Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1

Ho Kek Sian, Mamot Said, Osman Hassan, Kamarulzaman Kamaruddin, A. Fauzi Ismail, Roshanida A. Rahman, Nik Azmi Nik Mahmood, Rosli Md Illias

Research output: Contribution to journalArticle

49 Citations (Scopus)

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 languageEnglish
Pages (from-to)1101-1111
Number of pages11
JournalProcess Biochemistry
Volume40
Issue number3-4
DOIs
Publication statusPublished - Mar 2005

Fingerprint

Cyclodextrins
Bacilli
Bacillus
Purification
Enzymes
Starch
Proteins
Molecular Weight
Molecular weight
Affinity chromatography
Manihot
Isoelectric Point
Ammonium Sulfate
Substrates
cyclomaltodextrin glucanotransferase
Affinity Chromatography
Edetic Acid
Sepharose
Gel Chromatography
Ethylenediaminetetraacetic acid

Keywords

  • Cyclodextrin
  • Cyclodextrin glucanotransferase

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Engineering (miscellaneous)
  • Industrial and Manufacturing Engineering

Cite this

Sian, H. K., Said, M., Hassan, O., Kamaruddin, K., Ismail, A. F., Rahman, R. A., ... Illias, R. M. (2005). Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1. Process Biochemistry, 40(3-4), 1101-1111. https://doi.org/10.1016/j.procbio.2004.03.018

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 journalArticle

Sian, HK, Said, M, Hassan, O, Kamaruddin, K, Ismail, AF, Rahman, RA, Mahmood, NAN & Illias, RM 2005, 'Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1', Process Biochemistry, vol. 40, no. 3-4, pp. 1101-1111. https://doi.org/10.1016/j.procbio.2004.03.018
Sian, Ho Kek ; Said, Mamot ; Hassan, Osman ; Kamaruddin, Kamarulzaman ; Ismail, A. Fauzi ; Rahman, Roshanida A. ; Mahmood, Nik Azmi Nik ; Illias, Rosli Md. / Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1. In: Process Biochemistry. 2005 ; Vol. 40, No. 3-4. pp. 1101-1111.
@article{97227f3f29ff4d1f98ab832dfbc209cb,
title = "Purification and characterization of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. G1",
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.",
keywords = "Cyclodextrin, Cyclodextrin glucanotransferase",
author = "Sian, {Ho Kek} and Mamot Said and Osman Hassan and Kamarulzaman Kamaruddin and Ismail, {A. Fauzi} and Rahman, {Roshanida A.} and Mahmood, {Nik Azmi Nik} and Illias, {Rosli Md}",
year = "2005",
month = "3",
doi = "10.1016/j.procbio.2004.03.018",
language = "English",
volume = "40",
pages = "1101--1111",
journal = "Process Biochemistry",
issn = "0032-9592",
publisher = "Elsevier BV",
number = "3-4",

}

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

AN - SCOPUS:9644270574

VL - 40

SP - 1101

EP - 1111

JO - Process Biochemistry

JF - Process Biochemistry

SN - 0032-9592

IS - 3-4

ER -