The enzyme displayed many activities related to the degradation and transformation of starch. Gene encoding H. Sequence analysis revealed an open reading frame of bp that encodes a protein of amino acids. The CGTase is secreted to the extracellular medium by the Tat pathway.
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The YF mutant had the highest affinity which was indicated by the lowest K m of Increasing hydrophobicity around the catalytic center appeared to favor the cyclization activity of the mutants. Cyclodextrin glycosyltransferase EC 2. Catalysis of cyclization is the primary function of CGTase Leemhuis et al.
Contrarily, mutant AV CGTase in Bacillus circulans strain improved the hydrolytic activity but reduced cyclization activity Leemhuis et al. Additionally, Dijkhuizen and collages uncovered that mutation of tyrosine , which located in the central active site cleft of CGTase, significantly suppressed the cyclodextrin formation Penninga et al.
The amino acid side chain at conserved Phe residues also regulated the hydrolytic activity of CGTase. Beyond these findings, the conserved residue Asp was reported to be required for the proper conformation of amino residues in the catalytic sites and activity Leemhuis et al. Results from the Leemhuis.
All these findings suggested that amino acid mutations in the conserved and critical sites regulate the activity of CGTase. The function of domain D remains to be illustrated. These conserved regions contain the catalytic, specificity and substrate-binding residues Janecek Shanghai, China. Overlap extension PCR is a well-established PCR amplification method that can be used to introduce and recover mutations in specific DNA sequences without the requirement for restriction enzyme sites Angelaccio and di Patti Simple vector E.
Recovered plasmids with the correct restriction pattern were sent to Sangon Biotech for gene sequencing. Recombinant proteins were purified and subjected to western blotting using mouse antiHis monoclonal antibody.
The enzyme activity assays were conducted with soluble corn starch as a substrate and measured spectroscopically. The reaction medium 0. The optimum pH of the purified enzymes was determined by replacing 0. Reactions were performed following the previous procedures described for the enzyme activity assay. The pH stability of the enzymes was measured by incubating 0. The CGTase assay procedure described above was followed to determine the residual activity of the enzyme.
The thermal stability of the enzymes was measured by incubating 0. The standard CGTase activity assays described above was used to determine the residual activity of each enzyme Jeang et al.
The kinetic parameters of K m and V max were obtained by a nonlinear least-square fitting procedure using the Michaelis—Menten equation and the curve fitting software Origin 8.
As shown in Fig. Agarose electrophoresis analysis of the mutation genes showed that there were three specific bands of 2.
Verification of the mutant constructs generation. Xw The mutation sites Y, R and D are highlighted with red box. The recombinant expression plasmids were transformed into E. Western blotting for expression of the fusion His-tagged recombinant protein was shown in Fig. Experiments were performed in triplicate. The V max values in the range of Meanwhile, the observed k cat values varying between The ratio suggested how efficiently a catalytic enzyme converts a substrate into products.
A higher ratio indicates a higher efficiency of the enzyme. The increased ratio of 1. The highest activity was observed at pH 6. The activity of each enzyme without pre-incubation in the buffer at pH 6. In general, the cyclization activity of CGTases is much higher than the disproportionation and hydrolysis activities. Fujiwara et al. More significant enzyme activity enhancements 1. It was assumed that the cyclization was taken by nucleophilic attack, and the key amino acid residues in the cyclization reaction were aspartic acid in the cyclization reaction Wind et al.
When aspartic acid nucleophilic attack C1 as shown in Fig. Schematic representation of the reactions catalyzed by CGTase. After bond cleavage a covalently bound reaction enzyme glucosyl intermediate is formed. In the second step of the reaction the reaction intermediate is transferred to an acceptor molecule. This figure has been adapted from Ref. Leemhuis et al. In mutant of RF, phenylalanine with a pair of conjugate lone pair electrons was advantageous to the hydrophobic stacking effect and conducive to the stability of the seventh sugar molecules cyclization.
This result is similar with previous report Leemhuis et al. The mutant proteins were successfully expressed and purified. The optimum pH of the mutants was pH 6. G1 Cao et al. However, the mutants showed lower activity at pH 4. Extreme pH values were not suitable for the enzyme to carry out cyclization activity. Most of the reported CGTases exhibited optimum pH range from 5. The pH stability for cyclization is broad, in the range of pH 4. Studies by other researchers on CGTase from B.
And the enzymes had not a higher temperature stability compared to CGTase from alkalophilic Bacillus sp. Increasing hydrophobicity around the substrate-binding cleft of the catalytic center appeared to enhance the cyclization activity of the enzymes.
All the authors declare that they have no conflicts of interest regarding this paper. National Center for Biotechnology Information , U. Journal List 3 Biotech v. Published online Jun 8. Author information Article notes Copyright and License information Disclaimer.
Corresponding author. Received Sep 14; Accepted Apr 6. Introduction Cyclodextrin glycosyltransferase EC 2. Introduction of mutations by using overlap extension PCR Overlap extension PCR is a well-established PCR amplification method that can be used to introduce and recover mutations in specific DNA sequences without the requirement for restriction enzyme sites Angelaccio and di Patti Open in a separate window.
Enzyme activity assays The enzyme activity assays were conducted with soluble corn starch as a substrate and measured spectroscopically. Activity-pH profile The optimum pH of the purified enzymes was determined by replacing 0.
Thermal stability profile The thermal stability of the enzymes was measured by incubating 0. Compliance with ethical standards Conflict of interest All the authors declare that they have no conflicts of interest regarding this paper. Site-directed mutagenesis by the megaprimer PCR method: variations on a theme for simultaneous introduction of multiple mutations.
Anal Biochem. Factors controlling flavors binding constants to cyclodextrins and their applications in foods. Food Res Int. Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei. Enzymatic production of cyclodextrins.
Appl Microbiol Biot. Cyclomaltodextrin Glucanotransferase from Bacillus - Circulans E Action Patterns Biotechnol Appl Bioc. A novel cyclodextrin glycosyltransferase from an alkalophilic Bacillus species: purification and characterization.
Characterization of a thermostable cyclodextrin glucanotransferase isolated from Bacillus stearothermophilus ET1. J Agric Food Chem. Analysis of mutations in cyclodextrin glucanotransferase from Bacillus - stearothermophilus which affect cyclization characteristics and thermostability. J Bacteriol. Cyclization characteristics of cyclodextrin glucanotransferase are conferred by the nh2-terminal region of the enzyme.
Appl Environ Microb. Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS in batch, fed-batch, and continuous cultures. Biotechnol Prog. A predominant beta-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity.
J Mol Catal B-Enzym. Site-directed mutagenesis and functional analysis of maltose-binding site of beta-cyclodextrin glucanotransferase from Bacillus firmus var. Biotechnol Lett.
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To characterize this specificity we determined a 1. On this basis, a role of the individual residues during the cyclization reaction cycle is proposed. An especially interesting and unique family 13 enzyme is cyclodextrin glycosyltransferase CGTase. In addition, CGTase can perform hydrolysis very poorly and disproportionation reactions by using water or a free oligosaccharide as respective acceptors.