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

Ayadi, Dorra; Kammoun, Radhouane; Jemli, Sonia; Béjar, Samír

doi:10.2478/s11756-011-0122-2

Cited by 7 publications

(5 citation statements)

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“…Optimization of culture conditions yielded approximately eight‐fold increase in extracellular expression of recombinant active enzyme (Blamy‐I) as compared to its production under non‐optimized conditions. Similar observations were made by various researchers .…”

Section: Discussionsupporting

confidence: 91%

“…With the help of signal peptide, although E. coli cells were able to express recombinant α‐amylase outsides the cell membrane; however, a large fraction of it remained inside the cell. Furthermore, the influence of media components and cultivation parameters for the extracellular expression of recombinant proteins by E. coli are well recognized . Therefore, the four identified parameters viz, IPTG concentration, post induction time, incubation temperature and concentration of EDTA (Supporting Information Table S1), which were supposed to influence the extracellular expression of recombinant protein in E. coli , were optimized using our previous optimization techniques (Response Surface Methodology) for maximum extracellular expression of recombinant enzyme in culture media .…”

Section: Methodsmentioning

confidence: 99%

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Cloning and extracellular expression of a raw starch digesting α‐amylase (Blamy‐I) and its application in bioethanol production from a non‐conventional source of starch

Roy

Manhar

Nath

et al. 2015

J. Basic Microbiol.

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The aim of this study was to clone and efficiently express a raw starch-digesting α-amylase enzyme in the culture media and also to investigate the potential application of this recombinant enzyme in the digestion of non-conventional raw starch for bioethanol production. A raw starch digesting α-amylase gene isolated from Bacillus licheniformis strain AS08E was cloned and extracellularly expressed in E. coli cells using the native signal peptide. The mature recombinant α-amylase (Blamy-I) consisting of 483 amino acid residues was found to be homogenous with a mass of 55.3 kDa (by SDS-PAGE analysis) and a predicted pI of 6.05. Structural and functional analysis of Blamy-I revealed the presence of an extra Ca(2+) -binding region between the A and C domains responsible for higher thermostability of this enzyme. The statistical optimization of E. coli culture conditions resulted in an approximately eightfold increase in extracellular expression of Blamy-I as compared to its production under non-optimized conditions. Blamy-I demonstrated optimum enzyme activity at 80 °C and pH 10.0, and efficiently hydrolyzed raw starch isolated from a non-conventional, underutilized jack fruit seeds. Further utilization of this starch for bioethanol production using Blamy-I and Saccharomyces cerevisiae also proved to be highly promising.

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Section: Discussionsupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Cloning and extracellular expression of a raw starch digesting α‐amylase (Blamy‐I) and its application in bioethanol production from a non‐conventional source of starch

Roy

Manhar

Nath

et al. 2015

J. Basic Microbiol.

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“…NPST -10 10 , Bacillus lehensis 11 , Bacillus sp. A2-5a 12 , Paenibacillus pabuli US 132 13 and Thermoanaerobacter spp. P4 14 .…”

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confidence: 99%

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2014

IJPSR

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INTRODUCTION:Cyclodextrin glycosyl transferase (EC 2.4.1.9) is a unique enzyme capable of converting starch and related substrates into cyclomaltodextrins (CDs) 1 . CDs are cyclic oligosaccharides consisting of α-1, 4-linked 6, 7, or 8-glucopyranose units, usually referred to as α, β, or γ-CDs respectively.CDs possess a unique torus shape and the polar hydroxyl groups are oriented toward the outside, keeping the interior cavity relatively hydrophobic.Therefore, CDs are soluble in water and the hydrophobic environment of the cavity enables them to form inclusion complexes with many organic and inorganic molecules, thereby changing the physical and chemical properties of the included compounds. This is the basis of broad applications in the agriculture, textile, food, cosmetic, and pharmaceutical industries as well as in bioconversions and separation processes 2, 3, 4 .There are several known alkaliphilic Bacillus sp. that are able to produce CGTase.ABSTRACT: A Cyclodextrin glycosyltransferase (CGTase) producer was isolated from soil obtained from rice fields using alkaline Horikoshi II medium. The alkaliphile was characterized microscopically, biochemically and confirmed by 16S rRNA analysis as Bacillus oshimensis. CGTase production is dependent on the strain, medium composition and culture conditions; hence media optimization studies were carried out in shake flask using the statistical method. Tapioca starch was used as carbon source and a combination of yeast extract and peptone were used as nitrogen source in the media, along with MgSO 4 .7H 2 O, K 2 HPO 4 and Na 2 CO 3 . A 3 3 factorial design using Taguchi method has been chosen to elucidate the combined effect of these variables. The parameters were optimized by changing three independent variables, with fixed concentration of mineral sources. The optimal media composition for CGTase production was found to be comprised of Tapioca starch 1%; peptone 0.5%; yeast extract 0.5% and 0.14% Magnesium Sulphate, 0.2% ammonium dihydrogen phosphate according to the factorial designing of the experiment and was used for further studies. The CGTase activity was found to be 4.5 U/ml after media optimization. QUICK RESPONSE CODE

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“…In previous works, the conventional CGTase from Paenibacillus pabuli US132 (US132 CGTase) was characterized, cloned and overexpressed (Jemli et al 2008;Zouari Ayadi et al 2011). 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).…”

Section: Introductionmentioning

confidence: 99%

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

Cited by 7 publications

References 31 publications

Cloning and extracellular expression of a raw starch digesting α‐amylase (Blamy‐I) and its application in bioethanol production from a non‐conventional source of starch

Cloning and extracellular expression of a raw starch digesting α‐amylase (Blamy‐I) and its application in bioethanol production from a non‐conventional source of starch

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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

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