Enzyme solution preparation

Add 0.05 ml of the enzyme solution prepared in step 4-90 to the assay mixture, mix thoroughly, and begin timing the incubation with a stopwatch or suitable timer. 

The enzyme solution prepared from Pseudomonas aeruginosa 99 by Mitsuhashi and coworkers required magnesium ion, and had a substrate specificity similar to that of the enzyme solution prepared from Pseudomonas aeruginosa 130 as just described. The optimal pH was 6.5. These authors isolated the inactivated product of gentamicin Ci, and mass spectrometry thereof indicated an N-acetyl group present in the 2-deoxystreptamine moiety. The placement as a 3-N-acetyl group was shown by periodate oxidation of a methanolysis product, namely, iV-acetylgentamine, which consumes 2 moles of periodate per mole. 

Enzyme Solution. Prepare a fresh solution from a suitable source of phosphatase. (Taka-diastase, diluted with lactose, Parke Davis Co., London or Clarase, Takamine Laboratories, Clifton, N.J., U.S.A.,... 

Mix 3 g. of starch well with loml. of water in a test-tube and pour the mixture into 90 ml. of boiling water contained in a 300 ml. conical flask, stirring at the same time. Cool to about 70 and then place in a water-bath maintained at 65-70 , but not higher. Now add 2-3 ml. of the malt extract prepared as above, mix well and allow the hydrolysis to proceed. Take a series of test -tubes and in each put 10 ml. of water and 2 drops of a 1 % iodine solution. At intervals of about 4 minutes (depending upon the activity of the enzyme solution), remove 1 ml. of the reaction mixture, cool and add it to one of the test-tubes and note the colour obtained. At the beginning of the experiment the colour will be blue due to the starch alone. As the reaction proceeds, the colour gradually becomes violet, reddish, yellowish and finally colourless. 

Films or membranes of silkworm silk have been produced by air-drying aqueous solutions prepared from the concentrated salts, followed by dialysis (11,28). The films, which are water soluble, generally contain silk in the silk I conformation with a significant content of random coil. Many different treatments have been used to modify these films to decrease their water solubiUty by converting silk I to silk II in a process found usehil for enzyme entrapment (28). Silk membranes have also been cast from fibroin solutions and characterized for permeation properties. Oxygen and water vapor transmission rates were dependent on the exposure conditions to methanol to faciUtate the conversion to silk II (29). Thin monolayer films have been formed from solubilized silkworm silk using Langmuir techniques to faciUtate stmctural characterization of the protein (30). ResolubiLized silkworm cocoon silk has been spun into fibers (31), as have recombinant silkworm silks (32). 

Negatively charged species such as carboxylic acid group in acid-treated CNTs can attract positively charged enzymes from solution as long as the pH value of the enzyme solution is controlled to be lower than the iso-electric point of the enzyme thus, multilayer films of the enzyme can be formed by the layer-by-layer technique. For example, five layers of GOx can be immobilized on the electrode surface by alternatively dipping a poly(diallyldimethylammonium chloride (PDDA))-functionalized GC into a CNT solution and a GOx solution (pH 3.8). Figure 15.15 illustrates the preparation process for the formation of a multilayer film of GOx on the electrode. 

Formaldehyde solutions prepared by dissolving and depolymerization of paraformaldehyde (a homopolymer of formaldehyde with empirical formula HO (CH20)nH, where n > 6) are free of admixtures of methanol and formic acid. Depolymerized paraformaldehyde is useful in enzyme histochemistry, when the preservation of the enzyme activity is of crucial importance, but it has no advantage over formalin solutions routinely used in pathology and in immunohistochemistry. 

Their specimen of cholinesterase was prepared from horse serum by the method of Stedman and Stedman,1 and the method of estimation was that of Ammon.2 The enzyme solution was placed in the right-hand flask of a Barcrofb manometer, in a total volume of 3 ml. of 0-2 per cent NaHC03 solution the gas phase was 5 per cent C02 in Na. The reaction, carried out at 20°, was started by adding a solution containing 2 mg. of acetylcholine chloride. The C02 output was usually linear until about 100 fi. had been produced. 

Lastly, to prepare the enzyme solution, say 10 nM from a 200 nM stock enzyme solution, we use the same procedure, except that the dilution factor in this case is (1.00/ 0.10) in terms of the 0.1 mL enzyme solution volume cited above. 

Two microliters of pFlK-ORF plasmid solutions prepared with the Wizard SV 96 Plasmid DNA Purification System are treated with 5 U of S ifi and 5 U of Pmel in a 10 pL reaction volume containing lx Flexi Digest Buffer at 37°C for 60 min, and then the enzymes are inactivated by incubating at 65°C for 20 min. 

After ion exchange chromatography the enzyme preparation is in a more workable volume and is then ready for further purification. Depending on the requirement for the next step, the enzyme solution can be desalted by gel filtration or ultrafiltration or the buffer can be changed by diafiltration. 

Triton X-100, EDTA, dithiothreitol and electrolyte protect enzyme in dilute solution and against denaturation by heat or extreme pH-values [12, 48] <2>, at low dithioerythritol concentrations enzyme tends to aggregate [5] <2>, bovine serum albumin, 1 mg/ml, stabilizes dilute enzyme solutions [5] <2>, diadenosine pentaphosphate, i.e. AP5A, stabilizes during preparative electrophoresis [7]... 

Part A Instructor or teaching assistant prepares the enzyme solution... 

In vitro assays themselves take a few hours to complete. The protein nitrogen content of a sample must be known. Sample hydration can take one to several hours. Preparation of enzyme solutions takes vivo digestibility assays involve a feeding study, and the investigator faces similar issues as described for PER. 

As for Basic Protocol 1, the LOX enzyme is prepared according to the Support Protocol. However, more care should be taken to reduce turbidity and other UV-absorbing materials caused by suspended lipid and pigments, especially when low activity is expected (requiring more enzyme addition). Triton X-100 is a UV absorber, and it causes more lipid and pigments to be suspended. Partial purification may be necessary. HEPES and PIPES buffers are not used in this method because of excessive absorption at 234 nm (0.5 to 0.6 absorbance for 0.1 M solutions) MES is useful with limitations (0.2 absorbance for 0.1 M solution). 

Prior to assay, all LOX enzyme solutions should be kept ice-cold through all steps of the preparation procedure. The literature indicates that LOX solutions can be successfully frozen and stored after addition of 10% to 20% glycerol. In the author s laboratory, soybean LOX-1 is stored in the refrigerator ( 4°C) as a suspension in 2.3 M (NH4)2S04, and this procedure has proved to maintain good activity for years. 

Absolute Viscosimetric Method for the Determination of Endocellulase Activities. Experimental Setup of the Viscosimetric Measurements. The substrate, buffer, and enzyme solutions are prepared in the same manner as described in the section on light scattering. However, no clarification of the substrate solution by pressure filtration is needed. 

---