Purification, Some Properties And Modification Of Groups Of The Sucrase From Aspergillus Niger TH-2

Sucrase (EC,3.1.2.26), also called invertase orβ-fructofuranosidase, can hydrolysis of sucrose to produce glucose and fructose.It widely found in nature of animals, plants and microorganisms, is an important kind of organisms hydrolase. Studies have showen that microbial sources sucrase more stable than sucrase from animals and plants. In industrial often use yeast, aspergillus and bacteria as primary sources.The results were as follows:Screened high-yielding strain Aspergillus niger TH-2 from the 14 Aspergillus niger strains laboratory saved, and fermented the selected strain, collect mycelium. Electrophoresis pure Sucrase was obtained from Aspergillus niger TH-2 by a combination of grinding, extraction, ion exchange chromatography on DEAE-Sepharose column and Superdex-200 gel-filtration. The specific activity of the purified Sucrase was 135.2 U/mg,37.7% of the Sucrase activity was reserved, and the purification fold was 20.3.The molecular weight of this enzyme was worked out about 129Kd by Superdex-200 gel-filtration and 63.75Kd by SDS-PAGE, this explained that it has two subunits.The optimum temperature was 65℃and the optimum pH was 4.4, the sucrase appeared to be stable between pH 4～8. Its Michaelis-Menten Kinetics (Km) was 23.1mmol/L.Its activity was inhibited by ascorbic acid, but EDTA,hydrogen peroxide and urea had little effect on sucrase.Among several mental ions, Ag+,Hg+were the strongest inhibitors, Ca2+,Mg2+,K+,Ba2+,Co2+ have little effect on sucrase, but it activated by Zn2+,Cu2+,Mn2+. The sucrase was selectively modified by PMSF, NBS, IAc, pCMB, DTT, NAI, BD, Chloramine-T and maleic anhydride. The results showed that Met,Trp,Ser,Sulphur might in the active sites of sucrase, and His,Tyr,Arg, disulfide bond might not in the active sites of sucrase.