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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| Format: | Thesis |
| Language: | English |
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2005
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| Online Access: | http://eprints.utm.my/5094/ http://eprints.utm.my/5094/1/KamaleshRahmanMFKKKSA2005.pdf |
| _version_ | 1848890970054066176 |
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| author | Rahman, Kamalesh |
| author_facet | Rahman, Kamalesh |
| author_sort | Rahman, Kamalesh |
| building | UTeM Institutional Repository |
| collection | Online Access |
| 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. |
| first_indexed | 2025-11-15T20:50:31Z |
| format | Thesis |
| id | utm-5094 |
| institution | Universiti Teknologi Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T20:50:31Z |
| publishDate | 2005 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | utm-50942020-07-22T03:29:17Z http://eprints.utm.my/5094/ Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1 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 NonPeerReviewed application/pdf en http://eprints.utm.my/5094/1/KamaleshRahmanMFKKKSA2005.pdf Rahman, Kamalesh (2005) Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1. Masters thesis, Universiti Teknologi Malaysia, Faculty of Chemical and Natural Resources Engineering. |
| spellingShingle | TP Chemical technology Rahman, Kamalesh Molecular and enzymatic studies of cyclodextrin glucanotransferase gene from Bacillus sp. TS1-1 |
| title | 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_short | 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 |
| topic | TP Chemical technology |
| url | http://eprints.utm.my/5094/ http://eprints.utm.my/5094/1/KamaleshRahmanMFKKKSA2005.pdf |