Plants enzyme extraction from

Assays of soil enzyme activities are usually carried out in soil slurries, since efficiencies of enzyme extraction from soil and purification are still low (49). Such assays, under these conditions, will only give a measure of potential rather than actual activities moreover, they constitute integrated measures of activity as enzymes come from a variety of sources and are in several states in the soil (50). Enzyme activities may vary substantially with the season according to the synthesis, release into soil, and persistence of plant, animal, and microbial enzymes (57). 

Fig. 25.1 The three pathways for the preparation of natural flavours. The first two involve the extraction of the flavour or precursors from natural sources. The precursors can then be converted to the natural flavour by enzymes extracted from plants or microorganisms. The last method is the de novo synthesis of the flavour by microorganisms growing on simple substrates such as glucose and sucrose...

Biotransformation is the conversion of a compound into the product using living plant cells or enzymes extracted from plants [42,43]. This is the third option for using plant cell cultures for flavour production. Some examples are given in Table 25.3, but at present the yields are still low. 

The use of plant cells or enzymes extracted from the cells for the biotransformation of exogenous substances offers another method for producing flavours. This would be useful when compounds are not found in cell suspensions. Many studies have been carried out on the biotransformation of xenobiotics or pharmaceuticals by plant cell culture [43, 50]. 

Many enzymes extracted from higher plants have been tried for clotting cheese milk (Burnett 1976), however, attempts to use them have been unsuccessful. Most plant proteases are strongly proteolytic and cause extensive digestion of the curd, which has resulted in reduced yields, bitter flavors, and pasty-bodied cheese. 

Ziegelhoffer et al. (15) tested the stability of the apoplast-targeted Elcd in transgenic tobacco plants. In their study, the apoplast-targeted Elcd enzyme extracted from tobacco plants, along with the purified microbial Elcd, was subjected to different temperatures (60-90°C) for 10 min. Both enzymes showed similar high thermal stability throughout the experiment. Their results showed that at 60°C up to 95%, at 70°C up to 90%, at 80°C up to 80%, and at 90°C up to 40% of the enzymes activity was retained. However, as seen in Fig. 4, our heat stability test showed sur-... 

Figure 6.2 An example of the phylogenetic tree derived for peroxidase enzymes extracted from various plants. The digits refer to the particular plant species for which the DNA sequencing was carried out [1].

Activity for the enzymatic cleavage of disubstituted hydantoins useful in the synthesis of a-,a-disubstituted amino acids was recently detected in crude enzyme extracts from the plant Lens esculenta161 621 and in papain by Rai and Taneja[63i. 

One more enzyme belongs to this system, converting a free base directly into a ribosides -monophosphate (adenine phosphoribosyltransferase, EC 2.4.2.7). The enzyme partially purified from wheat germ [123] converted iPA into iPARMP moreover, the crude enzymes extracted from Arabidopsis thaliana and Lycopersicon esculentum plants were able to convert also BA into BARMP [124,125, respectively]. 

The cycle has been completely reconstructed in vitro by Racker (1955), who has thus achieved the reductive synthesis of a sugar from CO2 and plant enzyme extracts. The sequence of reactions is as follows ... 

Until about 1950, the predominant method of producing industrial enzymes was by extraction from animal or plant sources by 1993, this accounts for less than 10%. With the exception of trypsin, chymosin, papain [9001 -73-2J, and a few others, industrial enzymes are now produced by microorganisms grown in aqueous suspension in large vessels, ie, by fermentation (qv). A smaH (5%) fraction is obtained by surface culture, ie, soHd-state fermentation, of microorganisms (13). 

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). 

Pectins were extracted from isolated cell walls of 5-week-old wheat plants using different methods. Enzymic digestions of the cell walls involved pectinases such as a commercial pectolayse or recombinant endopolygalacturonase [Maness Mort, 1989]. Chemical extractions involved the chelating agent imidazole [Mort et al., 1991] or solvolysis with anhydrous HF at 0 °C in a closed teflon line [Mort et al., 1989] followed by imidazole extraction. 

This novel enzyme was the only esterase able to release acetyl from sugar beet pectin and removed about 30% of the total acetyl groups present. It also caused the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, in small amounts. PAE had an apparent molecular weight of 60 kDa and showed optimal activity at pH 5.5 and a temperature of 50 C. The enzyme is sensitive to buffer composition and requires a bivalent cation for optimal activity and stability. In purified form this enzyme proved unstable, especially in phosphate buffers. 

More than 3000 different enzymes have been extracted from animals, plants and microorganisms. Traditionally, they have been used in impure form since purification is expensive and pure enzymes may be difficult to store and use. There is usually an optimum temperature and pH for maximum activity of an enzyme. Outside these optimum conditions, activity may simply be held in check or the enzyme may become denatured , i.e. altered in such a way that activity is lost permanently, although some forms of denaturing are reversible. Many enzymes are also sensitive to transition-metal ions, the effect being specific to particular metal ions and enzymes. In some cases, certain metal ions are essential for the stability and/or activity of an enzyme. In other cases, metal ions may inhibit the activity of an enzyme. Similarly, certain organic compounds can act as enzyme inhibitors or activators. 

The cholinesterase to determine the toxic activity may be chosen (i) in pure form of commercial enzyme from animals in a water buffer solution or using biosensors, enzyme preparation impregnated into a rigid matrix that significantly activates the enzymic activity and (ii) in the form of crude extracts from plant or animal tissues. 

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