Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1

Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1

The 16s rRNA gene sequence from Bacillus sp. TS1-1 exhibited the closest match with Bacillus sp. NER (99%) and was identified as Bacillus sp. A cyclodextrin glucanotransferase (CGTase) gene of Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Beginning from the TTG codon, there was a...

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Main Author: Rahman, Kamalesh
Format: Thesis
Language:English
Published: 2005
Subjects:
TP Chemical technology
Online Access:http://eprints.utm.my/id/eprint/5094/1/KamaleshRahmanMFKKKSA2005.pdf
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spelling my-utm-ep.50942020-07-22T03:29:17Z Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1 2005-12 Rahman, Kamalesh TP Chemical technology The 16s rRNA gene sequence from Bacillus sp. TS1-1 exhibited the closest match with Bacillus sp. NER (99%) and was identified as Bacillus sp. A cyclodextrin glucanotransferase (CGTase) gene of Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Beginning from the TTG codon, there was an open reading frame composed of 2163bp (721 amino acids). The NH2 terminal position encoded a 46-amino acids signal peptide and followed by the mature enzyme of 675 amino acids. The deduced amino acid sequence of the mature CGTase from Bacillus sp. TS1-1 exhibited 98.7% homology with 96% identity to the CGTase sequence from alkalophilic Bacillus sp. 1-1. The recombinant CGTase of Bacillus sp. TS1-1 expressed in E.coli was successfully purified to homogeneity using ammonium sulfate precipitation, followed by a-cyclodextrin-bound-epoxy-activated Sepharose 6B affinity chromatography. The specific activity of the CGTase increased by approximately 280 fold, from 36.69 U/mg of proteins to 10289.23 U/mg of proteins. The purified CGTase enzymes exhibited a single band with molecular weight of 75kDa on SDS-PAGE. Biochemical characterization of the enzyme shows an optimum temperature of 60°C and optimum pH of 6.0. The enzyme was stable between pH 7 to pH 9 and temperature up to 70°C. The Km and Vmax values calculated were 0.52 mg/ml and 54.35 mg of ß-cyclodextrin/ml/min respectively. Sago starch was found to be the best substrate for cyclodextrin (CD) production among other starch sources (corn, rice, soluble and tapioca starch). Only β- and γ-CD were detected during the production of CDs. The CGTase produced about 86% of ß-CD from the total CDs production, using sago starch as substrate after 24 hours of incubation at 60oC, without adding any selective agent. The total ß-CD produced under the conditions mentioned above was 3.65 g/l. 2005-12 Thesis http://eprints.utm.my/id/eprint/5094/ http://eprints.utm.my/id/eprint/5094/1/KamaleshRahmanMFKKKSA2005.pdf application/pdf en public masters Universiti Teknologi Malaysia, Faculty of Chemical and Natural Resources Engineering Faculty of Chemical and Natural Resources Engineering
institution Universiti Teknologi Malaysia
collection UTM Institutional Repository
language English
topic TP Chemical technology
spellingShingle TP Chemical technology
Rahman, Kamalesh
Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1
description The 16s rRNA gene sequence from Bacillus sp. TS1-1 exhibited the closest match with Bacillus sp. NER (99%) and was identified as Bacillus sp. A cyclodextrin glucanotransferase (CGTase) gene of Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Beginning from the TTG codon, there was an open reading frame composed of 2163bp (721 amino acids). The NH2 terminal position encoded a 46-amino acids signal peptide and followed by the mature enzyme of 675 amino acids. The deduced amino acid sequence of the mature CGTase from Bacillus sp. TS1-1 exhibited 98.7% homology with 96% identity to the CGTase sequence from alkalophilic Bacillus sp. 1-1. The recombinant CGTase of Bacillus sp. TS1-1 expressed in E.coli was successfully purified to homogeneity using ammonium sulfate precipitation, followed by a-cyclodextrin-bound-epoxy-activated Sepharose 6B affinity chromatography. The specific activity of the CGTase increased by approximately 280 fold, from 36.69 U/mg of proteins to 10289.23 U/mg of proteins. The purified CGTase enzymes exhibited a single band with molecular weight of 75kDa on SDS-PAGE. Biochemical characterization of the enzyme shows an optimum temperature of 60°C and optimum pH of 6.0. The enzyme was stable between pH 7 to pH 9 and temperature up to 70°C. The Km and Vmax values calculated were 0.52 mg/ml and 54.35 mg of ß-cyclodextrin/ml/min respectively. Sago starch was found to be the best substrate for cyclodextrin (CD) production among other starch sources (corn, rice, soluble and tapioca starch). Only β- and γ-CD were detected during the production of CDs. The CGTase produced about 86% of ß-CD from the total CDs production, using sago starch as substrate after 24 hours of incubation at 60oC, without adding any selective agent. The total ß-CD produced under the conditions mentioned above was 3.65 g/l.
format Thesis
qualification_level Master's degree
author Rahman, Kamalesh
author_facet Rahman, Kamalesh
author_sort Rahman, Kamalesh
title Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1
title_short Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1
title_full Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1
title_fullStr Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1
title_full_unstemmed Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1
title_sort molecular and enzymatic studies of cyclodextrin glucanotransferase gene from bacillus sp. ts1-1
granting_institution Universiti Teknologi Malaysia, Faculty of Chemical and Natural Resources Engineering
granting_department Faculty of Chemical and Natural Resources Engineering
publishDate 2005
url http://eprints.utm.my/id/eprint/5094/1/KamaleshRahmanMFKKKSA2005.pdf
_version_ 1747814562229387264

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