Continuous hydrolysis of pullulan using covalently immobilized pullulanase in a packed bed reactor

Singh, Ram Sarup; Saini, Gaganpreet K.; Kennedy, John F.

doi:10.1016/j.carbpol.2010.08.037

Cited by 34 publications

(10 citation statements)

References 17 publications

(24 reference statements)

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“…Pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) is a kind of enzyme acting on branched substrates, generally used for hydrolysis of glycogen and amylopectin, by cleaving the α-1,6-glucosidic linkages in amylaceous polysaccharides [1-3], and hence belongs to the glycosyl hydrolases (GHase) family 13 that is also termed as the α-amylase family [4]. The most important industrial application of pullulanase is the production of glucose and maltose syrups from starch hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

Nie

Wang

et al. 2013

PLoS ONE

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Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)-pul and E. coli BL21(DE3)/pET-22b(+)-pul, were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)-pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)-pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)-pul. When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli.

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Section: Introductionmentioning

confidence: 99%

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

Nie

Wang

et al. 2013

PLoS ONE

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“…in many oral care products, for example, Listerine pocketpack oral care strips [13] and maltotriose syrup preparation [18][19][20]. Biomedical applications of pullulan include drug delivery, gene delivery [21,22], tissue engineering [23], vaccination [24,25], medical imaging [26], plasma expander [27], molecular chaperones [28], and film forming [29].…”

Section: Pullulan Is a Linear And Unbranched Exopolysaccharide Consismentioning

confidence: 99%

Biochemical and molecular characterization of a new pullulan producer Rhodosporidium paludigenum PUPY-06

2018

J App Biol Biotech

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Pullulan is a random coil glucan polymer produced by Aureobasidium pullulans. It is one of the commercially promising biopolymers frequently used in pharmaceutical, food and cosmetics industries. The aim of present study was to isolate an efficient fungal strain producing melanin-free pullulan. The fungal strains were isolated from fresh and decaying leaves of plants and screened for pullulan production. Fungal isolate PUPY-06 was the best producer of pullulan (2.12%, w/v). Morphological studies revealed the oval and cylindrical shape of its cells, budding and presence of septate as well as pseudohyphae. Colonies of the isolate on agar plate cultures were beige-to-orange colored with smooth, mucoid, and butyrous texture. Morphological and biochemical characterization established the fungal isolate as Rhodosporidium sp. 18s rRNA sequencing, and phylogenetic analysis revealed the isolate to be Rhodosporidium paludigenum. Fourier-transform infrared spectroscopy and nuclear magnetic resonance of crude pullulan confirmed its structural characteristics. Maximum production of pullulan was achieved after 7 days of cultivation in shake-flask fermentations. Agitation mode of cultivation supported the higher production of pullulan in comparison to the stationary mode. R. paludigenum PUPY-06 was found as a novel pullulan-producing strain. This is a first report on pullulan production by R. paludigenum PUPY-06.

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“…The pullulanase, as an important industrial enzyme, is widely employed in the combination with other amylolytic enzymes, such as the glucoamylase and a-amylase in the starch processing industry and the production of sugar syrups [14]. In addition, the pullulanase is used to improve the properties of starch materials, modify the functional properties of polysaccharides, prepare the maltotriose-rich syrup, and produce the low-calorie beers [15].…”

Section: Introductionmentioning

confidence: 99%

Debranching potato starch: synthesis, optimization and thermal property

Tang

Liu

et al. 2015

Polym. Bull.

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Pullulanase was used for the enzymatic hydrolysis of potato starch to increase the percentage of the amylose. Response surface methodology based on the Box-Behnken design was employed for the statistical optimization of the medium components for the hydrolysis of potato starch. The combined effects of four independent variables, i.e., the debranching temperature, debranching time, amount of pullulanase and pH were analyzed using a reduced cubic model, which was developed by Box-Behnken design in predicting the optimum debranching degree of potato starch. Under these conditions, the model predicted the best debranching environments: debranching temperature 52.79°C, debranching time 2.09 h, amount of pullulanase 58.16 U/g and pH 4.18. The differential scanning calorimeter (DSC), thermogravimetric analyzer (TGA) and Infrared spectrometer were used to evaluate the thermal properties, the structure of the native and debranching potato starch (DPS) so as to support the corresponding variation in the DPS. The debranching increased the onset temperature, peak temperature and end temperature of native potato starch, but decreased its melting enthalpy. The absorption peak intensity of the OH groups at wave number 3400 cm -1 in IR spectra was obviously strengthened after the native potato starch was debranched. It indicated that the amylose content in DPS was increased.

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Continuous hydrolysis of pullulan using covalently immobilized pullulanase in a packed bed reactor

Cited by 34 publications

References 17 publications

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

High-Level Expression of Bacillus naganoensis Pullulanase from Recombinant Escherichia coli with Auto-Induction: Effect of lac Operator

Biochemical and molecular characterization of a new pullulan producer Rhodosporidium paludigenum PUPY-06

Debranching potato starch: synthesis, optimization and thermal property

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