin, P. F., Sergeeva, E. G., Buzov, I. P., Mosichev, M. S. and Rubtosova, N. A. 1972. Characteristics of cheeses made with Mucor renninus 367 enzyme preparation. Prikl Biokhim. Mikrobiol. 8, 913-917. 

A. Whitehead. Study to compare the enzyme activity, add resistance and dissolution characteristics of currently available pancreatic enzyme preparations. Pham. Weekt. 

Lipolysis plays an important role in providing the characteristic flavor of many milk products. In particular, the ripening of most cheese varieties is accompanied by lipolysis due to microorganisms or to added enzyme preparations, and, in raw milk cheese, to the milk LPL. Lipolysis is not extensive, but is more pronounced in some cheeses (e.g., blue-veined and hard Italian varieties), than in others. Excessive lipolysis renders the cheese unacceptable (Fox and Law, 1991 Gripon et al., 1991). 

Enzyme Preparation. The purified enzyme from the squid liver was found to be approximately uniform judging from the SDS-PAGE pattern. This final preparation was purified about 470-fold from squid livers. The molecular weight of this enzyme was estimated at 42,000 by SDS-PAGE and gel filtration. The characteristics of this enzyme were studied with this purified material. 

The absorption spectrum of the copper amine oxidases shows a characteristic broad band at around 500 nm (460-510 nm) that confers a typical pink or yellow-pink color to highly purified enzyme preparations [10,12,32]. Absorption in the visible region is caused by the presence of the quinone cofactor and thus is not affected by removal of copper from the enzyme under non-reducing conditions [12,32], The quinone cofactor shows emission of fluorescence when excited at 280 and 365 nm [41,106]. Electron transition of Cu(II) is seen in circular dichroism spectra at 600-800 nm [12,32,107]. Under aerobic conditions, addition of the substrate leads to a temporary bleaching of the pink color of the enzyme, which is restored after complete consumption of the substrate. However, anaerobic conditions lead to the formation of a stable yellow intermediate... 

The cost of enzyme preparations has been decreasing in recent years however, it continues to affect considerably the price of ethanol obtained from cellulosic raw materials. Increased enzymatic hydrolysis efficiency is one way to reduce the enz)me cost in bioethanol production. Another method is enzyme recycle and reuse. Immobilization of biocatalysts allows for their economic reuse and development of continuous bioprocess. Although immobilization poses problems of substrate accessibility and binding for most endo- and exocellulases, P-glucosidase exhibits characteristics amenable to immobilization, such as activity on soluble substrates and the lack of a carbohydrate-binding module. Among the possible approaches, immobilization of (J-glucosidase is one prospective solution to the problem. 

Studies of immobilization on different carriers indicate that immobilization on silica gel or kaolin is the most prospective for application in the future, although the potential economic benefits appear much more favorable for kaolin. Comparison of immobilization characteristics of p-glucosidase immobilized on silica gel and on kaolin has shown that the kaolin method has more advantages, as it is considerably cheaper and more efficient. The feasibility of preliminary acetylation of the enzyme preparation requires further investigation. We have to take into consideration additional expenses necessary for acetylation and the issue of the decrease of the enzyme activity after acetylation. 

---