Extracellular enzymes crude

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. 

Hofrichter, M., and Fritsche, W., Depolymerization of Low-Rank Coal by Extracellular Fungal Enzyme Systems. 3. In Vitro Depolymerization of Coal Humic Acids by a Crude Preparation of Manganese Peroxidase From the White-Rot Fungus Nematoloma Frowardii B19. Applied Microbiology and Biotechnology, 1997. 47(5) pp. 566-571. 

Figure 6. HPLC kinetics of polygalacturonic acid depolymerization by extracellular pectate lyases from crude supernatants of Erwinia chrysanthemi and Lachnospira multiparus cultures. A panels are full scale representations of products found over the reaction time sequence. B panels have expanded ordinates to better demonstrate the kinetics of minor products. Area unit refers to integration from HPLC tracings of product absorbance at 235 nm. Numbers in the panels refer to the degree of polymerization for individual products. Conditions for enzyme assay and product detection were the same as described for Figure 5.

Milled wood lignin was mixed with the crude enzyme solution of Tram-ties versicolor extracellular phenoloxidases produced on spent sulfite liquor in a ratio of approximately 2 1. This comprised the main part of the two-component bio-adhesive. Industrial particles were bonded with 15% bioadhesive under conventional pressing conditions to have 19 mm particle boards (40 x 50 cm) of the properties described in Table IV. The bonding reaction (crosslinking) took place in aqueous solution at room temperature. If conventional pressing technology is applied, the temperature should be elevated in order to maintain water evaporation within a reasonable press time. 

Pullulanase is an extracellular enzyme of Aerobacter aerogenes that causes essentially quantitative hydrolysis of pullulan to maltotriose. The enzyme is readily prepared in a crude form that is free from other carbohydrases, and is important in structural studies because it debranches amylopectin and glycogen. When the A. aerogenes is grown in continuous culture, the enzyme is bound to the cells, but it can be released by detergents, and purified by ion-exchange chromatography. This purified enzyme has been crystallized. ... 

The crude extract or clarified broth containing the enzyme is then subjected to purification, conceived as a sequence of operations (see Fig. 2.1) aimed to remove all contaminants that can interfere with its intended use. Purification can also serve to the purpose of increasing the specific activity of the biocatalyst in the case of enzyme immobilization. The situation will be radically different for an intracellular than for an extracellular enzyme. In the first case, the enzyme extract is a complex mixture of proteins, nucleic acids and other cell constituents, while in the latter the enzyme concentrate contains only some extracellular proteins and small molecular weight solutes, since the cell membrane acts as a powerful barrier to retain most of the cell constituents aiding powerfully to purification. Many extracellular hydrolases... 

Mixtures or unpurified extracellular enzymes from various sources are used for degradation studies. Using cotton fibers and other substrates, Wilson and Wood [39] detected three different types of enzyme present in culture filtrates of Neocallimastix frontalis RK21. Haga et al. [51] used commercially available crude cellulases from the fungus Trichoderma viride to test the extents of biodegradation of fibers that had... 

Extracellular e/u/n-j3-l,6-D>glucanase was produced in high concentration when Bacillus circulans was grown on yeast cell walls (see also p. 368). This enzyme was separated from the e/idb-jS-l,3-D-glucanase by gel filtration, and was then purified by ion-exchange chromatography. The enzyme exhibited maximal activity at pH 5.5 but, in contrast to the crude culture fluid, had only a limited action on the cell walls of yeast or Schizosaccharomyces pombe. 

Resistance to Proteolysis. As the peptides were expected to be either secreted from plant cells in transgenic plants or applied to the external surfaces of plant tissues, the extracellular proteases present in the intercellular spaces of plant tissues were considered to be most important. Accordingly, peptides were tested against crude preparations of enzymes from the extracellular fluid (ECF) of plant leaves. Extracellular fluids from leaves of maize, tobacco and potato, and spent medium from cultured plant cells produced similar results tobacco ECF was used routinely. 

In an alternate process for the preparation of C-13 taxol side chain, the stereoselective enzymatic hydrolysis of racemic ci5-3-(acetyloxy)-4-phenyl-2-azetidinone 42 to the corresponding (S)-(—)-alcohol 43 has been demonstrated [53,54]. Lipase PS-30 from Pseudomonas cepacia (Amano International Enzyme Co.) and BMS lipase (extracellular lipase derived from the fermentation of Pseudomonas sp. SC 13856) catalyzed hydrolysis of the undesired enantiomer of racemic 42, producing S-(—)-alcohol 43 and the desired i -(+)-acetate 44 (Fig. 12). Reaction yields of more than 48% (theoretical maximum yield is 50%) and e.e. of more than 99.5% were obtained for the desired R-(+)-acetate. For a very efficient enzyme source (BMS lipase), a lipase fermentation using Pseudomonas sp. SG 13865 was developed. In a fed-batch process using soybean oil, the fermentation resulted in 1500 U/ml of extracellular lipase activity. Crude BMS lipase (1.7 kg containing 140,000 U/g) was recovered from the filtrate by ethanol precipitation. BMS lipase and lipase PS-30 were immobilized on Accurel polypropylene (PP). These immobilized lipases were reused (10 cycles) without loss of enzyme activity, productivity, or the e.e. of the product in the resolution process. The enzymatic process for the resolution of racemic acetate 42 was scaled up to 150 L at 10 g/L substrate concentration using inunobilized BMS lipase and lipase PS-30, respectively. From each reaction batch, 3-(R)-acetate 44 was isolated in 45 M% yield (theoretical maximum yield is 50%) and 99.5% e.e. 3-(R)-acetate 44 was chemically converted to 3-(R)-alcohol 45. The C-13 taxol side chain (41a or 45) produced... 

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