CYCLODEXTRIN GLYCOSYLTRANSFERASE PDF

The distances associated with the interactions are in Table III. Symm rel. It can be judged from the experimentally determined atomic B-factors. This indicates that the best hydrophobic stacking interactions 34 are made to Phe Furthermore, the glucose O6 atom is pointing toward Tyr, while the sugar O3 atom forms a good 2.

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This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC. Abstract The gene encoding the cyclodextrin glycosyltransferase CGTase of Paenibacillus pabuli US, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli.

Sequence analysis showed that the mature enzyme amino acids was preceded by a signal peptide of 34 residues. They have torus-shaped structures able to encapsulate a wide range of molecules, thereby modifying their physical and chemical properties.

Consequently, CDs are extensively used in pharmaceutical, chemical, agricultural, cosmetic, and food industries [ 1 , 2 ]. Domains A and B form the catalytic core, domains C and E are involved in substrate binding whereas domain D has unknown precise function [ 6 , 7 ].

Understanding the enzymatic features has been explored by cloning, sequencing, and comparing various cgtase genes [ 8 — 11 ]. The sequence data analysis has revealed the key amino acids residues that determine the reaction mechanism and product specificity, thereby enhancing genetic engineering techniques to provide modified CGTases [ 7 , 12 , 13 ].

However, the formation of inclusion bodies remains a significant barrier for expression of heterologous proteins in E. Refolding of inclusion bodies into soluble and active form needs high cost and tedious jobs.

Hence, maximizing the yield of soluble and active recombinant proteins in vivo by altering the culture conditions is an attractive alternative [ 14 ]. However, no universal approach has been established to minimize the formation of inclusion bodies and some empirical conditions must be screened on an individual basis.

To date, there are only a few reports that succeeded the overproduction of CGTase in E. In a previous study, the CGTase of Paenibacillus pabuli US was reported as good candidate for cyclodextrins production and efficient antistaling agent [ 18 ]. In this work, we described the molecular cloning of the US cgtase gene as well as the amino acid sequence inspection and the comparison with other related CGTases.

We also reported the production enhancement of the recombinant active enzyme in E. Bacterial strains, plasmids, and culture media Paenibacillus pabuli US strain, previously isolated [ 18 ], was used as source of chromosomal DNA. Paenibacillus pabuli US strain was grown as previously described [ 18 ]. Nucleotide sequences of the US cgtase gene carried by three independent pSJ8 plasmids obtained from different PCR reactions were determined.

For the promoter determination, the prokaryotic promoter prediction program NNPP2. All experiments were performed at least twice. The fermentor was inoculated, with an initial OD nm of 0. The pH, aeration and agitation were maintained constant at 7. The CGTase extract was prepared from the periplasm by the modified osmotic-shock procedure of Ausubel et al. The supernatant was recovered as periplasmic fraction and used for CGTase assay. The supernatant was then purified using hydrophobic interaction chromatography and starch adsorption as previously described for the native enzyme [ 18 ].

Samples were taken at regular time intervals and the reaction was stopped by boiling for 5 minutes. The initial speed was calculated for each substrate concentration and then represented according to the Lineweaver-Burk method [ 24 ].

Identification of the P. The cloning of this fragment in pCR2. The nucleotide sequence analysis of the fragment containing the US cgtase gene revealed the presence of a single open reading frame ORF with two potential initiation ATG codons Figure 1.

Consequently, this latter is most likely the true initiator codon. Therefore, the mature enzyme was consisted of amino acids with an estimated molecular mass of about

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Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC. Abstract The gene encoding the cyclodextrin glycosyltransferase CGTase of Paenibacillus pabuli US, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme amino acids was preceded by a signal peptide of 34 residues. They have torus-shaped structures able to encapsulate a wide range of molecules, thereby modifying their physical and chemical properties. Consequently, CDs are extensively used in pharmaceutical, chemical, agricultural, cosmetic, and food industries [ 1 , 2 ]. Domains A and B form the catalytic core, domains C and E are involved in substrate binding whereas domain D has unknown precise function [ 6 , 7 ].

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Cyclodextrin glycosyltransferase

CGTase from E. When the CGTase produced by B. Subtle differences in the conformation of the CGTase produced by E. Keywords: Cyclodextrin glycosyltransferase; expression; Bacillus macerans; Bacillus subtilis; Escherichia coli; conformation Cited By This article is cited by 35 publications. Journal of Agricultural and Food Chemistry , 65 11 , DOI:

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Help Wikipedia improve by adding precise citations! This peculiar enzyme is capable of catalyzing more than one reaction with the most important being the synthesis of non-reducing cyclic dextrins known as cyclodextrins starting from starch , amylose , and other polysaccharides. CGTase is an enzyme common to many bacterial species, in particular of the Bacillus genus e. Catalytic activities[ edit ] All of the CGTases can catalyze up to four reactions: cyclization, coupling, disproportionation and hydrolysis. All these activities share the same catalytic mechanism which is common to all glycosyl-hydrolases. The coupling reaction can be easily described as the reverse process of cyclization: the enzyme cleaves a cyclodextrin to produce a linear dextrin which is subsequently joined to a linear oligosaccharide.

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