scholarly journals 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

2013 ◽  
Vol 79 (24) ◽  
pp. 7562-7568 ◽  
Author(s):  
Ruizhi Han ◽  
Long Liu ◽  
Hyun-dong Shin ◽  
Rachel R. Chen ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Substrate Binding ◽  
Acid Synthesis ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Cyclodextrin Glycosyltransferase ◽  
Content Type ◽  
Paenibacillus Macerans ◽  
Ascorbic Acid Synthesis ◽  
Iterative Saturation Mutagenesis

ABSTRACT2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stablel-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to −7). In this study, iterative saturation mutagenesis (ISM) was performed on the −6 subsite residues (Y167, G179, G180, and N193) in the CGTase fromPaenibacillus maceransto improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites—Y167, G179, G180, and N193—was first performed and revealed that four mutants—Y167S, G179R, N193R, and G180R—produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the −6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.

scholarly journals 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

2012 ◽  
Vol 79 (2) ◽  
pp. 672-677 ◽  
Author(s):  
Ruizhi Han ◽  
Long Liu ◽  
Hyun-dong Shin ◽  
Rachel R. Chen ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Systems Engineering ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Cyclodextrin Glycosyltransferase ◽  
Triple Mutant ◽  
Site Saturation ◽  
Starting Point ◽  
Glycosyl Donor ◽  
Paenibacillus Macerans

ABSTRACTIn this work, the site saturation mutagenesis of tyrosine 195, tyrosine 260 and glutamine 265 in the cyclodextrin glycosyltransferase (CGTase) fromPaenibacillus maceranswas 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.

scholarly journals 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

2014 ◽  
Vol 80 (15) ◽  
pp. 4717-4724 ◽  
Author(s):  
Ruizhi Han ◽  
Jianghua Li ◽  
Hyun-dong Shin ◽  
Rachel R. Chen ◽  
Long Liu ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Binding Capacity ◽  
Acid Synthesis ◽  
Soluble Starch ◽  
Wild Type ◽  
Cyclodextrin Glycosyltransferase ◽  
Protein Fusion ◽  
Content Type ◽  
Self Assembling ◽  
Fusion Enzymes

ABSTRACTIn this study, we fused six self-assembling amphipathic peptides (SAPs) with cyclodextrin glycosyltransferase (CGTase) fromPaenibacillus maceransto catalyze 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) production with cheap substrates, including maltose, maltodextrin, and soluble starch as glycosyl donors. The results showed that two fusion enzymes, SAP5-CGTase and SAP6-CGTase, increased AA-2G yields to 2.33- and 3.36-fold that of wild-type CGTase when soluble starch was used as a substrate. The cyclization activities of these enzymes decreased, while disproportionation activities increased. Enzymatic characterization of the two fusion enzymes was performed, and kinetics analysis of AA-2G synthesis confirmed the enhanced soluble starch specificity of SAP5-CGTase and SAP6-CGTase compared to that in the wild-type CGTase. As revealed by structure modeling of the fusion and wild-type CGTases, enhanced substrate-binding capacity may result from the increased number of hydrogen bonds present after fusion. This study demonstrates an effective protein fusion approach to improving the substrate specificity of CGTase for AA-2G synthesis. Fusion enzymes, especially SAP6-CGTase, are promising starting points for further development through protein engineering.

scholarly journals Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite −7

2016 ◽  
Vol 82 (8) ◽  
pp. 2247-2255 ◽  
Author(s):  
Lei Wang ◽  
Xuguo Duan ◽  
Jing Wu
Keyword(s):  
Active Site ◽  
Wild Type ◽  
Cyclodextrin Glycosyltransferase ◽  
Wild Type Enzyme ◽  
Widespread Application ◽  
Content Type ◽  
Hydrogen Bonding Interactions ◽  
Paenibacillus Macerans ◽  
Site Blocking ◽  
Cyclodextrin Production

ABSTRACTCyclodextrin glycosyltransferases (CGTases) (EC 2.4.1.19) catalyze the conversion of starch or starch derivates into mixtures of α-, β-, and γ-cyclodextrins. Because time-consuming and expensive purification procedures hinder the widespread application of single-ingredient cyclodextrins, enzymes with enhanced specificity are needed. In this study, we tested the hypothesis that the α-cyclodextrin selectivity ofPaenibacillus maceransα-CGTase could be augmented by masking subsite −7 of the active site, blocking the formation of larger cyclodextrins, particularly β-cyclodextrin. Five single mutants and three double mutants designed to remove hydrogen-bonding interactions between the enzyme and substrate at subsite −7 were constructed and characterized in detail. Although the rates of α-cyclodextrin formation varied only modestly, the rate of β-cyclodextrin formation decreased dramatically in these mutants. The increase in α-cyclodextrin selectivity was directly proportional to the increase in the ratio of theirkcatvalues for α- and β-cyclodextrin formation. The R146A/D147P and R146P/D147A double mutants exhibited ratios of α-cyclodextrin to total cyclodextrin production of 75.1% and 76.1%, approximately one-fifth greater than that of the wild-type enzyme (63.2%), without loss of thermostability. Thus, these double mutants may be more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The production of β-cyclodextrin by these mutants was almost identical to their production of γ-cyclodextrin, which was unaffected by the mutations in subsite −7, suggesting that subsite −7 was effectively blocked by these mutations. Further increases in α-cyclodextrin selectivity will require identification of the mechanism or mechanisms by which these small quantities of larger cyclodextrins are formed.

scholarly journals Effect of Fluorosis on Liver Cells of VC Deficient and Wild Type Mice

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Wei Wei ◽  
Yan Jiao ◽  
Yonghui Ma ◽  
John M. Stuart ◽  
Xiudian Li ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Gene Networks ◽  
Acid Synthesis ◽  
Liver Cells ◽  
C Cells ◽  
Wild Type ◽  
Key Enzyme ◽  
Ascorbic Acid Synthesis ◽  
Deficient Mice

For decades, mouse and other rodents have been used for the study of oxidative or related studies such as the effect of fluoride. It is known that rodents normally synthesize their own vitamin C (VC) due to the presence of a key enzyme in ascorbic acid synthesis, l-gulono-lactone-γ-oxidase (Gulo), while humans do not have the capacity of VC synthesis due to the deletion of most parts of the GULO gene. The spontaneous fracture (sfx) mouse recently emerged as a model for study of VC deficiency. We investigated the effect of fluoride on liver cells from wild type Balb/c andsfxmice. We found that activities of SOD, GPx, and CAT were reduced in both wild type andsfxmice; however, the amount of reduction in thesfxcells is more than that in Balb/c cells. In addition, while both cells increased MDA, the increase in thesfxcells is greater than that in Balb/c cells. Gene networks ofSod,Gpx, andCatin the liver of humans and mice are also different. Our study suggests that reaction to fluoride in vitamin C deficient mice might be different from that of wild type mice.

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

2013 ◽  
Vol 79 (10) ◽  
pp. 3234-3240 ◽  
Author(s):  
Ruizhi Han ◽  
Jianghua Li ◽  
Hyun-Dong Shin ◽  
Rachel R. Chen ◽  
Guocheng Du ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Soluble Starch ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Efficient Synthesis ◽  
Wild Type ◽  
Cyclodextrin Glycosyltransferase ◽  
Content Type ◽  
Fusion Enzymes ◽  
Glycosyl Donor

ABSTRACTIn 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) fromAlkalimonas amylolyticaα-amylase (CBMAmy) to cyclodextrin glycosyltransferase (CGTase) fromPaenibacillus macerans. One fusion enzyme, CGT-CBMAmy, was constructed by fusing the CBMAmyto the C-terminal region of CGTase, and the other fusion enzyme, CGTΔE-CBMAmy, was obtained by replacing the E domain of CGTase with CBMAmy. 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 CGTΔE-CBMAmyand CGT-CBMAmywere 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.

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