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 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 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 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.

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis

2001 ◽  
Vol 358 (2) ◽  
pp. 423-430 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Pichia Pastoris ◽  
Association Constant ◽  
Mutational Analysis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Glycosylation Sites ◽  
High Mannose

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc2Man8 to GlcNAc2Man14. The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

scholarly journals Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

2007 ◽  
Vol 406 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Lavinia Cicortas Gunnarsson ◽  
Cedric Montanier ◽  
Richard B. Tunnicliffe ◽  
Mike P. Williamson ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Hydrophobic Interactions ◽  
Molecular Engineering ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Rhodothermus Marinus ◽  
Binding Properties ◽  
Carbohydrate Binding Modules

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

scholarly journals Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

2012 ◽  
Vol 78 (7) ◽  
pp. 2230-2240 ◽  
Author(s):  
Xiaoyun Su ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Catalytic Efficiency ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Ph Optimum ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Type Protein ◽  
Wild Type Protein ◽  
Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

scholarly journals Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

2012 ◽  
Vol 78 (11) ◽  
pp. 3923-3931 ◽  
Author(s):  
Susana Valeria Valenzuela ◽  
Pilar Diaz ◽  
F. I. Javier Pastor
Keyword(s):  
Catalytic Activity ◽  
Kinetic Parameters ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Dimensional Structure ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Content Type ◽  
Catalytic Module

ABSTRACTXyn30D from the xylanolytic strainPaenibacillus barcinonensishas been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing aKmof 14.72 mg/ml and akcatvalue of 1,510 min−1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.

scholarly journals Exploration of Two Pectate Lyases from Caldicellulosiruptor bescii Reveals that the CBM66 Module Has a Crucial Role in Pectic Biomass Degradation

2020 ◽  
Vol 86 (16) ◽  
Author(s):  
Hamed I. Hamouda ◽  
Nasir Ali ◽  
Hang Su ◽  
Jie Feng ◽  
Ming Lu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plant Biomass ◽  
Degradation Products ◽  
Amino Acid Identity ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Content Type ◽  
Acid Identity ◽  
Pectate Lyases ◽  
Caldicellulosiruptor Bescii

ABSTRACT Pectin deconstruction is the initial step in breaking the recalcitrance of plant biomass by using selected microorganisms that encode pectinolytic enzymes. Pectate lyases that cleave the α-1,4-galacturonosidic linkage of pectin are widely used in industries such as papermaking and fruit softening. However, there are few reports on pectate lyases with good thermostability. Here, two pectate lyases (CbPL3 and CbPL9) from a hyperthermophilic bacterium, Caldicellulosiruptor bescii, belonging to family 3 and family 9 polysaccharide lyases, respectively, were investigated. The biochemical properties of the two CbPLs were shown to be similar under optimized conditions of 80°C to 85°C and pH 8 to 9. However, the degradation products from pectin and polygalacturonic acids (pGAs) were different. A family 66 carbohydrate-binding module (CbCBM66) located in the N terminus of the two CbPLs shares 100% amino acid identity. A CbCBM66-truncated mutant of CbPL9 showed lower activities than the wild type, whereas CbPL3 with a CbCBM66 knockout portion was reported to have enhanced activities, thereby revealing the different effect of CbCBM66. Prediction by the I-TASSER server revealed that CbCBM66 is structurally close to BsCBM66 from Bacillus subtilis; however, the COFACTOR and COACH programs indicated that the substrate-binding sites between CbCBM66 and BsCBM66 are different. Furthermore, a substrate-binding assay indicated that the catalytic domains in the two CbPLs had strong affinities for pectate-related substrates, but CbCBM66 showed a weak interaction with a number of lignocellulosic carbohydrates. Finally, scanning electron microscopy (SEM) analysis and a total reducing sugar assay showed that the two enzymes could improve the saccharification of switchgrass. The two CbPLs are impressive sources for the degradation of plant biomass. IMPORTANCE Thermophilic proteins could be implemented in diverse industrial applications. We sought to characterize two pectate lyases, CbPL3 and CbPL9, from a thermophilic bacterium, Caldicellulosiruptor bescii. The two enzymes share a high optimum temperature, a low optimum pH, and good thermostability at the evaluated temperature. A family 66 carbohydrate-binding module (CbCBM66) was identified in the two CbPLs, sharing 100% amino acid identity. The deletion of CbCBM66 dramatically decreased the activity of CbPL9 but increased the activity and thermostability of CbPL3, suggesting different roles of CbCBM66 in the two enzymes. Moreover, the degradation products of the two CbPLs were different. These results revealed that these enzymes could represent potential pectate lyases for applications in the paper and textile industries.

scholarly journals Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a Xylanase That Contains a Family 3 Carbohydrate-Binding Module

2011 ◽  
Vol 77 (12) ◽  
pp. 4260-4263 ◽  
Author(s):  
Makiko Sakka ◽  
Yurika Higashi ◽  
Tetsuya Kimura ◽  
Khanok Ratanakhanokchai ◽  
Kazuo Sakka
Keyword(s):  
Recombinant Proteins ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Content Type ◽  
Paenibacillus Curdlanolyticus ◽  
Biochemical Analyses ◽  
Hydrolysis Of

ABSTRACTPaenibacillus curdlanolyticusB-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass.

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