Studies On The Separation, Purification And Properties Of Alliinase

Garlic, Allium sativum L., is one of the edible plants that has generated a lot of interests throughout human history as a medicinal panacea. The garlic alliinase (EC 4.4.1.4) catalyzes the synthesis of allicin, which is the main functional flavor component of garlic. The outputs of volatile aroma components of garlic bulbs essential oil highly depends on the activity of alliinase, so it's of great significance to study its properties. In this article, alliinase was purified to homogeneity successfully from fresh bulbs using various steps, including tissue homogenate, PEG8000 method, affinity chromatography on ConA-sepharose, chromatography on hydroxylapatite. The alliinase gene was acquired by RT-PCR, and the amino acids sequence was predicted by its nucleotide sequence. Furtherly, properties and kinetic characteristics of alliinase were researched. Primarily study results as follows:1.Separation and purification of alliinase At first, precipitation with 20% PEG8000 was selected to separate alliinase, because this method had higher purification fold of 5.19 and higher recovery of 90.93% than precipitation with 30~45% ammonium sulfate did. Futher purification was achieved by ConA-sepharose chromatography; purification fold was 20.40 and recovery was 78.2%. Ultimately, alliinase was purified up to 20.68 times with a recovery of 38.53% by HA chromatography. Urea-SDS-PAGE showed that no unpurposed propein was found after ConA-sepharose chromatography.2. Identification of alliinaseâ… , alliinaseâ…¡ Two different alliinase, named alliinaseâ… and alliinaseâ…¡, were found in ConA-sepharose chromatography. Alliinaseâ…¡ is the preponderant one, while activity of alliinase â…  was lower. Alliinaseâ…¡ was purified up to 20.68 times with a recovery of 38.53%.3. Predicted amino acids sequence of alliinase Total RNA was extrated from garlic bulbs, then the alliinase gene was acquired by RT-PCR, and the amino acids sequence was predicted by its nucleotide sequence. 4. The properties of alliinase The relative molecular weight of subunit of alliinaseâ… , alliinaseâ…¡ was 44300 and 47500 respectively. Alliinaseâ…¡ had a carbohydrate content of 5.31% and the glycosidic linkage was inferred to be N-type. Spectrogram of purified alliinase proved that there was PLP in alliinase as cofactor. Kinetic characteristics of alliinase as follows: Its optimum pH and temperature was 6.5 and 35oC respectively. Alliinase was stable comparatively at pH value of 6~7, and the stability has great relation to temprature. Using synthetic S-allylcysteine sulfoxide as substract, its Km was 4.17 mmol/L and Vmax was 156.25 units/mg pr.