title: Raffinose in the Sugarbeet (Beta vulgaris): I. Biosynthesis and Degradation in the Root; II. Hydrolysis in Molasses With Sweet Almond Emulsin
Creators: name: Chuang, Te-chao
Publisher: name: Utah State University
Year: 1970

description: PART I The precursors for the biosynthesis of raffinose in sugarbeets were studied. An enzyme preparation was obtained from the sugarbeet (Beta vulgaris) root. Incubation of this enzyme with (X-galactose-1-phosphate, Uridine triphosphate (UTP), Adenosine triphosphate (ATP), Magnesium chloride (MgC12), and L- cysteine at pH 5, 10°c for 6 hours formed a compound which was identified as raffinose by a hydrolysis method and thin- layer chromatography. The same result was obtained when Uridinediphosphate (UDP)-galactose was incubated with sucrose, ATP , MgC12 , and L- cysteine in the presence of the beet enzyme preparation. These reactions suggested that the sugarbeet contained an enzyme system capable of transferring a galactose unit from X-galactose-1-phosphate or UDP-galactose to sucrose, forming raffinose. The galactosylation of sucrose via UDP- galactose was further confirmed using sucrose-UL-C14 as one of the substrates. Radioactive raffinose was identified on a radio-autograph. The involvement of UDP-galactose in the raffinose synthesis is believed to be of more significance in vivo. A preliminary study indicated there are at least five main categories of nucleotide compounds in beet root tissues. It is suggested that a sugar nucleotide pool is present in the sugarbeet for transglycosylation among various sugars. A reaction similar to the above using UDP-glucose-UL-c14 and Nicotinamide adenine dinucleotide instead of UDP-galactose resulted in the formation of labeled raffinose. This suggested that an UDP-glucose-4-epimerase activity in beet tissues may be responsible for the conversion of UDP-glucose to UDP-galactose for the raffinose synthesis. The possible participation of galactinol in the raffinose synthesis in sugarbeets was investigated. An experiment using galactinol, sucrose-UL-c14 and ATP as reactants with the sugarbeet enzyme did not give positive results. The presence of X-galactosidase activity in the beet enzyme preparation was noted. Incubation of the enzyme at 37 C with raffinose and stachyose respectively liberated galactose as a common product. It is suggested that X-galactosidase is the enzyme responsible for the breakdown of raffinose in the sugarbeet. The turn-over of galactose liberated from raffinose by this hydrolytic enzyme is discussed. The separation of X-galactosidase from raffinose synthetase was attempted. Some difficulties involved are discussed. A study of the effects of pH and temperature on the enzyme activities showed that pH 5 and low temperatures (0-15°C) are favorable for the raffinose synthesis, while high temperatures (above 15°C) favored X-galactosidase activity. Raffinose was formed when UDP-galactose and sucrose were incubated with the enzyme preparation at 0C for 24 hours. This would account for the accumulation of raffinose during cold storage since at low temperatures X-galactosidase activity is retarded while raffinose synthetase seems to be unaffected. PART II Incubation of 30 milligrams commercial sweet almond emulsin (800 units/mg) with 2.5 BX molasses which contained 1. 5 mg raffinose in 3 milliliters 0.1 M acetate buffer at pH 6, 35C for one day resulted in a complete hydrolysis of raffinose in the digest. No sucrose inversion occurred under the above conditions.