The Study Of Purification Process In Pilot-Plant Scale, Enzymatic Properties And Quality Standard Of Alliinase From Garlic

Garlic (Allium Sativum L.) is used world-wild as a spice as well as remedy in folk medicine for the prevention and treatment of infectiouse diseases, cardiovascular disease as well as cancer. Numerous clinical trials were undertaken to elucidate the benefits of garlic. Sulfur containing compounds like the extremely unstable allicin [(2-propenyl)-2- propenethiosulfinate] and ajoene [(E, Z) -4, 5, 9-trithiadodeca-1, 6, 11-triene-9-oxide] were suggested to be responsible for most of these effects. Allicin itself does not occur in fresh garlic but gets rapidly liberated from the more stable precursor alliin [S-(+)-2- propenyl-L-cysteine sulfoxide sulfoxide] in the cytosol if cells of fresh garlic are disrupted. The reaction is catalysed by the enzyme alliinase (EC 4. 4.1. 4) present in vacuoles. Beacause of its instability,allicin is not suitable for use as clinical drug directly. As an alternative strategy proposed by Professor Chenjian, a binary medical product may be prepared consisting of predetermined amounts of stable alliinase purified and precursor alliin, which are kept physically separated by a special formulation. In vivo, the pro-drug alliin would be enzymatically converted to a predictable amount of allicin. The study of purification process in pilot-plant scale as drug component, enzymatic properties and quality standard of alliinase from garlic were accomplished in this paper. Primarily study methods and results were as follows:Firstly, easy, sensitive, fast, noncostly, and highly efficient throughput assay methods for alliinase activity in its purification process were developed. The enzymatic activity of alliinase was assayed from the amount of enzymatically formed pyruvate using natural alliin as substrate. Protein concentrations of alliinase preparations were determined following the method described by Bradford using BSA as standards. And then, the processing conditions for the extraction and purification of alliinase in the lab were optimized:①it was in Na/K phosphate buffer (pH 6.5,10% (v/v) glycerol, 20μmol/L pyridoxal 5'-phosphate and 1mmol/L CaCl2 that peeled garlic cloves were homogenized in a blender;②precipitation with solid ammonium sulfate to give 35% saturation or 15% (m/v) PEG8000 was selected to isolate alliinase;③it was by ultrafiltration that supernatant solution containing alliinase was desalted and concentrated;④the protein isoelectric point was found at pH4.9. Under these processing conditions in laboratory, three lots alliinase were isolate from fresh bulbs by ammonium sulfate salting out and PEG8000 precipitation methods, respectively. Alliinase activity in the product was determined by developed HPLC. Using natural alliin as substrate, the enzymatic activity of alliinase was assayed from the amount of enzymatically cleaved natural substrate. SDS-Page gave evidence that the alliinase obtained from fresh garlic consisted of two identical subunits with molecular weights of 54.0kDa.Second, the processing conditions for the extraction and purification of alliinase in pilot-plant scale were optimized: the times for the extraction of aliinase from fesh garlic was optimized to be 2 using production equipment in the ratio of substance and liquid of 1:1 and 1:0.5, respectively; precipitation with solid ammonium sulfate to give 45% saturation was selected to separate alliinase over again because the concentration of impure enzyme in solution had been different from that in laboratory;it was suggested in Na/K phosphate buffer (pH 6.5) that precipitated protein was resuspended. Underlying these optimizing processing conditions, alliinase had been extracted and purified for three lots with ammonium sulfate method in pilot-plant scale. In the stage of alliinase isolation, simple and sensitive analytical method was successfully adopted to control the whole process. As a result, the yield and activity of alliinase from garlic attained the requirement of predesign. The molecular weight of garlic enzyme, as determined by SDS-PAGE, was found to be 107.8 kDa. And the processing transition from small scale in lab to pilot-plant scale in factory had been realized at the first time.In addition, underlying the processing conditions and production equipment for the extraction and purification of alliinase in pilot-plant scale, alliinase was isolate from fresh bulbs by PEG8000 precipitation method, which would be applied to final purification step of the standard preparation of alliinase.Thirdly, enzymatic properties and kinetic charactristics of homogeneous alliinase from Garlic were researched. Highest enzyme activity for the purified alliinase was found to be at 35℃. The enzyme had pH optima at pH7.0,but it was the most stable at pH6.5. The Km values for alliinase from garlic was estimated to be 2.139mmol/L for natural L-(+)-alliin. Vmax using natural L-(+)-alliin as substrate was at 41.67 U/mg(protein). When the injection with NaCl and without glucos was used as solvent, there was a stimulation of enzyme activity, but injection with glucos gave a strong inhibition. The lyase solved in 75% or 95% alcohol completely lost its activity rapidly. Obviously, alliinase was sensitive to pH or temperatures. Injection containing glucos was not suitable as solvent for this enzyme. Using 75% or 95% alcohol as disinfectant should avoid lowering the enzyme activity. The present study gave directions to furtherly optimizing purification procedure of enzyme and its clinical application.At last, the quality standard of this garlic enzyme was set down, involving characters, identification, tests, titration, classification, and storage, et al. This quality standard of alliinase from garlic would make solid base for in-depth quality study of this garlic enzyme. Along with development of research for this enzyme, the quality standard should be unceasing revised and perfected.