A β-cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: Purification, properties and potential use in bread-making

Abstract:The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24• C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).

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“…Bacterial strains, plasmids and media Paenibacillus pabuli US132 strain (Jemli et al 2007) was used in this work.…”

Section: Methodsmentioning

confidence: 99%

“…• C for 10 min as described previously (Jemli et al 2007). One activity unit was defined as the amount of enzyme able to decrease 10% of amylose-iodine complex optical density per min under the assay conditions.…”

Section: Cgtase Enzyme Assaymentioning

confidence: 99%

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

et al. 2011

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“…The dextrinization activity of the CGTase was monitored by measuring the decrease in absorbance (blue value) of iodineamylose complex as described in a previous work (Jemli et al 2007). The cyclization activity was measured as β-CD forming activity according to the method reported by Volkova et al (2000).…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…In addition, the enzyme has been reported as a potential good candidate for application as an anti-staling agent particularly because of its action on bread firmness (Jemli et al 2007). This study aims to investigate the possibility of decreasing the cyclization activity of the US132 CGTase so as to provide it with an antistaling property comparable to that of the commercial α-amylase, Novamyl.…”

Section: Introductionmentioning

confidence: 99%

“…Among these enzymes, the maltogenic α-amylase of Geobacillus stearothermophilus (commercially available as Novamyl) is widely used in the baking industry to improve the shelf-life of bread (Olesen 1991). CGTases could also be used as anti-staling enzymes since they decrease the firmness of bread (Jemli et al 2007). In addition, the increase of the hydrolytic activity of CGTase, by random or site-directed mutagenesis, makes the enzyme more useful for the baking industry (Lee et al 2002;Shim et al 2004).…”

Section: Introductionmentioning

confidence: 99%

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Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

et al. 2012

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Abstract:The cyclodextrin glucanotransferase from Paenibacillus pabuli US132 (US132 CGTase) was engineered using a rational approach in an attempt to provide it with anti-staling properties comparable to those of the commercial maltogenic amylase (Novamyl). The study aimed to concurrently decrease the cyclization activity and increase the hydrolytic activity of US132 CGTase. A five-residue loop (PAGFS) was inserted, alone or with the substitution of essential residues for cyclization (G180, L194 and Y195), mimicking the case of Novamyl. The findings indicate that, unlike the case of the CGTase of Thermoanerobacterium thermosulfurigenes strain EM1 whose initial high hydrolytic activity was exceptional, these mutations completely abolished the cyclization and hydrolytic activities of the US132 CGTase. This suggests that those mutations are not able to convert conventional CGTases, whose hydrolytic activities are very weak, into hydrolases. Accordingly, and for the first time, a structural barrier at subsite −3 was advanced as an influential factor which might explain the low hydrolytic activity of conventional CGTases.

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Enzymatic Polysaccharide Degradation

Munteanu¹,

Ritter²

2010

Biocatalysis in Polymer Chemistry

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A β-cyclodextrin glycosyltransferase from a newly isolated Paenibacillus pabuli US132 strain: Purification, properties and potential use in bread-making

Cited by 47 publications

References 26 publications

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

Enzymatic Polysaccharide Degradation

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