Systems Engineering of Tyrosine 195, Tyrosine 260, and Glutamine 265 in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Enhance Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

Abstract

In this work, the site saturation mutagenesis of tyrosine 195, tyrosine 260 and glutamine 265 in the cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase for maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G). Specifically, the site-saturation mutagenesis of three sites-tyrosine 195, tyrosine 260, and glutamine 265-was performed, and it was found that the resulting mutants (containing the mutations Y195S [tyrosine ¡ serine], Y260R [tyrosine ¡ arginine], and Q265K [glutamine ¡ lysine]) produced higher AA-2G yields than the wild type and the other mutant CGTases when maltodextrin was used as the glycosyl donor. Furthermore, double and triple mutations were introduced, and four mutants (containing Y195S/ Y260R, Y195S/Q265K, Y260R/Q265K, and Y260R/Q265K/Y195S) were obtained and evaluated for the capacity to produce AA-2G. The Y260R/Q265K/Y195S triple mutant produced the highest titer of AA-2G at 1.92 g/liter, which was 60% higher than that (1.20 g/liter) produced by the wild-type CGTase. The kinetics analysis of AA-2G synthesis by the mutant CGTases confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, all seven mutants had lower cyclization activities and higher hydrolysis and disproportionation activities. Finally, the mechanism responsible for the enhanced substrate specificity was explored by structure modeling, which indicated that the enhancement of maltodextrin specificity may be related to the changes of hydrogen bonding interactions between the side chain of residue at the three positions (195, 260, and 265) and the substrate sugars. This work adds to our understanding of the synthesis of AA-2G and makes the Y260R/ Q265K/Y195S mutant a good starting point for further development by protein engineering.

Fusion of Self-Assembling Amphipathic Oligopeptides with Cyclodextrin Glycosyltransferase Improves 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis with Soluble Starch as the Glycosyl Donor

“…As shown in Fig. 2A, the optimal temperature for AA-2G production with the wild-type CGTase was 36°C, which was also reported in our previous studies (8)(9)(10) and is similar to the optimal temperature for ␣-CGTase-catalyzed AA-2G synthesis with ␤-cyclodextrin as the glycosyl donor (6). However, SAP5-CGTase and SAP6-CGTase produced the highest AA-2G titer at 28°C, and this optimal temperature is the same as that of production with the chimeric enzymes CGT-CBM Amy and CGT⌬E-CBM Amy (11).…”

“…To date, many attempts have been made to find an inexpensive and soluble substrate to replace ␣-and ␤-cyclodextrin as glycosyl donors for AA-2G production. For instance, we have enhanced the transformation efficiency of maltodextrin for AA-2G production through site-directed saturation mutagenesis of the Ϫ3 subsite (8), ϩ2 subsite (9), and Ϫ6 subsite residues (10) in the CGTase from Paenibacillus macerans. Furthermore, we improved the transformation efficiency of soluble starch to AA-2G by fusing the carbohydrate-binding module from Alkalimonas amylolytica ␣-amylase with CGTase from P. macerans (11).…”

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

“…Control incubations were prepared by adding the same amount of Tris-HCl buffer (pH 8.3, 0.25 M) to 5 ml DTNB reagent. The cyclodextrin glycosyltransferase Y195C from Paenibacillus macerans (17) was used as a positive-control protein, as it contains one free cysteine residue.…”

Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

“…significantly enhanced for AA-2G synthesis (9,10,28). The ϩ2 subsite is considered to be responsible for the stabilization of the glucose ring with phenyl rings at these sites (29)(30)(31)(32), and the Ϫ3 subsite has important effects on the four reactions (cyclization, coupling, disproportion, and hydrolysis) of CGTase because it is near the active site (17,33).…”

“…In previous work, we have attempted to replace ␣-and ␤-cyclodextrins with an inexpensive and soluble substrate as a glycosyl donor for AA-2G production. For instance, we enhanced the transformation efficiency of maltodextrin for AA-2G production through site-directed saturation mutagenesis of residues K47 (9), Y195, Y260, and Q265 (10) in CGTase from Paenibacillus macerans. This outcome suggested that mutagenesis was effective for improving the specificity of CGTase toward maltodextrin by affecting the hydrogen bond interactions between the enzymes and linear substrates.…”