Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii beta-glucosidase II.
The biosynthetic activity of yeast Pichia etchellsii beta-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate:1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethyl acetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.
Bachhawat
Preeti
P
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New Delhi 110016.
Mishra
Saroj
S
Bhatia
Yukti
Y
Bisaria
V S
VS
D044602:Cellulases / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli; D005960:Glucosides / Q000096:biosynthesis* / Q000737:chemistry; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D010843:Pichia / Q000201:enzymology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013730:Terphenyl Compounds / Q000737:chemistry / Q000378:metabolism*
D005960:Glucosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D013730:Terphenyl Compounds; D044602:Cellulases