Bonded enzymes found more active, stable Enzyme molecules are attached to polymer matrices
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fructose-1,6- diphosphate and NAD
aldolase on AEC
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—Na 2 HAs0 4 solution
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-GAPD on AEC
methylene blue • and diaphorase
44 C&EN SEPT. 22, 1969
BIOLOGICAL Sufferers of failing kidneys or enzyme deficiency diseases may soon take a day's supply of needed enzymes by mouth. Astronauts on extended jour neys will perhaps consume foods made by passing metabolic wastes through columns packed with enzymes. Dr. Jackson Lynn and Dr. Richard D. Falb of Battelle Memorial Insti tute report attaching of enzyme mole cules to polymer matrices using link ing agents. The polymer-enzyme sys tems are insoluble in water, more ac tive than the usual soluble enzyme preparations, and more stable. Aldo lase, glyceraldehyde-3-phosphate de hydrogenase (GAPD), and fructose1,6-diphosphatase are bonded to aminoethylated cellulose (AEC) us ing glutaraldehyde. Aldolase bonded to AEC is stable for 21 days at 4° C , whereas aldolase solution preparations are only 50% active after 15 days. GAPD bonded to AEC retains most of its activity after one day, but a solu tion is completely inactivated. By matching polarity of the poly mer to that of the substrate molecule, the Columbus group achieves lower Km values for the bonded enzymes than for the same enzymes in soluble form. Km is the molar concentration of substrate needed for half the maxi mum possible reaction rate. Lower Km means a more active enzyme toward the substrate. To demonstrate ability of sequential syntheses using cellulose-bonded en zymes, the Battelle workers pass fructose-l,6-diphosphate and nicotin amide adenine dinucleotide (NAD) in solution through a column packed with aldolase on AEC. The effluent solution containing the products dihydroxyacetone phosphate and glyceraldehyde-3-phosphate passes through a second column packed with GAPD on AEC. GAPD catalyzes oxidation of glyceraldehyde-3-phosphate to 1,3diphosphoglyceric acid. NAD serves as coenzyme for the oxidation, be ing reduced to NADH. The effluent from the second column containing all products including NADH flows into an indicator solution of methylene blue and diaphorase. NADH reduces diaphorase which reduces methylene blue from the blue to the colorless form. Decolorization of methylene blue indicates the presence of NADH, NADH indicates oxidation of glyceraldehyde-3-phosphate, and glyceraldehyde-3-phosphate indicates breakdown of fructose-l,6-diphos-
phate. This proves the success of sequential enzyme-catalyzed synthesis. Aldolase, GAPD, and fruotose-1,6diphosphatase catalyze reactions in the glycolysis series, the system which breaks glycogen and hexose sugars down to pyruvic and lactic acids for energy and other purposes. Fructose1,6-diphosphatase catalyzes hydrolysis of fructose-1,6-diphosphate to fructose-6-phosphate, a reverse step in gly colysis. Dr. Lynn and Dr. Falb have hopes of eventually reproducing the Calvin cycle, which is the dark phase of photosynthetic conversion of carbon dioxide and water to sugars. The group is also working on en closing polymer-bound enzymes in microcapsules, small spheres 1 to sev eral hundred microns in diameter, made from semipermeable membrane. Patients with failing kidney action might take an oral dose of microcap sules containing urease, aspartase, and fumaric acid. Urea would be hydrolyzed by urease to carbon dioxide and ammonia. Toxic ammonia would be combined with fumaric acid by as partase to produce nontoxic aspartic acid. Urease and aspartase are pro teins which would be denatured al most at once by stomach acid if not protected by bonding to cellulose and enclosure by microcapsules. Children with histidinemia suffer mental retardation due to toxic build up of histidine. Doses of histidase in microcapsules by mouth might be used to supply the missing enzyme and protect such children from poison ing by the histidine in their diets. The Battelle workers also use diazotized p-aminobenzylated cellulose and copolymers of ethylene and maleic anhydride to bind and stabilize aldo lase in insoluble form.
Virus makes proteins from larger peptides Polio virus proteins are made by cut ting sections out of larger molecules, whereas proteins made by bacterio phage viruses are made by joining preformed subunits to produce the final molecule. This has implications not only for knowledge of notorious disease-causing polio virus, but the finding may shed light on the whole problem of protein synthesis in mam malian cells, as opposed to the same process in bacteria. Dr. David Balti more, MIT biologist, focuses on noncapsid viral polypeptides NCVP-1, NCVP-2, and NCVP-X. In work with Michael F. Jacobson, Jean Asso, and
