Extracellular enzymes purified

Relationships Between Purified Extracellular Enzymes from Induced or Cellulose-Grown Cells... 

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).

A number of laboratories have been successfiil in obtaining semipurified/purified PLAj preparations fi om a number of sources, including platelets (47-70). These studies have been helpful to understand the molecular size as well as the nature of active sites of these enzymes. Several reports have demonstrated the presence of two substrate specific and distinct etjzymes, including secretory PLA (sPLA,) and cytosolic PL (cPLAj) in platelets. cPL appears to have a molecular mass of 85 kDa that is structurally distinct fi-om that of the mammalian Type II, a 14 kDa non-pancreatic PLAj, which exists in an extracellular form in many tissues, including platelets, inflammatory joint fluid, spleen and placenta (47-70). The human non-pancreatic PLA enzyme purified is also present in platelets and is enriched in rheumatoid synovial fluid. 

In a recent study, a serine-phospholipid-selective phospholipase A has been purified and cloned its cDNA from rat platelets, which secrete two types of phospholipases upon stimulation (71). The purified enzyme from extracellular medium of activated rat platelets, yielded a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. The presence of active serine residues was confirmed by labeling the 55-kDa protein with pHJDiisopropyl fluorophosphate, an inhibitor of the enzyme. Based on cDNA for the enzyme cloned from a rat megakaryocyte cDNA library, the 456-amino acid... 

The maximum rates of hydrolysis of synthetic a-D-glucopyranosides and isomaltose by a purified extracellular a-D-glucosidase (pH optimum 5—6, temperature optimum 75°C) from the thermophile B. thermoglucosidus have been compared. Tris, 4-nitrophenyl a-D-xylopyranoside, D-glucose, and D-glucono-1,5-lactone inhibited the hydrolysis of 4-nitrophenyl a-D-glucopyranoside by the enzyme, whose activity was also inhibited by heavy-metal ions but not by H edta, 4-chloromercuribenzoate, and iodoacetate. 

Factors such as polymer crystallinity also influence the degradation rate. Studies involving the degradation of films using purified extracellular PHB depolymerase enzymes demonstrated that low crystallinity solution-cast films degraded substantially faster than higher crystallinity melt-cast materials [55]. 

A purified extracellular cyclodextrin D-glucanotransferase (pH optimum 4.6, pi 5A, mol. wt. 8.8 x 10 ) from an alkaliphilic Bacillus species has been shown to be a single homogeneous protein by polyacrylamide gel electrophoresis and ultracentrifugation. The Km values for cyclohexa-, cyclohepta-, and cyclo-octa-amyloses at a constant concentration of sucrose are 5.88, 0.39, and 0.25 mmol 1, respectively. The enzyme converted starch, amylopectin, glycogen, and amylopectin j8-limit dextrin into cyclodextrins. 

A purified extracellular enzyme from D. pneumoniae specifically hydrolysed 2-acetamido-2-deoxy-3-0-j8-D-galactopyranosyl-o -D-glucopyranosyl units attached to either threonine or serine residues. ... 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

Although the exact mechanism of degradation at metabolic level for each compound or group of compounds is not well known, the involvement of extracellular oxidative enzymes such as LAC, MnP, LiP, and versatile peroxidase (VP) (see Tables 1 and 2 of Chap. 6) and intracellular monooxygenases as cytochrome P-450 is well documented for pollutants such as hydrocarbons, dyes, and halogenated solvents [25]. To determine the actual role of the extracellular enzymes, many studies are performed in vitro experiments with purified enzymes. In the case of cytochrome P-450, usually inhibitors are used. 

Beginning with Eduard Buchner s discovery (c. 1900) that an extract of broken yeast cells could convert glucose to ethanol and C02, a major thrust of biochemical research was to deduce the steps by which this transformation occurred and to purify and characterize the enzymes that catalyzed each step. By the middle of the twentieth century, all ten enzymes of the glycolytic pathway had been purified and characterized. In the next 50 years much was learned about the regulation of these enzymes by intracellular and extracellular signals, through the kinds of allosteric and covalent mechanisms we have described in this chapter. The conventional wisdom was that 1860 1917 in a linear pathway such as... 

Because a-L-arabinofuranosidase is an extracellular enzyme, a crude preparation may be made simply by fractionation of the culture filtrate with ammonium sulfate. The enzyme can be purified from the crude enzyme-preparation by some suitable combination of ion-exchange chromatography, gel filtration, and similar techniques. Three examples of purification procedures, two from micro-organisms and one from a plant, are given here. 

Modler, H., Brunner, J. R. and Stine, C. M. 1974. Extracellular protease of Pencillium roqueforti. II. Characterization of a purified enzyme preparation. J. Dairy Sci. 57, 528-534. 

---