Enzymes Involved in the Biosynthesis and Degradation of Cyclic Maltosyl-maltose in Arthrobacter globiformis M6

Abstract

Enzymatic syntheses of various nonreducing oligosaccharides from starch have been reported by many researchers. Nonreducing oligosaccharides are generally divided into two groups: linear and cyclic oligosaccharides. Trehalose (α D glucopyranosyl α D glucopyranoside) is a typical linear nonreducing oligosaccharide occurring in bacteria, yeasts, fungi, plants and invertebrates. Mass production of trehalose from starch has been developed using two bacterial enzymes, maltooligosyltrehalose synthase (EC 5.4.99.15) and maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141), 1 3) and now this saccharide is used in the food, cosmetics and the pharmaceutical industries. Cyclomaltohexaose (α cyclodextrin), one of the most well known cyclic oligosaccharides, is produced from linear α 1,4 glucans by the intramolecular α 1,4 transglycosylation reaction of a cyclomaltodextrin glucanotransferase (EC 2.4.1.19).4) The cyclic oligosaccharide has a hydrophobic cavity in the center of the structure. Guest molecules of suitable size can enter the cavity, and the formation of the inclusion complex is used for stabilizing labile materials, 5) masking odors 6) and modifying viscosity. 7) Co te and co workers first reported that a cyclic tetrasaccharide consist-(1 }, was produced from a dextran like polysaccharide, alternan, by its degrading enzyme.8 10) The cyclic oligosaccharide was designated cycloalternan (CA). Recently, we discovered two novel enzymes, 6 α glucosyltransferase and 3 α isomaltosyltransferase, in Bacillus globisporus, and succeeded in the mass production of this saccharide from starch by the joint reaction of the two enzymes.11 13) Although the characteristics of CA has not yet been totally clarified, potential applications of the saccharide in food, cosmetics and medicines are anticipated because of its unique structure. Hence nonreducing oligosaccharides pro- )-α-D-Glcp-(1 4)-α-D-Glcp-(1 6)-α-D-Glcp-(1 4)-α-D-Glcp-(1 }, was designated cyclic maltosyl-maltose (CMM).CMM was not hydrolyzed by various amylases, such as α-amylase, β-amylase, glucoamylase, isoamylase, pullulanase, maltogenic α-amylase and α-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. A glycosyltransferase involved in the synthesis of CMM from starch was purified to homogeneity from the culture supernatant of A. globiformis M6. The enzyme acted on maltooligosaccharides that have degrees of polymerization more than 3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan and dextran. The CMM-forming enzyme catalyzed both intermolecular and intramolecular α-1,6-maltosyl transfer reaction and found to be a novel maltosyltransferase (6MT). To reveal the degradation pathway of CMM, we identified two enzymes, CMM hydrolase (CMMase) and α-glucosidase, as the responsible enzymes from the cell-free extract of the strain. CMMase hydrolyzed CMM to maltose via maltosyl-maltose as intermediates; however, it did not hydrolyze CMM to glucose, suggesting that it is a novel hydrolase that hydrolyzes...

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

“…Microbial metabolism of cyclic glucooligosaccharides ( e.g. , cyclodextrin, cyclic α-maltosyl-(1→6)-maltose, and cycloalternan) is often part of a monopolizing metabolic pathway that is advantageous in the competition for starch acquisition by transiently changing the polysaccharide into special cyclic forms ( 53 , 54 , 55 , 56 , 57 ). Such metabolic pathways generally consist of three stages: synthesis of the cyclic oligosaccharides outside the cells by an extracellular enzyme, uptake across the cellular membrane by a transporter, and degradation by intracellular enzymes.…”

Structural features of a bacterial cyclic α-maltosyl-(1→6)-maltose (CMM) hydrolase critical for CMM recognition and hydrolysis

“…A. globiformis M6 can grow on CMM as the sole carbon source, but no CMM decomposition product was detected in the culture supernatant. A further study revealed that an intracellular enzyme, CMM hydrolase (CMMase, EC 3.2.1.-), plays a key role in the CMM catabolism (31)(32)(33). CMMase degrades CMM into two maltose molecules by a two-stage hydrolysis of the ␣-1,6 linkages via maltosyl-maltose (MM, Fig.…”

Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway

“…The gene cluster of the CMM metabolic pathway consisted of seven open reading frames ( cmmA – G ) [ 11 ], and three enzymes belonging to glycoside hydrolase family 13 are involved in the formation and degradation of CMM. An extracellular enzyme, 6-α-maltosyltransferase (CmmA), produces CMM by inter- and intramolecular α-1,6-transglucosylation [ 10 ], and two intracellular enzymes, CMM hydrolase (CmmF) [ 12 ] and α-glucosidase (CmmB), synergistically degrade CMM to glucose [ 13 ]. We previously reported the mechanistic details of the hydrolysis of CMM by CMM hydrolase with its crystal structures [ 14 ].…”