Enhancement of α-cyclodextrin product specificity by enriching histidines of α-cyclodextrin glucanotransferase at remote subsite −6

Yue, Yang; Song, Binghong; Xie, Ting; Sun, Yan; Chao, Yapeng; Qian, Shijun

doi:10.1016/j.procbio.2013.11.002

Cited by 5 publications

(4 citation statements)

References 29 publications

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“…In general, the products of CGTase reactions contain α-, β-, and γ-CD. , The purification of a single type of cyclodextrin from a CD mixture requires several steps, which can be time-consuming and disadvantageous for human health and the environment . Therefore, to enable an efficient purification process, CGTases with high product specificity have been developed. ,,− However, compared to other kinds of CGTases, α-CGTases show relatively low expression levels and their product ratio is usually lower than 90%, which limits the potential application of α-CD in many fields. ,,, To overcome this problem, another approach, “the solvent process”, has been developed. This method involves the addition of organic solvent as a complexing agent to extract target CDs selectively. − For example, in the presence of 1-butanol, the α:β CD formation ratio of the CGTase increased dramatically to 97:3 with a 42% (w/w) conversion rate.…”

Section: Discussionmentioning

confidence: 99%

“…However, the market share of α-CD is still smaller than that of other CDs because of its low production yield and high price caused by its purification procedure . To solve these problems, genetically modified mutant CGTases, which increase the α-CD production ratio, have been developed; however, they result in the production of β- and γ-CD as well. ,− …”

Section: Introductionmentioning

confidence: 99%

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Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

Koo

Jeong

et al. 2017

J. Agric. Food Chem.

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Cyclodextrins (CDs) are produced from starch by cyclodextrin glucanotransferase (CGTase), which has cyclization activity. Specifically, α-CD is an important biomolecule, as it is a molecular carrier and soluble dietary fiber used in the food industry. Upon inspection of the conserved regions of the glycoside hydrolase (GH) 13 family amylases, the amino acids K232 and H233 of CGTase were identified as playing an important role in enzyme reaction specificity. A novel CD hydrolyzing enzyme, cyclodextrin glycosyl transferase (CGTase)-alpha, was developed using site-directed mutagenesis at these positions. Action pattern analysis using various substrates revealed that CGTase-alpha was able to hydrolyze β- and γ-CD, but not α-CD. This selective CD hydrolyzing property was employed to purify α-CD from a CD mixture solution. The α-CD that remained after treatment with CGTase-alpha and exotype glucoamylase was purified using hydrophobic interaction chromatography with 99% purity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

Koo

Jeong

et al. 2017

J. Agric. Food Chem.

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“…The supernatant was collected via centrifugation at 13 000 × g for 10 min and filtered through a 0.45‐µm ultrafiltration membrane. The proportions of α‐CD, β‐CD, and γ‐CD in the filtered product were estimated by high‐performance liquid chromatography (HPLC; Waters 1525) with a refractive index detector (Waters 2414) . The column was an Xbndge™ Amide Column (3.5 μm, 4.6 × 250 mm), and the mobile phase was acetonitrile:ddH 2 0 (60:40).…”

Section: Methodsmentioning

confidence: 99%

Purification and biochemical characterization of a cyclodextrin glycosyltransferase from Geobacillus thermoglucosidans CHB1

Jia

Chen

et al. 2017

Starch Stärke

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A strain named Geobacillus thermoglucosidans CHB1 was isolated from stockpiled hen dung compost. The cyclodextrin glycosyltransferase (CGTase) from the fermentation broth of this microorganism was purified 350.50-fold at a yield of 9.33% through ammonium sulfate precipitation, DEAE-Sepharose Fast Flow chromatography, Sephadex G-100 chromatography, and preparative electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified enzyme was approximately 70 kDa. The CGTase maintained more than 50% cyclization activity between 50 and 80°C and optimal activity at 65-70°C and remained stable at 50°C. Its optimal pH was 5.5, and its activity showed stability between pH 5.5 and 9.5. The activity of the enzyme was significantly enhanced by LiCl, NiCl 2 , and MgSO 4 but inhibited by AlCl 3 , CoCl 2 , FeCl 3 , HgCl 2 , BaCl 2 , SDS, and ZnSO 4 . The K m and V max of the reaction of this enzyme with soluble starch as the substrate were 12.5 mg/mL and 23.7 mmol/min, respectively. This enzyme produced a-cyclodextrin (CD), b-CD, and g-CD at a ratio of 0.57:1:0.21 from soluble starch as the substrate, and the conversion rate reached 60.3% from 3% soluble starch within 21 h. This CGTase also produced CDs from maltodextrin and potato starch as the substrate. To the best of our knowledge, this manuscript constitutes the first report of a CGTase purified from G. thermoglucosidans, and the results show that this enzyme might have potential for use in industrial production processes requiring stable thermal conditions, but further work is required to optimize its affinity and activation conditions.

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“…Reviews of recent progress in the artificial modifications of CGTases and their impact on biotechnological processes have been published (2,3). While some progress has been made, particularly through modifications at subsite Ϫ3 or Ϫ6 (13,14), improvements in the ␣-cyclodextrin specificity of ␣-CGTases, particularly via modifications at subsite Ϫ7, remain underexplored.…”

mentioning

confidence: 99%

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

Wang

Duan

2016

Appl Environ Microbiol

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, ␤-, and ␥-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 ␣-cyclodextrin selectivity of Paenibacillus macerans ␣-CGTase could be augmented by masking subsite ؊7 of the active site, blocking the formation of larger cyclodextrins, particularly ␤-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 ␣-cyclodextrin formation varied only modestly, the rate of ␤-cyclodextrin formation decreased dramatically in these mutants. The increase in ␣-cyclodextrin selectivity was directly proportional to the increase in the ratio of their k cat values for ␣-and ␤-cyclodextrin formation. The R146A/D147P and R146P/D147A double mutants exhibited ratios of ␣-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 ␣-cyclodextrin than the wild-type enzyme. The production of ␤-cyclodextrin by these mutants was almost identical to their production of ␥-cyclodextrin, which was unaffected by the mutations in subsite ؊7, suggesting that subsite ؊7 was effectively blocked by these mutations. Further increases in ␣-cyclodextrin selectivity will require identification of the mechanism or mechanisms by which these small quantities of larger cyclodextrins are formed. C yclodextrin glycosyltransferase (EC 2.4.1.19, CGTase), which belongs to glycohydrolase (GH) family 13, catalyzes the cyclization, coupling, disproportionation, and hydrolysis of starch or linear ␣-(1,4)-linked glucans (1-3). Industrial interest in these enzymes arises from their preference for transglycosylation over hydrolysis (2,4,5) and from their ability to catalyze the synthesis of circular ␣-(1,4)-linked oligosaccharides, which are known as cyclodextrins (3, 6). All known CGTases catalyze the formation of mixtures of cyclodextrins containing 6 (␣-cyclodextrin), 7 (␤-cyclodextrin), or 8 (␥-cyclodextrin) glucose units. If these reactions were allowed to reach equilibrium, the ␣-cyclodextrin/␤-cyclodextrin/␥-cyclodextrin ratio in the mixture would become 27:58:15 (7). In practice, the ratio of these products is determined by the kinetics of their production, which varies with each individual enzyme (2, 8). The CGTases are classified as ␣-, ␤-, or ␥-CGTases or mixed CTGases on the basis of the cyclodextrin that they produce in the main proportion (1-3).Cyclodextrins are unique natural materials. They adopt the structure of a truncated cone with a hydrophilic external surface and a hydrophobic internal cavity (9) that allow them to bind hydrophobic molecules within their internal cavities and carry them into aqueous solution...

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Enhancement of α-cyclodextrin product specificity by enriching histidines of α-cyclodextrin glucanotransferase at remote subsite −6

Cited by 5 publications

References 29 publications

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

Development and Application of Cyclodextrin Hydrolyzing Mutant Enzyme Which Hydrolyzes β- and γ-CD Selectively

Purification and biochemical characterization of a cyclodextrin glycosyltransferase from Geobacillus thermoglucosidans CHB1

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

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