Studies On Identification Of Neokestose Produced By Xanthophyllomyces Den Drorhous And Enzyme Related To The Oligosaccharide's Prodution

In this paper,oligosaccharides produced by Xanthophyllomyces dendrorhous as the starting strain was investigated according to the patents for inventions,"a oligosaccharides-neokestose fermentation production methods"[open,CN1873016A].The main contents include:the analysis of the structure of oligosaccharide,the purification of enzyme produced in the fermentation process,and research on its nature.There may be neokestose in the fermentation by high-performance liquid chromatography analysis and the activity was determinated in the broth , a certain activity.The products that hydrolyzed by enzyme include sucrose,neokestose,glucose,fructose and others.Compared with the material in the fermentation broth,content of sucrose decreased significantly,the content of neokestose increased,while glucose and fructose produced.It preliminarily notes the enzyme plays fructose-transferring role.The major and specific oligosaccharide produced by Xanthophyllomyces dendrorhous was purified by high performance liquid chromatography (HPLC) method.Its structure was identified by mass chromatography (MS) method associated with nuclear magnetic resonance (NMR) method.The results indicated that the molecular weight of oligosaccharide was 504Da, and its structure wasβ-D-fructofuranose (2→6)-a-D-glucopyranose (2→1)-β-D-fructofuranose, namely called neokestose.The enzyme in the broth was purified by a successive procedure including Ammonium sulfate precipitation,AKTA chromatography protein purification system chromatography .The purified enzyme was confirmed to be one subunit by denaturing polyacrylamide electrophoresis,the molecular weight was estimated to be 70541Da.The optimal temperature range of hydrolysis reaction is 18-30℃,with the temperature increasing, the activity decreases quickly,complete inactivation in 60℃,the enzyme is fairly stable in 18-30℃.The optimum pH is 6.5,stability of activity is at pH 5-6.5.The effect of K+(2mmol/L)on the enzyme activity is only a little,or even activated.Ca2+and Mg2+ (2mmol/L)have little effect on the enzyme activity.The effect of Zn2+and Al3(+2mmol/L)on the enzyme activity is inhibited.2mmol/L urea and SDS have no impact on the enzyme activity,but when the concentration of SDS increased to 10mmol/L,the enzyme activity is strongly inhibited.