Abstract
A cyclodextrin glucanotransferase (CGTase) was successively purified by ammonium sulphate precipitation, and affinity chromatography on α-CD (epoxy)-Sepharose 6B column. The specific activity of the CGTase was increased approximately 2200-fold, from 8.43 U/mg protein to 18,866 U/mg protein. SDS-PAGE showed that the purified CGTase was homogeneous and the molecular weight of the purified CGTase was about 75 kDa. The molecular weight of the enzyme that was estimated by gel filtration under native condition was 79 kDa. This has indicated that Bacillus sp. G1 CGTase is a monomeric protein. The isoelectric point (pI) of the enzyme was about 8.8. Characterization of the enzyme exhibited optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme was stable from pH 7.0 to 9.0 and retained its high activity up to 60°C. However, in the presence of 20 mM Ca 2+, the purified CGTase is able to prolong its thermal stability up to 70°C. CGTase was strongly inhibited by ZnSO 4, CuSO 4, CoCl 2, FeSO 4, FeCl 3 and EDTA. K m and V max for the purified enzyme were 0.15 mg/ml and 60.39 mg β-cyclodextrin/(ml min), respectively, with soluble starch as substrate. In cyclodextrin production, tapioca starch was found to be the best substrate used to produce CDs. The enzyme produced γ- and β-CD in the ratio of 0.11:0.89 after 24 h incubation at 60°C, without the presence of any selective agents.
| Original language | English |
|---|---|
| Pages (from-to) | 1101-1111 |
| Number of pages | 11 |
| Journal | Process Biochemistry |
| Volume | 40 |
| Issue number | 3-4 |
| DOIs | |
| Publication status | Published - Mar 2005 |
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Keywords
- Cyclodextrin
- Cyclodextrin glucanotransferase
ASJC Scopus subject areas
- Biochemistry
- Organic Chemistry
- Engineering (miscellaneous)
- Industrial and Manufacturing Engineering
Cite this
Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1. / Sian, Ho Kek; Said, Mamot; Hassan, Osman; Kamaruddin, Kamarulzaman; Ismail, A. Fauzi; Rahman, Roshanida A.; Mahmood, Nik Azmi Nik; Illias, Rosli Md.
In: Process Biochemistry, Vol. 40, No. 3-4, 03.2005, p. 1101-1111.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1
AU - Sian, Ho Kek
AU - Said, Mamot
AU - Hassan, Osman
AU - Kamaruddin, Kamarulzaman
AU - Ismail, A. Fauzi
AU - Rahman, Roshanida A.
AU - Mahmood, Nik Azmi Nik
AU - Illias, Rosli Md
PY - 2005/3
Y1 - 2005/3
N2 - A cyclodextrin glucanotransferase (CGTase) was successively purified by ammonium sulphate precipitation, and affinity chromatography on α-CD (epoxy)-Sepharose 6B column. The specific activity of the CGTase was increased approximately 2200-fold, from 8.43 U/mg protein to 18,866 U/mg protein. SDS-PAGE showed that the purified CGTase was homogeneous and the molecular weight of the purified CGTase was about 75 kDa. The molecular weight of the enzyme that was estimated by gel filtration under native condition was 79 kDa. This has indicated that Bacillus sp. G1 CGTase is a monomeric protein. The isoelectric point (pI) of the enzyme was about 8.8. Characterization of the enzyme exhibited optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme was stable from pH 7.0 to 9.0 and retained its high activity up to 60°C. However, in the presence of 20 mM Ca 2+, the purified CGTase is able to prolong its thermal stability up to 70°C. CGTase was strongly inhibited by ZnSO 4, CuSO 4, CoCl 2, FeSO 4, FeCl 3 and EDTA. K m and V max for the purified enzyme were 0.15 mg/ml and 60.39 mg β-cyclodextrin/(ml min), respectively, with soluble starch as substrate. In cyclodextrin production, tapioca starch was found to be the best substrate used to produce CDs. The enzyme produced γ- and β-CD in the ratio of 0.11:0.89 after 24 h incubation at 60°C, without the presence of any selective agents.
AB - A cyclodextrin glucanotransferase (CGTase) was successively purified by ammonium sulphate precipitation, and affinity chromatography on α-CD (epoxy)-Sepharose 6B column. The specific activity of the CGTase was increased approximately 2200-fold, from 8.43 U/mg protein to 18,866 U/mg protein. SDS-PAGE showed that the purified CGTase was homogeneous and the molecular weight of the purified CGTase was about 75 kDa. The molecular weight of the enzyme that was estimated by gel filtration under native condition was 79 kDa. This has indicated that Bacillus sp. G1 CGTase is a monomeric protein. The isoelectric point (pI) of the enzyme was about 8.8. Characterization of the enzyme exhibited optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme was stable from pH 7.0 to 9.0 and retained its high activity up to 60°C. However, in the presence of 20 mM Ca 2+, the purified CGTase is able to prolong its thermal stability up to 70°C. CGTase was strongly inhibited by ZnSO 4, CuSO 4, CoCl 2, FeSO 4, FeCl 3 and EDTA. K m and V max for the purified enzyme were 0.15 mg/ml and 60.39 mg β-cyclodextrin/(ml min), respectively, with soluble starch as substrate. In cyclodextrin production, tapioca starch was found to be the best substrate used to produce CDs. The enzyme produced γ- and β-CD in the ratio of 0.11:0.89 after 24 h incubation at 60°C, without the presence of any selective agents.
KW - Cyclodextrin
KW - Cyclodextrin glucanotransferase
UR - http://www.scopus.com/inward/record.url?scp=9644270574&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=9644270574&partnerID=8YFLogxK
U2 - 10.1016/j.procbio.2004.03.018
DO - 10.1016/j.procbio.2004.03.018
M3 - Article
VL - 40
SP - 1101
EP - 1111
JO - Process Biochemistry
JF - Process Biochemistry
SN - 0032-9592
IS - 3-4
ER -