Enzymological Characteristics Of Polygalacturonase And Its Products Analysis

In this paper, a polygalacturonase produced strain was isolated firstly. The fermentation medium and producing conditions of polygalacturonase were investigated to enhance enzyme activity. Subsequently, enzymological characteristics of polygalacturonase and hydrolysis products of pectin what Oligosaccharides were researched.⑴Strain and productionA polygalacturonase produced strain M-8 was isolated from rotten raisin and was identified as Aspergillus niger. The fermentation medium and conditions of enzyme production under flask shaking were investigated. The optimal medium was determined as follow(%): pectic 0.7, NaNO3 0.1, MgSO4 0.02, FeSO4 0.001, KH2PO4 0.03, bean powder 1.0 and potato 20.0. The optimal fermentation condition was: culture temperature at 28℃, initial pH at 7.0, the culture medium volume at 50mL, inoculum concentration of 7%(v/v). Under the obtained optimum conditions, the strain reached a maximum polygalacturonase activity of 964U/mL in shaking flask after 48h cultivation.⑵Enzymological characteristicsA polygalacturonase was purified from Aspergillus niger M-8 by acetone fractionation and FPLC ion exchange techniques. The enzyme has a relative molecular mass of 68.8KDa by SDS polyacrylamide gel electrophoresis. The optimal temperature was 50℃. The polygalacturonase activity was well in the range of temperature from 40 to 60℃. The polygalacturonase was stable in 35℃and kept 90.5% enzyme activity when treated for 120min. After incubation at 50℃for 60min or at 60℃for 10min, the component of polygalacturonase remained 30.3% and 16.1% of the total respectively. The optimal pH was 3.2. The polygalacturonase was stable in the range of pH from 2.4 to 5.2 and exceeded 80% of the total activity. Metal ions such as Zn2+ and Ca2+ could stimulate polygalacturonase activity especially. Cu2+, Pb2+, Hg2+ and Ag+ were inhibitors of polygalacturonase activity. The Michaelis-Menten kinetic parameters of the polygalacturonase (Km and Vmax) were 2.04×10-3(g/L) and 1.567×103(μg/min), respectively. The polygalacturonase showed store stability to some extend.⑶Oligosaccharides analysisCitrus pectin was decomposed by the polygalacturonase.A series of oligosaccharide mixtures could be obtained by ethanol precipitation and the recovery reached 87.2%. ESI-MS experiments of oligosaccharide mixtures were performed to analyze component. Tetrasaccharide composed by three galacturonic acid and one esterifiable galacturonic acid was the main product in the hydrolysate prepared by two-fold ethanol precipitation. Trisaccharide composed by three galacturonic acid was the main product prepared by four-fold ethanol precipitation. Disaccharide composed by two galacturonic acid was the main product in the hydrolysate prepared from apple pectin and beet pectin.Currently, compositions and structure of pectin-oligosaccharides is infrequent in domestic research. In this paper, pectin-oligosaccharides can be obtained through hydrolysis of polygalacturonase from Aspergillus niger M-8 and the compositions and structure of oligosaccharides were studied. This work will be helpful for the biology activity research and application of pectin-oligosaccharides.