Purification And Properties Of Fructan Exohydrolase From Gartic

Garlic(Allium Sativum L.) is perennial herb, belongs to the liliaceae allium plants. Its bulb which is edible, has a unique flavor, high nutritional value and medicinal value. Therefore, garlic is known as "natural health products". Because of its low price, high sweetness, low calorie, unique flavor and suitable taste,high fructose syrup is widely used in food and pharmaceutical industries. A large number of fructan accounts for more than 75% of the dry weight of garlic. High fructose syrup can be made from garlic. Fructan hydrolase in garlic can break down the fructan stored. The process of production is simple, and the technology has a high conversion rate, pure product, high yield, and great potential in industry of fructose and fructose syrup. Currently, research on garlic fructan hydrolase is relatively fewer, so it has an important economic value and practical significance for high fructose syrup production on properties of garlic fructan hydrolase.Garlic fructan hydrolase was isolated and purified by DE-52 ion-exchange chromatography, Sephadex G-200 gel filtration chromatography and Phenyl Sepharose CL-4B hydrophobic interaction chromatography that uses garlic as the raw material, then its properties of enzymatic were studied. In order to provide a theoretical basis for black garlic processing conditions and blackening mechanism, the conditions which garlic fructan crude extracts spontaneously decompose under thermal conditions is explored. The main findings are as follows:1. High-purity fructan hydrolase was isolated and purified by ammonium sulfate precipitation, DE-52 ion-exchange chromatography, Sephadex G-200 gel filtration chromatography and Phenyl Sepharose CL-4B hydrophobic interaction chromatography. Three isozymes whose molecular weight was 56.3kDa, 47.9kDa and 69.0kDa were obtained by SDS-PAGE. Fructan hydrolase was purified while the purification fold was 17.09 times and the recovery rate was 14.98%.2. Study on properties of fructan hydrolase enzyme showed the optimum temperature of garlic fructan hydrolase is 50℃, its optimum pH is 5.0. The enzyme activity can be maintained relatively stable below 80℃, in the pH range of 5.0 to 8.0. The kinetic constants Km of fructan hydrolase is 1.161 mg / mL, and the Vmax is 0.474 mg ? h-1 ? m L-1. Fructan hydrolase activity can be promoted by metal ions Mn2 +, Fe2 + and bovine serum albumin, sodium sulfite, mercaptoethanol and ascorbic acid and other chemical substances, while its activity can be inhibited by metal ions Cu2 +, Fe3 + and alcohol, salicylic acid, sorbic acid and hydrogen peroxide and other reagents. Enzyme activity was not significantly affected by metal ions Ca2 +, Ba2 +, Mg2 +, Zn2 +, EDTA, gelatin and sodium carboxymethyl cellulose, etc. The results of modifing fructan hydrolase showed that fructan hydrolase contains lysine, and that the glutamic acid, histidine, tryptophan, cysteine are essential in fructan hydrolase, while arginine, serine, tyrosine and disulfide are not essential for fructan hydrolase.3. The study on conditions of garlic fructan spontaneously decompose under thermal conditions showed that garlic fructan can spontaneously hydrolysis under thermal conditions, and the process cannot be performed below 80℃ and 50% relative humidity. There is no obvious connection between the rate of hydrolysis and concentration, but as the passage of hydrolysis time, the rate of hydrolysis may decrease proportionally.