Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

Abstract

c Corynebacterium glutamicum amylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The cloned CgAM gene was ligated into the pET-17b vector and used to transform Escherichia coli BL21(DE3). Site-directed mutagenesis of Tyr-172 in CgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. The k cat /K m of the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WT CgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novel CgAM.

Identification of essential tryptophan in amylomaltase from Corynebacterium glutamicum

“…In addition, cyclization activity which results from the intramolecular transfer of glucosyl group was disappeared in the mutated enzymes. For coupling and hydrolysis, low activities were obtained even in the WT, the result was corresponded to general characteristics of amylomaltases as previously reported [16,29]. This experiment thus demonstrated the importance of both tryptophan residues lie close to the active site region, W425 and W673, in CgAM catalysis.…”

“…The purified WT and mutated enzymes were compared for different activities of AM (Table 4). WT-CgAM had high activities of starch transglucosylation, disproportionation, and cyclization reactions, the values were about two fold higher than those reported previously for the recombinant WT-CgAM without his-tag [29]. For starch transglucosylation activity, W425A showed total loss of activity while W673A showed 87% decrease in activity.…”

Optimization of large-ring cyclodextrin production from starch by amylomaltase fromCorynebacterium glutamicumand effect of organic solvent on product size

“…CD yields produced from CGTase are limited by enzyme product inhibition (Leemhuis et al 2003;Gaston et al 2009) and breakdown of CDs into linear oligosaccharides through the coupling reaction. While CGTase exhibited high coupling activity (Ibrahim et al 2012;Ara et al 2015), coupling activity of CgAM was low or not detectable (Srisimarat et al 2012). In addition, CGTase linearized large cycloamyloses at higher rate, compared to CD7 and CD8 (Terada et al 2001).…”

“…), coupling activity of Cg AM was low or not detectable (Srisimarat et al . ). In addition, CGTase linearized large cycloamyloses at higher rate, compared to CD7 and CD8 (Terada et al .…”

“…Starch transglycosylation activity was measured by the iodine method as described in Srisimarat et al (2012). Enzymes were incubated with 0Á2% (w/v) soluble potato starch and 1% (w/v) maltose in 50 mmol l À1 phosphate buffer pH 6Á0 at 30°C for 10 min.…”