General Properties, Occurrence, and Preparation
1.2
95
15.6 Separation and Purification of Cyclomaltodextrins
The cyclomaltodextrins (α-CD, β-CD, and γ -CD) can be selectively obtained from a fermentation culture or an enzyme digest of cyclomaltodextrin glucanotransferase reaction with solubilized starch. The majority of the cyclomaltohexaose (α-CD) can be separated from cyclomaltoheptaose (β-CD) and γ -CD by their selective precipitation with p-cymene from the culture
supernatant or from an enzyme digest [168]. The α-CD can then be precipitated from the
supernatant with cyclohexene, which is extracted with acetone to remove the cyclohexene and
the α-CD can be crystallized from water or a propanol-1/water solution [169]. The p-cymene
precipitates of β-CD and γ -CD are put into a water solution and β-CD selectively precipitated from γ -CD with fluorobenzene. The γ -CD is then precipitated with anthracene saturated
in diethyl ether. After the removal of the fluorobenzene from β-CD with acetone or ethanol
extraction, β-CD can be crystallized from water, and after the removal of anthracene with
acetone or ethanol extraction from γ -CD, it can also be crystallized from water [170,171].
The selective precipitations of the cyclomaltodextrins with various organic molecules is based
on the selective formation of complexes of the organic molecules with the specific sizes of
the cyclomaltodextrins and the relatively hydrophobic interior cavities of the cyclomaltodextrins [166,167,168].
15.7 Release of Oligosaccharides from Glycoproteins
The O-linked oligosaccharides are relatively easily released from the protein by a β-elimination reaction, using mild alkali (0.05–0.5-M NaOH) and temperatures of 0–45 °C for
15–216 h [172,173,174]. A standard procedure is 0.1-M NaOH at 37 °C for 48 h. Conditions, however, must be determined for each glycoprotein. The asparagine N-linked glycosides can be cleaved by hydrazinolysis [175]. The glycoprotein is heated at 100 °C with
anhydrous hydrazine for 8–12 h in a sealed tube. Various endoglycosidases, such as endoβ-N-acetylglucosaminidase have also been used to release the oligosaccharides from glycoproteins [176,177]. A chemical method that releases both O- and N-linked oligosaccharides
from glycoproteins involves trifluoromethane sulfonic acid (TFMS) [178]. TFMS reactions
are performed at 0 °C for 0.5–2 h under nitrogen. After reaction, the mixture is cooled below
−20 °C in a dry ice-ethanol bath and slowly neutralized by the addition of 60% (v/v) aqueous
pyridine that is previously cooled to −20 °C. More information on the structure and analysis
of glycoproteins may be found in Chap. 8.
References
1. Bonner WA (1991) Origins Life Evolut Biosph
21:72
2. Bailey J, Chrysostomou A, Hough JH, Gledhill
TM, MeCall A, Clark S, Ménard F, Tamura M
(1998) Science 281:672
3. Lobry de Bruyn CA, Alberda van Ekenstein
W (1895) Rec Trav Chim 14:156; 203; (1896)
15:92; (1897) 16:241, 262, 274, 282; (1899)
18:147
4. French D (1954) Adv Carbohydr Chem 9:149
5. Avigad G, Feingold DS, Hestrin S (1956)
Biochim Biophys Acta 20:129
6. Feingold DS, Avigad G, Hestrin S (1957) J Biol
Chem 224:295
1.2
95
15.6 Separation and Purification of Cyclomaltodextrins
The cyclomaltodextrins (α-CD, β-CD, and γ -CD) can be selectively obtained from a fermentation culture or an enzyme digest of cyclomaltodextrin glucanotransferase reaction with solubilized starch. The majority of the cyclomaltohexaose (α-CD) can be separated from cyclomaltoheptaose (β-CD) and γ -CD by their selective precipitation with p-cymene from the culture
supernatant or from an enzyme digest [168]. The α-CD can then be precipitated from the
supernatant with cyclohexene, which is extracted with acetone to remove the cyclohexene and
the α-CD can be crystallized from water or a propanol-1/water solution [169]. The p-cymene
precipitates of β-CD and γ -CD are put into a water solution and β-CD selectively precipitated from γ -CD with fluorobenzene. The γ -CD is then precipitated with anthracene saturated
in diethyl ether. After the removal of the fluorobenzene from β-CD with acetone or ethanol
extraction, β-CD can be crystallized from water, and after the removal of anthracene with
acetone or ethanol extraction from γ -CD, it can also be crystallized from water [170,171].
The selective precipitations of the cyclomaltodextrins with various organic molecules is based
on the selective formation of complexes of the organic molecules with the specific sizes of
the cyclomaltodextrins and the relatively hydrophobic interior cavities of the cyclomaltodextrins [166,167,168].
15.7 Release of Oligosaccharides from Glycoproteins
The O-linked oligosaccharides are relatively easily released from the protein by a β-elimination reaction, using mild alkali (0.05–0.5-M NaOH) and temperatures of 0–45 °C for
15–216 h [172,173,174]. A standard procedure is 0.1-M NaOH at 37 °C for 48 h. Conditions, however, must be determined for each glycoprotein. The asparagine N-linked glycosides can be cleaved by hydrazinolysis [175]. The glycoprotein is heated at 100 °C with
anhydrous hydrazine for 8–12 h in a sealed tube. Various endoglycosidases, such as endoβ-N-acetylglucosaminidase have also been used to release the oligosaccharides from glycoproteins [176,177]. A chemical method that releases both O- and N-linked oligosaccharides
from glycoproteins involves trifluoromethane sulfonic acid (TFMS) [178]. TFMS reactions
are performed at 0 °C for 0.5–2 h under nitrogen. After reaction, the mixture is cooled below
−20 °C in a dry ice-ethanol bath and slowly neutralized by the addition of 60% (v/v) aqueous
pyridine that is previously cooled to −20 °C. More information on the structure and analysis
of glycoproteins may be found in Chap. 8.
References
1. Bonner WA (1991) Origins Life Evolut Biosph
21:72
2. Bailey J, Chrysostomou A, Hough JH, Gledhill
TM, MeCall A, Clark S, Ménard F, Tamura M
(1998) Science 281:672
3. Lobry de Bruyn CA, Alberda van Ekenstein
W (1895) Rec Trav Chim 14:156; 203; (1896)
15:92; (1897) 16:241, 262, 274, 282; (1899)
18:147
4. French D (1954) Adv Carbohydr Chem 9:149
5. Avigad G, Feingold DS, Hestrin S (1956)
Biochim Biophys Acta 20:129
6. Feingold DS, Avigad G, Hestrin S (1957) J Biol
Chem 224:295