Enzymes extraction

Biotechnology Cell disruption Yeast disruption for enzyme extraction... 

Electrolyte leakage due to the action of endogenous enzymes. Extracts from tub ... 

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... 

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). 

Fan YYD (2003) Preliminary study of an enzyme extracted from AlcaUgenes sp. Strain YF11 capable of degrading pesticides. Bull Environ Contam Toxicol 70 367-371... 

Aspergillus niger was the biocatalyst of choice for the biohydrolysis of para-nitrostyrene oxide (see above). A selective kinetic resolution using a crude enzyme extract of this biocatalyst followed by careful acidification of the cooled crude reaction mixture afforded the corresponding (i )-diol in high chemical yield (94%) and good ee (80%). This key intermediate could then be transformed via a four-step sequence (Scheme 11) into enantiopure (i )-nifenalol, a molecule with -blocker activity, which was obtained in 58% overall yield [88]. 

SpribiUe R, Forkmann G (1984) Conversion of dihydroflavonols to flavonols with enzyme extracts from flower buds of Matthiola incana. Z Naturforsch 39C 714-719... 

Stotz G, Forkmann G (1982) Hydroxylation of the B-ring of flavonoids in the 3 - and 5 -position with enzyme extracts from the flowers of Verbena hybrida. Z Natuforsch 37C 19-23... 

The mean lignocellulose particle size in the growth medium effects the efficiency and timing for extraction. The high relative surface area of small particles provides for increased enzyme extraction and earlier extraction (increased nutrient accessibility results in enhanced growth). However, if the particles are too small they can limit aeration and suppress fungal growth. 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Fig. 7. Chemical structure of azumamide A-E and activity against the crude enzyme extract from K562 cells. ...

Only 5% ee results when using the enzyme extract. 

Five- to six-month-old tobacco plants (Nicotiana tabacum var. Samsun) grown in a glasshouse at 20°C were used for this study. Commercial synthetic substrates employed both for histochemical and biochemical assays were guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), 3-3 di-aminobenzidine (DAB), tetramethylbenzidine (TMB) and syringaldazine. Isopropylamine and monosodium salts of ferulic acid were also used as substrates as well as their / -fluorinated analogues substituted with a fluorine atom on the / -carbon (Fig. 1). Histochemical observations were done on hand-made transverse sections of fresh tobacco stems. Biochemical assays were performed separately on bark (inner cortical parenchyma, phloem and fibres) and xylem fractions. Technical data of incubation, enzyme extraction, spectrophotometric and electrophoretic assays were given elsewhere (5-7). Synthesis of fluorinated compounds was performed as previously described (4). 

Controls with the anti-glycohydrolases IgG, first incubated with the enzymic extract before being applied in thin sections, showed no labeling (Fig. 2B). Other controls performed, i.e., replacement of the primary antibody by normal rabbit serum or preimmune serum, suppression of the first step corresponding to the primary antibody, or labeling of uninfected wood specimen, were also negative. 

Thanks are expressed to Dr. K.-E. Eriksson (STFI, Stockholm, Sweden) for the gift of the enzyme extract and inoculation of the wood samples to Professor R. Guinet (Institut Pasteur, Lyon Lentilly, France) for the preparation of the IgG directed against the crude protein extracted and to Dr. E. Odier (INA P.G., France) for providing the anti-ligninase antiserum. 

In vitro studies were conducted with enzymes extracted from peanut (7), pea (. ), and onion (9). The enzymes were fractionated by ammonium sulfate precipitation, dialyzed, and stored frozen until used. The enzymes were assayed for various activities as described In the Results and Discussion. 

The following spectrophotometric methods are conceptually related to the POV however, they are intended for other purposes. HPLC-UVD at 234 nm was applied to detect lipid hydroperoxides, obtained in a set of experiments for assessing the effectiveness of lipoxygenase enzymes extracted from various microorganisms. The absorbance. A, measured at three different wavelengths is linearly correlated to the concentrations in xM units of lipid hydroperoxides, Clh, 7-oxocholesterol, Cqc and dienals, Cde according to the set of simultaneous equations 60. This method was used to track the Cu(II)-induced oxidation of LDL" °. 

Biocatalysis. Biocatalysis, also termed biotransformation and bioconversion, makes use of natural or modified isolated enzymes, enzyme extracts, or whole-cell systems for the production of small molecules. A starting material is converted by the biocatalyst in the desired product. Enzymes are differentiated from chemical catalysts particularly with regard to stereoselectivity. 

Much of the information on ANS has come not from studies on enzyme extracts but from analysis of DNA sequences and recombinant proteins. Sequences for the ANS were first isolated using transposon generated mutant lines of A. majus and Z. mays They encoded proteins of 40 to 41 kDa that were found to have similarity to 20GDs, during a study on a nonflavonoid enzyme. This sequence-based identification was confirmed by the in vitro assay of the recombinant Perilla frutescens protein, and subsequent assays on recombinant ANS from a range of species that confirmed the requirement for Fe, 20G, and ascorbate. Sequence comparisons show that ANS is more closely related to flavonol synthase (FLS), another 20GD, than to F3H. 

---