Chemoenzymatic Synthesis of Branched Oligo‐ and Polysaccharides as Potential Substrates for Starch Active Enzymes

Abstract

Oligo- and polysaccharides embodying the alpha-maltotriosyl-6(II)-maltotetraosyl structure were readily synthesized by transglycosylation of maltosyl fluoride onto panose and pullulan catalysed by the bacterial transglycosylase cyclodextrin glycosyltransferase (CGTase). The two products obtained proved useful for increasing the knowledge of substrate binding and processing at the active site of barley limit dextrinase that is involved in the metabolism of amylopectin by acting upon its branch points.

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

“…S1). 4 However, the observed preference of HvLD for the hydrolysis of β-limit dextrins derived from amylopectin over amylopectin itself 5 is probably caused, in part, by the difference in the lengths of the branch chains extending beyond subsite −2/−3. Finally, the side chain of the solvent-exposed HvLD Phe620 showed no significant electron density, indicative of a high level of flexibility.…”

“…5 LD is also highly active towards pullulan, a linear polysaccharide of α-1,6-linked maltotriose repeats, 4,5 while the highest catalytic efficiency described for LD is for its activity towards a simple branched maltoheptaose (four Glc residues in the main chain and three Glc residues in the branch). 4 LD is grouped into GH13 subfamily 13 together with a subset of bacterial pullulanases, while other bacterial pullulanases are assigned to subfamilies 12 and 14. 6 LD has also been referred to as plant pullulanase.…”

Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase

“…The result is in perfect agreement with kinetic data for HvLD purified from malt that shows K m values in the 0.5-0.8 mM range for small limit dextrins (6-7 Glc units), with k cat varying from 85 s -1 , when there were three main chain Glc units on the non-reducing side of the a-1,6-linkage, to approximately 215 s -1 when there are one or two Glc units on the non-reducing side [28,30].…”

New Insight into Structure/Function Relationships in Plant .ALPHA.-Amylase Family GH13 Members

“…23) Previously LD have been purified in very small amounts only from germinating barley seeds (malt) and this LD preparation was prone to proteolytic degradation, which hampered thorough analysis of structure function relationships and biochemical properties. 20,21) Very recently, however, successful heterologous production of barley LD was achieved by a strategy involving secretory expression of this large multi domain enzyme during high cell density fermentation of transformed Pichia pastoris. 22,33) A fed batch fermentation enabled efficient production of LD and 34 mg electrophoretically homogenous LD was purified from the culture supernatant from 2 L starting batch by affinity chromatography on β CD Sepharose and Sephadex G200 gel filtration resulting in a yield of 85%.…”