Isolation,Purification,Some Properties And Modification Of Groups Of The β-D-xylosidase From Chinese Chive

β-D-xylosidase(EC 3.2.1.37) is a type of glycoside hydrolases, which widely exists in plants, bacteria and fungi. It can hydrolyze not only catalytically by reacting synergistically with xylanase, but also xylooligosaccharide, as well as xylo oligosaccharide structurally higher than xylobiose, from the non-reducing end by excision, and finally decompose into xylose. As one of the key enzymes in the xylooligosaccharide enzyme system, β-D-xylosidase has been widely used in various fields like food processing, papermaking, energy, etc. especially in food industry, we can decompose β-D-xylosidase to extract xylose, which is applicable as the raw material for xylitol, food ingredient or flavour improving agent. Currently, relative studies on β-D-xylosidase throughout the globe focus mainly on its microbial origin, while its botanical origin is still rarely involved, not to mention its isolation and purification. Chinese chive belongs to liliaceous vegetable of perennial root. Adaptable and weatherable, it is planted all over China and therefore easily available. We hereby choose Chinese chive as the research subject in order to provide a method of purification by extracting the botanical-origin β-xylosidase, and explore the nature of β-xylosidase. We hope such a study will help provide a reference for the future industrial production and research. Following are the main results:1. The isolation and purification of β-D-xylosidase in Chinese chiveβ-xylosidase of electrophoresis purity can be extracted from the fresh chive through homogenization, sodium acetate buffer extraction, ammonium sulfate precipitation, carboxymethyl-Sepharose ion exchange chromatography and then Superdex-200 gel filtration chromatography. After purification, the specific activity of β-xylosidase reaches up to 18.25 U/mg, its purification fold is 12.59, and its recovery rate is 1.83%.2. Part of enzymatic properties of β-D-xylosidase in Chinese chiveAccording to the study, the relative molecular mass of relative molecular mass of β-D-xylosidase β-D-xylosidase is 123.02 k D, and its subunit molecular mass is 63.14 k D. The study on its enzymatic properties shows that the optimum temperature of β-D-xylosidase is 65 °C, and that its optimum p H is 4. This enzyme shows a better stability under the conditions of 25-55 °C and p H 3.0-5.0; in optimal conditions its Michaelis constant(Km) is 0.28 mmol/L.3. Effects of some chemical reagents and metal ions on β-D-xylosidase in Chinese chiveMethanol, ethanol, isopropanol, sodium dodecyl sulfate(SDS), EDTA and Ag+ have inhibiting effects on β-D-xylosidase, but Mn2+ and Co2+ have some activation effects on it; while Li+, Cd2+, Ca2+, Zn2+, KSCN and carbamide has basically no effect on its activity.4. Analysis on groups of β-D-xylosidase in Chinese chiveResults of the chemical reaction of different chemical modifiers to β-D-xylosidase in Chinese chive show that: Chloramine-T, p CMB and NBS have very strong inhibitory effects on β-D-xylosidase, which illustrates that sulfide group of methionine, mercapto-group of protein molecule and indolyl of tryptophan have contributed to the formation of the activity center of β-D-xylosidase and therefore could probably be the necessary groups of its activity center in Chinese chive. Correspondingly, chemical modifiers like DTT, Br Ac, BD, SUAN, NAI, IAc and PMSF have no obvious effects on the enzyme activity of β-D-xylosidase in Chinese chive, which means those chemical groups corresponding to the above modifiers have probably no direct relationship with activity center of β-D-xylosidase.