Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme

Rahman, Kamalesh; Illias, Rosli Md.; Hassan, Osman; Mahmood, Nik Hasnaa Nik; Rashid, Noor Aini Abdul

doi:10.1016/j.enzmictec.2005.09.014

Cited by 32 publications

(22 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In terms of β-CD production, 542 mg was produced in 100 mL reaction using 1% w/v gelatinized sagoo starch and at pH 6 is from Bacillus sp. TS1-1, G1 and 17-1 (Rahman et al 2006;Ong et al 2008;Kaneko et al 1989). rCGTase retained >50% of its activity at pHs 5.0-10.0, while native CGTase A2-5a retains 50% of its activity at pHs 9.0-10.0 (Komentani et al 1994).…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Characterization of Recombinant Cyclodextrin Glycosyltransferase from Bacillus sp. a2-5a using Sagoo Starch as Substrate

et al. 2012

View full text Add to dashboard Cite

Cyclodextrin (CD) is a cyclic oligosaccharide molecule and depending on the number of glucose molecules, three types of CDs are commonly used, α-CD, β-CD, and γ-CD. CDs can be produced enzymatically using starch as substrate catalyzed by CD glycosyltransferase (CGTase). In current research, recombinant CGTase production from the synthetic gene was optimized for its production using three growth media and two induction temperatures. The highest yield was obtained in Luria Bertani medium at 25 °C. The rCGTase protein was affinity purified as a 76.39 kDa protein which showed α-cyclization and starch hydrolysis activities using zymography method. The optimum temperature, pH and incubation time was 55 °C, 6, and 24 h, respectively. The enzyme was stable at a wide pHs in the range of 5-10, retained its half activity at 56 °C for 30 min and had cyclization ratio for α-CD : β-CD : γ-CD was 4 : 81 : 15. An amount of 542 mg β-cyclodextrin was produced from 100 mL reaction of 1% (b/v) sagoo starch using 38.4 μg rCGTase in optimum condition. This work reports for the first time the character of rCGTase from Bacillus sp. A2-5a using sagoo starch as a substrate

show abstract

Section: Discussionmentioning

confidence: 99%

Enzymatic Characterization of Recombinant Cyclodextrin Glycosyltransferase from Bacillus sp. a2-5a using Sagoo Starch as Substrate

et al. 2012

View full text Add to dashboard Cite

show abstract

“…The CGTases from various sources exhibit different Km values for soluble starch ranging from 0.52 to 21.2 mg/ml (Gawande and Patkar, 2001;Martins and Hatti-Kaul, 2002;Rahman et al, 2006;Stavn and Granum, 1979). Km value of CGTase from Thermoanaerobacter sp.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a thermostable cyclodextrin glycosyltransferase from Thermoanaerobacter sp. P4

Avcı¹,

Dönmez²

2012

Afr. J. Biotechnol.

View full text Add to dashboard Cite

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from a thermophilic anaerobic bacterium, Thermoanaerobacter sp. P4, was purified by ammonium sulfate precipitation followed by α-cyclodextrin epoxy activated-sepharose 6B column chromatography. Enzyme was purified 141 fold and had the specific activity of 143.8 U/mg proteins. Purification yields after ammonium sulfate precipitation and affinity chromatography were 25.8 and 17.8%, respectively. SDS-PAGE analysis showed that enzyme was purified successfully and had a single band. Molecular weight of the enzyme was determined as 68.7 kDa. The enzyme had optimum cyclization activity at 80 to 90°C and hydrolyzing activity at 90°C and maintained 87 and 95% of these activities at 95°C, respectively. Optimal pH was found as 7.0. It retained full activity at 80°C for 4 h. Enzyme was strongly inhibited by HgSO 4 and AgNO 3 . Addition of 1 mM CaCl 2 increased the enzymatic activity up to 7%. This novel enzyme could be a good candidate for industrial applications according to its characteristic found in the current study.

show abstract

“…Optimal temperature was determined by incubating the same amount of enzyme with 10% (w/v) pregelatinized soluble starch at different temperature from 30-100°C in pH 7. Reaction was performed using the same treatment represented in cyclization assay (Rahman et al, 2006).…”

Section: Zymography Assaymentioning

confidence: 99%

“…Assay of optimal pH and temperature: Optimal pH was determined by reacting 0.05 U of enzyme with 10% (w/v) pregelatinized soluble starch dissolved in several buffers with varying pH (Rahman et al, 2006). Optimal temperature was determined by incubating the same amount of enzyme with 10% (w/v) pregelatinized soluble starch at different temperature from 30-100°C in pH 7.…”

Section: Zymography Assaymentioning

confidence: 99%

“…The pH stability was determined by incubating 0.05 U enzyme in several buffers at different pH (Rahman et al, 2006) without substrate at 60°C for 30 min. Subsequently, the residual activity of the enzyme was measured after reacting 0.05 U enzyme mixture with 10% (w/v) pregelatinized soluble starch in 0.05 M TrisHCl buffer (pH 7) and incubated at 60°C for 30 min.…”

Section: Assay Of Ph and Temperature Stabilitymentioning

confidence: 99%

See 1 more Smart Citation

Y93F Substitution of Cyclodextrin Glucanotransferase from Bacillus sp. A2-5a and its Enzyme Characterization

Rostinawat¹,

Riani²,

Elfahmi³

et al. 2015

Biotechnology

View full text Add to dashboard Cite

A B S T R A C TIn this recent study, Tyr93 of Bacillus sp. A2-5a (BA2-5a) cyclodextrin glucanotransferase (CGTase) was replaced with Phe residue by site-directed mutagenesis to study its role on the product specificity and kinetic properties. Molecular docking approach was also applied to acquire a detailed analysis of enzyme-substrate interaction. The Y93F was overproduced in Escherichia coli BL21 (DE3) and purified by Ni-NTA resin. The purified Y93F CGTase was 76.39 kDa based on 10% SDS-PAGE analysis and showed both β-cyclization and starch hydrolysis activities in a zymography assay. There was no major structural conformational change in the Y93F since, its optimum and stability of temperature and pH were the same as the wild type. The substitution did not alter the cooperativity property of the enzyme. Our work provided the new evidence that Y93F substitution caused no α-cyclodextrin (CD) formation in BA2-5a CGTase. In addition, the Y93F produced more β-CD and decreased its k cat and k cat /K m . In conclusion, Y93F substitution of BA2-5a CGTase alters the enzyme kinetic property and product specificity.

show abstract

Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. TS1-1 and characterization of the recombinant enzyme

Cited by 32 publications

References 25 publications

Enzymatic Characterization of Recombinant Cyclodextrin Glycosyltransferase from Bacillus sp. a2-5a using Sagoo Starch as Substrate

Enzymatic Characterization of Recombinant Cyclodextrin Glycosyltransferase from Bacillus sp. a2-5a using Sagoo Starch as Substrate

Purification and characterization of a thermostable cyclodextrin glycosyltransferase from Thermoanaerobacter sp. P4

Y93F Substitution of Cyclodextrin Glucanotransferase from Bacillus sp. A2-5a and its Enzyme Characterization

Contact Info

Product

Resources

About