Purified enzymes

Finally, a number of studies146"149 have demonstrated that a variety of enzymes purified from tissues of older animals have a decreased specific activity when compared to the enzyme purified from younger animals. These proteins, however, appear to be identical as measured by a variety of physical and chemical parameters. It is possible that one of the differences is due to the accumulation of Met(O) residues in the proteins of the older animals. 

So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca +/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca + and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca + in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca +/calmodulin protein kinase inhibitor. However, whereas depolarisation... 

This enzyme was shown to be specific for xylan oligomers and small acetylated synthetic substrates. Many characteristics have been published recently about this type of enzyme, purified from Trichoderma reesei, and A. oryzae [6], and a different A. n/ger preparation[7]... 

Lendenmann U, JC Spain (1996) 2-aminophenol 1,6-dioxygenase a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS 45. J Bacteriol 178 6227-6232. 

Denger K, THM Smits, AM Cook (2006) L-Cysteate sulpho-lyase, a widespread pyridoxal 5 -phosphate-coupled desulfonative enzyme purified from Silicibacter pomeroyi DSS-3. Biochem J 394 657-664. 

Dsz enzymes from R. erythropolis strains IGTS8, KA2-5-1, and D-l as well as the desulfurization enzymes from Paenibacillus All-2 and B. subtilis WU-S2B have been purified and characterized. These enzymes have been reported to be associated with the soluble fraction of the cell extract, indicating that these are soluble enzymes. The characteristics of the enzymes purified from various organisms and their comparison is given below (Table 6). 

The first two patents [150,151] protect the biocatalytic system and its use in the form of whole cells or cell-free extracts or enzymes purified from the organisms. The other claimed organisms in the patent include the thermophilic culture Aneurinibacillus sp. IGTN4T (ATCC N° PTA-4581), P. stutzeri, Yokenella sp. and P. nitroreducens. The following text gives the details of the strain PTA-806, since the others have not been reported in much detail. 

Histochemical studies of bone marrow samples show that peroxidase-containing granules are detectable in promyelocytes. The human promyelo-cytic leukaemia cell line HL-60 grows easily in culture, and the cells resemble promyelocytes both structurally and functionally. Furthermore, they can be induced to differentiate in vitro upon addition of various agents, such as retinoic acid and phorbol esters, and these differentiated cells resemble more mature forms of neutrophils. HL-60 cells possess almost the same amount of myeloperoxidase (4.4 fig per 106 cells) as mature neutrophils, and the enzyme purified from these cells has the same subunit structure. The cells thus actively synthesise the enzyme only until they are induced to differentiate. This cell line has been extensively used to study the molecular events controlling the expression of enzymes such as myeloperoxidase, and also to investigate the molecular controls that lead to a cessation of their expression. 

Y.-H. Park, S. S. Lee, Identification and Characterization of Capsaicin-Hydrolyzing Enzymes Purified from Rat Liver Microsomes , Biochem. Mol. Int. 1994, 34, 351-360. 

The only major accessory enzyme purified thus far for the xylanase was an a-(l,3)-L-arabinosidase with a molecular weight of 66,(XX) and isoelectric point of 4.1 (publication in preparation). The enzyme was not capable of acting on intact xylans. However, it acted on the end-products of arabinoxylan (but not glucuronoxylan) degradation by the xylanase, doubling the total extent of degradation. 

P-Mannanases are generally prepared by conventional chromatographic procedures, which can lead to high yields of enzyme, but in some cases only a poor state of purity. Small quantities of highly purified p-mannanases have been prepared by substrate affinity chromatography of partially purified enzymes on a column of glucomannan immobilised on aminohexane Sepharose 4B (6). The action patterns of enzymes described in this paper were determined using enzymes purified by this latter procedure. 

In a different, but related, approach a lysine cyclodeaminase gene was identified in the rapamycin gene cluster, and subsequently cloned, overexpressed and the enzyme purified (Scheme 2.17). The enzyme catalyzes the conversion of L-lysine... 

Beta-Glucocerebrosidase, recombinant, glycoprotein enzyme purified from human placenta tissues... 

Iduronidase solution lyophilized lysosomal enzymes purified from bovine testis (Moscerdam Substrates) are reconstituted in 2.2 ml demineralized water and aliquots are stored at -80°C. 

Lipoxygenase (EC 1.13.11.12) is an enzyme that catalyzes the hydroperoxidation of polyunsaturated fatty acids and esters containing a cis-cis-l, 4-pentadiene system (Table 6). In 1947, Theorell et al. obtained the enzyme in a crystalline form from soybeans and reported that the enzyme neither contained nor required a metal cofactor192. Subsequent studies from three groups of investigators have demonstrated that the enzyme purified from soybeans in an iron-containing dioxygenase74-76 ... 

The enzyme purified to homogeneity from Hansenula mrakii (1FO 0895) has a molecular weight of approximately 62,000 and consists of two subunits non-identical in molecular weight (39,000 and 25,000). The enzyme exhibits absorption... 

This is what was found by Englehardt et al. (1973) for this compound using a hydrolyzing enzyme purified from a Bacillus sp. ... 

FIGURE 15-33 Dependence of glycolytic flux in a rat liver homogenate on added enzymes. Purified enzymes in the amounts shown on... 

More recently the biotransformation of limonene by another Pseudomonad strain, P. gladioli was reported [76,77]. P. gladioli was isolated by an enrichment culture technique from pine bark and sap using a mineral salts broth with limonene as the sole source of carbon. Fermentations were performed during 4-10 days in shake flasks at 25°C using a pH 6.5 mineral salts medium and 1.0% (+)-limonene. Major conversion products were identified as (+)-a-terpineol and (+)-perillic acid. This was the first time that the microbial conversion of limonene to (+)-a-terpineol was reported, see pathway 4. The conversion of limonene to a-terpineol was achieved with an enzyme, a-terpineol dehydratase (a TD), by the same group [78]. The enzyme, purified more than tenfold after cell-disruption of Pseudomonas gladioli, stereospecifically converted (4 )-(+)-limonene to (4/ )-(+)-a-terpineol or (4S)-(+)-limonene to (4S)-(+)-a-terpineol. a-Terpineol is widely distributed in nature and is one of the most commonly used perfume chemicals [27]. 

An enzyme purified from rye grass capable of specifically hydrolyzing 3 -nucleotides has been available for some years (84)- The enzyme has also been purified (50-fold) from germinating rye seedlings (85) and seems quite specific for 3 -nucleotides. 3 -Deoxymononucleotides are not attacked (86) nor is arabinonucleoside 3, 5 -diphosphate (87). Enzymic activity increases 10-fold during germination (85). 

Figure 12. Polyacrylamide disc gel electrophoretic patterns of enzymes purified from the extracellular protein produced by T. reesei QM 9414 in response to ImM sophorose. To the gels, from left to right, were applied 175 fig extracellular protein mixture, 45 fig CBH II, 45 fig endoglucanase and 80 fig CBH I (D).

Brevibacterium albidum cuts the sequence TGGCCA (Figure 7.4). There are over 400 different restriction enzymes purified from about 250 different microorganisms. The restriction enzymes break the double-stranded DNA molecules in two different ways as shown in Figure 7.4 ... 

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