Enzymes fractionation

Analysis of reaction mixtures for 1-propanol and 2-propanol following incubation of NDPA with various rat liver fractions in the presence of an NADPH-generating system is shown in Table I ( ). Presence of microsomes leads to production of both alcohols, but there was no propanol formed with either the soluble enzyme fraction or with microsomes incubated with SKF-525A (an inhibitor of cytochrome P450-dependent oxidations). The combined yield of propanols from 280 ymoles of NDPA was 6.1 ymoles and 28.5 ymoles for the microsomal pellet and the 9000 g supernatant respectively. The difference in the ratio of 1- to 2-propanol in the two rat liver fractions may be due to differences in the chemical composition of the reaction mixtures (2) Subsequent experiments have shown that these ratios are quite reproducible. For comparison, Table I also shows formation of propanols following base catalyzed decomposition of N-propyl-N-nitrosourea. As expected (10,11), both propanol isomers were formed, the total yield in this case being almost quantitative. 

AE catalyses the cleavage of acetyl groups from different substrates. The enzyme activity was determined by measuring the release of acetic acid. The amount of acetic acid was measured spectrophotometrically using an acetic acid analysis kit (Boehringer, Mannheim). The activity of AE was measured in 0.6% sugar beet pectin solubilised in 25 mM Na-succinate pH 6.2 and incubated with enzyme fraction in total 500 nl assay. The samples were incubated at 40°C and aliquots were examined after 0, 1, 2 and 3 hours of incubation. The enzyme reaction was stopped by incubating the samples at lOO C for 5 min. Precipitated... 

AE was also detected by using the substrate triacetin. The enzyme fraction was incubated with 80 mM triacetin in 25 mM Na-succinate pH 6.2. The samples were incubated at 40°C for 30 min. After boiling for 5 min. the samples were analysed for released acetic acid. During purification triacetin was used as substrate. 

As the reaction went on, the activity in the tissue fell (12 % after 30 min and 3 % after 6 h) while it increased in the pulp (55 %) and in the juice (from 32 % to 40 % between 30 min and 6 h of maceration) thus limiting the enzyme fraction available for the continuation of the tissue maceration. 

Enzyme fractions obtained by linear gradient elution and stepwise elution were pooled and used later in this work. 

When appreciable cation-independent transferase activities were found (F3, HIO), double reciprocal plots of enzyme activity against concentration of added Mg + were nonlinear, but became linear when only the stimulated enzyme fractions were plotted. The apparent values so obtained were independent of the concentration of UDP-glucuronic acid, and vice versa (HIO), suggesting that Km for Mg + represents the dissociation constant of an Mg +-enzyme complex (D5). 

Quartey, E.G.K., Hustad, J.A., Faber, K. and Anthonsen, T. (1996) Selectivity enhancement of PPL-catalysed resolution by enzyme-fractionation and mediumengineering Synthesis of both enantiomers of tetrahydropyran-2-methanol. Enzyme. Microb. Technol., 19, 361-366. 

Stability. Some discussion regarding stability of milk lipases was presented in the preceding section. Egelrud and Olivecrona (1973) found that the enzyme fractions from heparin-Sepharose can be stored frozen at -20°C with less than 10% loss of activity in 2 weeks. The purified enzyme had only moderate stability at 4°C high concentrations of salt or a pH below 6.5 or above 8.5 increases the rate of inactivation. 

At present, only few data from studying of purified enzyme fractions are available. This could either be due to a very low level of enzyme activity, or due to the application of unsuitable substrates that are not metabolized. 

In 1964, Anraku (2, S) reported the isolation of an enzyme from Escherichia coli B which hydrolyzed ribonudeoside 2, 3 -cyclic phosphates. Enzyme fractions representing a 900-fold purification also possessed 3 -nucleotidase activity. Similar activities have subsequently been purified from Proteus mirabilis (4, 5), halophilic Vibrio algino-lyticus (6, 7), Bacillus subtilis (8), and various Enterobacteriaceae, specifically, Shigella sonnei, Salmonella heidelberg, Serratia marcescens, Proteus vulgaris (9), and others (10). The enzyme from each organism is strikingly similar, but some differences are apparent. 

T. reesei yields one major endoglucanase and one major cellobiohydrolase. The multiplicity of these components is minimal when culture conditions are carefully controlled. The widely reported multiplicity of these components might possibly be due to post-translational modification of one endoglucanase and one cellobiohydrolase. The third major component produced by T. reesei, cellobiase, is present intracellularly as well as extracellularly. Whether or not these two enzyme fractions are the same enzyme, remains to be determined. 

Endoglucanase and cellobiohydrolase enzyme fractions, obtained from DEAE-Sepharose column chromatography of crude enzyme from in-house-grown T. reesei culture filtrates, were examined for activity on cellulose. As expected, cellobiohydrolase action resulted in cellobiose formation while cellotriose as well as cellobiose was formed by the action of endoglucanase. In both cases, the formation of glucose was minimal. Examination of the combined activity of these components on celluloses, showed a certain degree of synergism does exist. 

Enzyme Fractionation. Buffer-soluble and -insoluble cellulases from auxin-treated apices of pea epicotyls were extracted in a crude form and purified to homogeneity as previously described (3). For fractionation, a Sephadex G-100 column (1.6 X 100 cm) was prepared and equilibrated with 20mM sodium phosphate, pH 6.2, containing 5% glycerol and 0.03% sodium azide at 2°C. Crude enzyme preparations (1.5 mL) containing... 

In more recent investigations, the assumed multienzyme involved in cyclosporin A biosynthesis could be isolated from T. inflatum. A partially purified enzyme fraction was indeed capable of forming enzyme-substrate complexes by thioester linkage. Although de novo synthesis (in vitro) of cyclosporin A has not yet been achieved, the formation of a partial sequence, namely, the diketopiperazine cyclo(DAla-MeLeu), from D-alanine and L-leucine was observed under consumption of ATP and S-adenosyl-L-methio-nine [25]. 

Studies in this laboratory for the past several years have been concerned with the elucidation of the latter steps in this series of reactions. Specifically, efforts have been directed toward the characterization of the reaction involving the transfer of aminoacyl sRNA to mammalian ribonucleoprotein particles, the enzymatic and cofactor requirements, and possible intermediates in this process. The evidence obtained indicates that aminoacyl transfer is an enzymatic reaction requiring at least two enzyme fractions, which have been resolved and partially purified, GTP and a sulfhydryl compound further, the possibility exists that a ribosome-bound sRNA-amino acid (or peptide) compound is formed as an intermediate in this reaction. 

Experimentally, C14-aminoacyl sRNA was incubated with rat liver microsomes or ribosomes, GTP, various fractions obtained from the nonparticulate portion of rat liver homogenates, and buffered salt-sucrose medium in a total volume of approximately 2 ml. (6-10). The C14-aminoacyl sRNA was prepared by the phenol-extraction procedure from the pH 5 amino acid-activating enzymes, fraction of rat liver after incubation with C14-L-amino acids (9, 13). C14-leucyl sRNA (approximately 1000 c.p.m.), having a specific radioactivity of approximately 55,000 c.p.m. per mg. of RNA, and containing a complement of endogenous, unlabeled, bound amino acids, was used in most of these studies. The microsomes were sedimented from the post-mitochondrial supernatant at 104,000 x g (10) and the ribosomes were prepared from them by extraction with deoxycholate (16). 

The time-dependent incorporation of amino acid into ribosomal protein is shown in Figure 2. When the crude pH 5 Supernatant fraction was used, incorporation was very rapid and essentially complete in 2 to 4 minutes. Incorporation was usually slower when the more purified enzyme fractions, transferases I and II, were used incorporation was complete after approximately 20 minutes. A similar rate of incorporation was observed when the transferase n used was isolated either from the microsomes or the post-microsomal supernatant. 

---