Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-α-glucoside formation

Liu

Shin

et al. 2013

Appl Environ Microbiol

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.

Section: Resultsmentioning

confidence: 91%

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confidence: 99%

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confidence: 99%

See 1 more Smart Citation

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

Liu

Shin

et al. 2013

Appl Environ Microbiol

“…CGTase is considered to be the best enzyme for large-scale production of AA-2G due to its high substrate specificity (Tanaka et al 1991). Microbial fermentation is the main source for CGTase production, and the main microbes include Paenibacillus macerans, Bacillus sp., and Bacillus circulans.…”

Section: Production Of Aa-2g By Biotransformationmentioning

confidence: 99%

Functions, applications and production of 2-O-d-glucopyranosyl-l-ascorbic acid

Appl Microbiol Biotechnol

Liu

et al. 2012

Vitamin C (VC) is an essential nutrient that cannot be synthesized by the human body. Due to its extreme instability, various VC derivatives have been developed in an attempt to improve stability while retaining the same biological activity. One of the most important VC derivatives, 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G), has attracted increasing attention in recent years with a wide range of applications in cosmetics, food, and medicine. In this mini-review, we first introduce the types and properties of different VC glycosyl derivatives. Next, we provide an overview of the functions and applications of AA-2G. Finally, we discuss in-depth the current status and future prospects of AA-2G production by biotransformation.

“…Many saccharides, such as ␣-and ␤-cyclodextrins, maltodextrin, and starch, can be used as glycosyl donors (10). It has been demonstrated that ␣-and ␤-cyclodextrins are the best glycosyl donors for AA-2G production (10)(11)(12). However, because of the high cost of ␣-cyclodextrin and the low solubility of ␤-cyclodextrin in aqueous solution, neither is suitable for the large-scale production of AA-2G (9).…”

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confidence: 99%

Carbohydrate-Binding Module–Cyclodextrin Glycosyltransferase Fusion Enables Efficient Synthesis of 2- O - d -Glucopyranosyl- l -Ascorbic Acid with Soluble Starch as the Glycosyl Donor

Shin

et al. 2013

Appl Environ Microbiol

In this study, we achieved the efficient synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) from soluble starch by fusing a carbohydrate-binding module (CBM) from Alkalimonas amylolytica ␣-amylase (CBM Amy ) to cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans. One fusion enzyme, CGT-CBM Amy , was constructed by fusing the CBM Amy to the C-terminal region of CGTase, and the other fusion enzyme, CGT⌬E-CBM Amy , was obtained by replacing the E domain of CGTase with CBM Amy . The two fusion enzymes were then used to synthesize AA-2G from soluble starch as a cheap and easily soluble glycosyl donor. Under the optimal conditions, the AA-2G yields produced using CGT⌬E-CBM Amy and CGT-CBM Amy were 2.01 g/liter and 3.03 g/liter, respectively, which were 3.94-and 5.94-fold of the yield from the wild-type CGTase (0.51 g/liter). The reaction kinetics of the two fusion enzymes were analyzed and modeled to confirm the enhanced specificity toward soluble starch. It was also found that, compared to the wild-type CGTase, the two fusion enzymes had relatively high hydrolysis and disproportionation activities, factors that favor AA-2G synthesis. Finally, it was speculated that the enhancement of soluble starch specificity may be related to the changes of substrate binding ability and the substrate binding sites between the CBM and the starch granule.