Cultures enzyme extraction

Enzyme extraction and purification steps The crude enzyme was extracted from the solid state culture with 100 ml of 0.33% toluene at 4°C. The enzyme was concentrated with 60-90% (NH4)2S04, then the dialysed enzyme... 

The first evidence that supplementation of exogenous ADA may be helpful in SCID patients came from the 1975 report of Potmar et al. [8], demonstrating that addition of bovine-intestinal ADA or human-erythrocyte ADA to cultures of lymphocytes of a SCID patient restored their ability to proliferate when stimulated with mitogens. The ability to respond to mitogens is an indicator of immune function restoration. Therapeutic use of enzyme extracted from calf tissue revealed that this form of ADA has a short half-life. 

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]. 

Fig. 7 Chemo-enzymatic formation of tetrahydroalstonine from tryptamine and secologanin by an enzyme extract from engineered E. coli harbouring a dual vector expressing both, STR1 and SG cDNAs from R. serpentina cell suspension cultures followed by chemical reduction. Due to simultaneous expression of STR1 and SG cDNAs, the intermediate strictosidine does not accumulate...

Saccharomyces cerevisiae Live cultures, yeast extracts, enzymes... 

Gao, J., Garrison, A.W., Hoehamer, C., Mazur, C., and Wolfe, N.L., Phytotransformations of organophosphate pesticides using axenic plant tissue cultures and tissue enzyme extract. In situ and on-site bioremediation. The Fifth International Symposium, San Diego, 19-22 April 1999. 

The source of enzyme was the human malaria parasite Plasmodium falciparum, cultured in red blood cells. Intact parasites were obtained by lysis of the cells with saponin, and the enzyme extract was obtained by freezing and thawing. 

Early experiments concerned the enzymatic hydrogenation of fumaric acid to succinic acid, catalyzed by either yeast 24,55 or enzyme extracts from yeast cells56. Much later, strains of Escherichia coli, Aerobacter aerogenes and Aerobacter cloacae were shown to have the same ability 57,58. An interesting mixed culture fermentation has been developed, where the fungus Rhizopus chinensis produces fumaric acid from glucose and a selected E. coli converts the previously formed fumaric acid into succinic acid58. 

For C. roseus suspension-cultured cells, elicitation with fungal elicitors results in the induction of TDC activity (99,186,202,203,284,329-331). This is due to the induction of expression of the Tdc gene. Similarly, SSS activity is induced (202,203,284,329,330). The induction by the Pythium aphanider-matum or yeast elicitor of the transcription of both genes is not affected by cycloheximide that is, the induction is independent of de novo protein biosynthesis, and thus follows an already available signal-transduction chain. The response is quite fast, for the enhanced transcription can already be measured 15 min after elicitation (202,203). Also, the NADPH cytochrome P-450 reductase mRNA level is induced by elicitation with fungal elicitors (113). Moreno et al (99,151) measured activities of a number of enzymes involved in secondary metabolism in C. roseus before and after elicitation with a P. aphanidermatum preparation. GlOH activity was found to be slightly decreased by elicitation and IPP-isomerase showed similar behavior. The pattern of terpenoids formed by the crude enzyme extracts from elicited and nonelicited cells was different. The total incorporation decreased, that is, the activities of the enzymes of the terpenoid pathway were lower. The relative incorporation decreased particularly for squalene. 

Fig. 3 shows activity of poly(ADP-ribose) glycohydrolyase as a function of temperature. In each case, the ceU cultures or extracts were held 30 min at each temperature. Values are shown for heated extracts and for ceUs subjected to hyperthermia in culture and either harvested immediately for assay or aUowed to recover for 24 hr prior to assay. Over the entire temperature range studied activity was more temperature resistant in heated extracts. The activities in heated extracts were very similar to those obtained from extracts of cultures made following 24 hr of recovery from hyperthermia. The enzyme activity decreased with increasing temperature in all cases in a biphasic pattern with a breakpoint around 42°C. At temperatures above 42°C the decrease in enzyme activity with increasing temperature suggested thermal inactivation of the enzyme. Utilizing the... 

Different approaches have been carried out to investigate the biosynthesis of EAs. Early feeding experiments with isotope-labeled precursors, e.g., [ C]-L- tryptophan 1, to rye ears infected with C. purpurea or to fermentation cultures of different Claviceps species proved its incorporation into 2 [100-102]. Further biochemical experiments with crude and purified enzyme extracts fi om producers revealed the determinant step in the biosynthesis of EAs by the key enzyme 4-dimethylallyltransferase (4-DMATS) [103, 104], i.e., the prenylation of 1 at C-4 position [17, 104, 105]. 

Nutmeg E. coli was transformed with vector only (Ctrl) or with the Mf FatBl expression plasmid. Liquid cultures were grown to equal density and crude extracts prepared. Enzyme extracts representing equal proportions of cultures were assayed with the different acyl-ACP substrates. Elm E. coli was transformed with His-tag Ua FatBl expression plasmid. The affinity-purified Ua FatBl enzyme was diluted to a concentration appropriate for the assay conditions and measured. At this dilution the vector-only control did not show any activity above background... 

Enzyme extracts collected from microbial cultures and purified have been considered for in-situ detoxification. One cell-free enzyme that has been used for detoxification of organophosphate insecticides is parathion hydrolase. The hostile environment of a chemical-waste landfill, including the presence of enzyme-inhibiting heavy-metal ions, is detrimental to many biochemical approaches to in-situ treatment. Furthermore, most sites contain a mixture of hazardous constituents, which might require several different enzymes for their detoxification. 

In tobacco cell cultures the extractable levels of ATP-sulfurylase and cysteine synthase are very low when the cells are subject to nitrogen stress but increase rapidly upon alleviation of the stress, suggesting that a product of nitrogen assimilation derepresses the levels of these enzymes. In Lemna (and possibly in cultured Rosa cells) it appears that this role is fulfilled by APS sulfotransferase and that ATP-sulfurylase and cysteine play unimportant roles in coordinating the sulfate assimilation pathway with the nitrate assimilation pathway. A further regulatory mechanism known to occur in cultured tobacco cells is that excessively high concentrations of cysteine induce the synthesis of cysteine desulfiiydrase (see Section VI). 

A breakthrough with regard to the availability of the enzyme was achieved by its successful overexpression in several microorganisms [116]. The most efficacious expression was accomplished in the yeast Pichea pastoris. Yields of more than 20 g of pure HNL protein per liter of culture volume could be obtained. It was subsequently shown that the crude enzyme extract (140 lU/ml) can be used for efficient conversion of aU substrates. Under standard conditions 100 lU of enz3mie is used per mmol of aldehyde. Substrates that gave unsatisfactory e.e. s under these conditions were converted with a 20-fold quantity of enzyme at pH 4.5. All reactions were carried out at 0°C. 

Biopolymer Extraction. Research interests involving new techniques for separation of biochemicals from fermentation broth and cell culture media have increased as biotechnology has grown. Most separation methods are limited to small-scale appHcations but recendy solvent extraction has been studied as a potential technique for continuous and large-scale production and the use of two-phase aqueous systems has received increasing attention (259). A range of enzymes have favorable partition properties in a system based on a PGE—dextran—salt solution (97) ... 

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). 

---