Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme
A cyclodextrin glucanotransferase (CGTase) gene from Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Starting from TTG codon, there was an open reading frame composed of 2163 bp (721 amino acids). The NH2 terminal position encoded a 46-amino acid of a signal peptide and followed by...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier Inc.
2006
|
| Subjects: | |
| Online Access: | http://eprints.utm.my/168/1/EMTNAR2006.pdf |
| _version_ | 1796853039822274560 |
|---|---|
| author | Rahman, Kamalesh Mohd Illias, Rosli Hassan, Osman Nik Mahmood, Nik Azmi Abdul Rashid, Noor Aini |
| author_facet | Rahman, Kamalesh Mohd Illias, Rosli Hassan, Osman Nik Mahmood, Nik Azmi Abdul Rashid, Noor Aini |
| author_sort | Rahman, Kamalesh |
| collection | ePrints |
| description | A cyclodextrin glucanotransferase (CGTase) gene from Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Starting from TTG codon, there was an open reading frame composed of 2163 bp (721 amino acids). The NH2 terminal position encoded a 46-amino acid of a signal peptide and followed by the mature enzyme (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 alpha-cyclodextrin-bound epoxy-
activated Sepharose 6B affinity chromatography. The purified CGTase enzymes exhibited a single band with molecular weight of 75 kDa on SDS-PAGE. Biochemical characterization of the enzyme shows an optimum temperature of 60 â—¦C and optimum pH of 6.0. The enzyme was stable between pH 7 and 9 and temperature up to 70 â—¦C. The Km and Vmax values calculated were 0.52 mg/ml and 54.35 mg of beta-cyclodextrin/ml/min. The yield of the products from soluble starch as the substrate were 86% for gamma-cyclodextrin and 14% for -cyclodextrin after 24 h incubation at 60 â—¦C, without adding any selective agent. The total beta-CD produced under the conditions mentioned above was 3.65 g/l. |
| first_indexed | 2024-03-05T17:53:43Z |
| format | Article |
| id | utm.eprints-168 |
| institution | Universiti Teknologi Malaysia - ePrints |
| language | English |
| last_indexed | 2024-03-05T17:53:43Z |
| publishDate | 2006 |
| publisher | Elsevier Inc. |
| record_format | dspace |
| spelling | utm.eprints-1682010-06-01T02:41:06Z http://eprints.utm.my/168/ Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme Rahman, Kamalesh Mohd Illias, Rosli Hassan, Osman Nik Mahmood, Nik Azmi Abdul Rashid, Noor Aini Q Science (General) A cyclodextrin glucanotransferase (CGTase) gene from Bacillus sp. TS1-1 was isolated and cloned into Escherichia coli. Starting from TTG codon, there was an open reading frame composed of 2163 bp (721 amino acids). The NH2 terminal position encoded a 46-amino acid of a signal peptide and followed by the mature enzyme (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 alpha-cyclodextrin-bound epoxy- activated Sepharose 6B affinity chromatography. The purified CGTase enzymes exhibited a single band with molecular weight of 75 kDa on SDS-PAGE. Biochemical characterization of the enzyme shows an optimum temperature of 60 â—¦C and optimum pH of 6.0. The enzyme was stable between pH 7 and 9 and temperature up to 70 â—¦C. The Km and Vmax values calculated were 0.52 mg/ml and 54.35 mg of beta-cyclodextrin/ml/min. The yield of the products from soluble starch as the substrate were 86% for gamma-cyclodextrin and 14% for -cyclodextrin after 24 h incubation at 60 â—¦C, without adding any selective agent. The total beta-CD produced under the conditions mentioned above was 3.65 g/l. Elsevier Inc. 2006-02 Article PeerReviewed application/pdf en http://eprints.utm.my/168/1/EMTNAR2006.pdf Rahman, Kamalesh and Mohd Illias, Rosli and Hassan, Osman and Nik Mahmood, Nik Azmi and Abdul Rashid, Noor Aini (2006) Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme. Enzyme and Microbial Technology, 39 . pp. 74-84. ISSN 0141-0229 http://dx.doi.org/10.1016/j.enzmictec.2005.09.014 10.1016/j.enzmictec.2005.09.014 |
| spellingShingle | Q Science (General) Rahman, Kamalesh Mohd Illias, Rosli Hassan, Osman Nik Mahmood, Nik Azmi Abdul Rashid, Noor Aini Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme |
| title | Molecular cloning of a cyclodextrin glucanotransferase gene
from alkalophilic Bacillus sp. TS1-1 and characterization
of the recombinant enzyme |
| title_full | Molecular cloning of a cyclodextrin glucanotransferase gene
from alkalophilic Bacillus sp. TS1-1 and characterization
of the recombinant enzyme |
| title_fullStr | Molecular cloning of a cyclodextrin glucanotransferase gene
from alkalophilic Bacillus sp. TS1-1 and characterization
of the recombinant enzyme |
| title_full_unstemmed | Molecular cloning of a cyclodextrin glucanotransferase gene
from alkalophilic Bacillus sp. TS1-1 and characterization
of the recombinant enzyme |
| title_short | Molecular cloning of a cyclodextrin glucanotransferase gene
from alkalophilic Bacillus sp. TS1-1 and characterization
of the recombinant enzyme |
| title_sort | molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic bacillus sp ts1 1 and characterization of the recombinant enzyme |
| topic | Q Science (General) |
| url | http://eprints.utm.my/168/1/EMTNAR2006.pdf |
| work_keys_str_mv | AT rahmankamalesh molecularcloningofacyclodextringlucanotransferasegenefromalkalophilicbacillusspts11andcharacterizationoftherecombinantenzyme AT mohdilliasrosli molecularcloningofacyclodextringlucanotransferasegenefromalkalophilicbacillusspts11andcharacterizationoftherecombinantenzyme AT hassanosman molecularcloningofacyclodextringlucanotransferasegenefromalkalophilicbacillusspts11andcharacterizationoftherecombinantenzyme AT nikmahmoodnikazmi molecularcloningofacyclodextringlucanotransferasegenefromalkalophilicbacillusspts11andcharacterizationoftherecombinantenzyme AT abdulrashidnooraini molecularcloningofacyclodextringlucanotransferasegenefromalkalophilicbacillusspts11andcharacterizationoftherecombinantenzyme |