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Crude garlic extract contains one Mn-superoxide dismutase designated as SOD1 and two Cu,Zn superoxide dismutases as SOD2 and SOD3. The major isoform SOD2 was purified to homogeneity by Sephacryl S200-HR gel filtration, DEAE Sepharose ion exchange chromatography, and chromatofocusing using PBE 94. SOD2 was purified 82-fold with a specific activity of 4,960 U/mg protein. This enzyme was stable in a broad pH range from 5.0 to 10.0 and at various temperatures from 25 to 60 degrees C. The native molecular mass of SOD2 estimated by high performance liquid chromatography on TSK gel G2000SW column was 39 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a single band near 18 kDa, suggesting that native enzyme was homodimeric. The isoelectric point as determined by chromatofocusing was 5. Analysis of its N terminal amino acid sequence revealed high sequence homology with several other cytosolic Cu,Zn-SODs from plants. Exposure of cancer cell lines to garlic Cu,Zn-SOD2 led to a significant decrease in superoxide content with a concomitant rise in intracellular peroxides, indicating that the enzyme is active in mammalian cells and could, therefore, be used in pharmacological applications.

Analysis of peroxidase activity by native polyacrylamide gel electrophoresis (PAGE) from a garlic bulb (Allium sativum L) extract showed two major activities (designated POX1 and POX2). The POX2 isoenzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. The purified enzyme was found to be monomeric with a molecular mass of 36.5 kDa, as determined by sodium dodecyl sulfate-PAGE. The optimum temperature ranged from 25 to 40 degrees C and optimum pH was about 5.0. The apparent Km values for guaiacol and H2O2 were 9.5 and 2 mM, respectively. POX2 appeared highly stable since 50% of its activity was conserved at 50 degrees C for 5 h. Moreover POX2 was stable over a pH range of 3.5-11.0. Immobilization of POX2 was achieved by covalent binding of the enzyme to an epoxy-Sepharose matrix. The immobilized enzyme showed great stability toward heat and storage when compared with soluble enzyme. These properties permit the use of this enzyme as a biosensor to detect H2O2 in some food components such as milk or its derivatives.

The objective of this study was to obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features are the use of a mild nonionic detergent, 40-80% saturation with (NH4)2SOm4, maintained at low temperature to remove impurity, and the use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10- 15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. After homogenization, the supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4 degrees C, followed by a threestep fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as a green precipitate that was dissolved in a small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion-exchange chromatography (DEAE cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40-fold from the starting material. The specific activity of the pure enzyme was 168 U/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase: optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH was 5.7 and the optimum temperature was 50 degrees C. The enzyme was stable at pH 4.0-10.0 and 40-60 degrees C. Activation energy was between 10 and 20 kcal, and as Michaelis Menten coefficients, Vm values were 100 and 20 mM/s and Km values were 21.27 and 8.33 mM for paranitrophenylphosphate and paranitrophenyl, respectively. Studies of the effect of metal ions on enzyme activity showed both an activating and a deactivating effect. While Cu, Mo, and Mn showed strong inhibitory effects, Na, Ca, and K were the significant activators of acid phosphatase.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.