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 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 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.

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

2013 ◽  
Vol 80 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Long Liu ◽  
Zhuangmei Deng ◽  
Haiquan Yang ◽  
Jianghua Li ◽  
Hyun-dong Shin ◽  
...  
Keyword(s):  
Systems Engineering ◽  
Rational Design ◽  
Catalytic Domain ◽  
Disulfide Bridge ◽  
Biochemical Properties ◽  
Bridge Engineering ◽  
High Catalytic Activity ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Triple Mutant

ABSTRACTHigh thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase fromAlkalimonas amylolyticathroughin silicorational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (kcat/Km) increased from 1.8 × 104to 2.4 × 104liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

Biosynthesis of 2-O-d-glucopyranosyl-l-ascorbic acid from maltose by an engineered cyclodextrin glycosyltransferase from Paenibacillus macerans

2013 ◽  
Vol 382 ◽  
pp. 101-107 ◽  
Author(s):  
Long Liu ◽  
Ruizhi Han ◽  
Hyun-dong Shin ◽  
Jianghua Li ◽  
Guocheng Du ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Cyclodextrin Glycosyltransferase ◽  
Paenibacillus Macerans
Sign in / Sign up

Export Citation Format

Share Document