Production Of Pullulanase Research Articles

Production of extracellular Pullulanase by NP 7 bacteria using OFAT methodology

Pullulanase is a debranching enzyme that spilts explicitly α-1,6 glycosidic bonds. It is widely used in starch saccharification, in production of glucose, fructose as well as maltotriose syrup and bioethanol production. Pullulanase is produced by submerged fermentation process that requires optimization of physical and chemical parameters to achieve higher activity and make this process cost effective. The present study aimed the optimization of physical as well as chemical parameters of production medium. The optimization includes physical parameters such as inoculum size, incubation time, temperature, pH and agitation rate while chemical parameters include carbon source, nitrogen source and different metal ions. From the optimization study, it was found that highest pullulanase production was achieved after 48 hours of incubation at 45ºC temperature, pH 7 and 120 rpm. Optimization of chemical parameters reveals that incorporation of starch (1%) and 0.2mMole/L of calcium chloride in the media gave significant increase in enzyme activity. The maximum productivity achieved after optimization was 139.33 U/ml using OFAT method which was four times higher.

Production, characterization, and application of novel fungal pullulanase for fruit juice processing

The present study aimed to produce, characterize, and apply pullulanase from Aspergillus flavus (BHU-46) for fruit juice processing, assessing its enzymatic properties and impact on juice quality. Pullulanase was produced via solid-state fermentation using wheat bran as the substrate. Purification and characterization included specific activity, molecular weight, pH and temperature optima, and substrate specificity. The enzyme was immobilized in sodium alginate beads and used for clarifying mosambi, apple, and mango juices. Parameters such as yield, clarity, reducing sugar, total soluble solids (TSS), total phenol, and enzymatic browning were evaluated pre-and post-treatment. The purified pullulanase had a specific activity of 652.2 U/mg and a molecular weight of 135 kDa. Optimal pH values were 6.5 and 10, with maximum activity at 50 °C. Pullulanase showed a high affinity for pullulan and starch, indicating Pullulanase type II classification. Immobilized pullulanase improved yield, clarity, reducing sugar, TSS, and total phenol in fruit juices. The highest yield and clarity were observed in mosambi juice. Additionally, the enzyme reduced enzymatic browning, increasing the lightness of the juice. This study provides a significant contribution to the juice processing industry and represents the first report on the application of pullulanase for fruit juice processing.

Pullulanase: unleashing the power of enzyme with a promising future in the food industry.

Pullulanases are the most important industrial group of enzymes in family 13 glycosyl hydrolases. They hydrolyze either α-1,6 and α-1,4 or both glycosidic bonds in pullulan as well as other carbohydrates to produce glucose, maltose, and maltotriose syrups, which have important uses in food and other related sectors. However, very less reports are available on pullulanase production from native strains because of low yield issues. In line with the increasing demands for pullulanase, it has become important to search for novel pullulanase-producing microorganisms with high yields. Moreover, high production costs and low yield are major limitations in the industrial production of pullulanase enzymes. The production cost of pullulanase by using the solid-state fermentation (SSF) process can be minimized by selecting agro-industrial waste. This review summarizes the types, sources, production strategies, and potential applications of pullulanase in different food and other related industries. Researchers should focus on fungal strains producing pullulanase for better yield and low production costs by using agro-waste. It will prove a better enzyme in different food processing industries and will surely reduce the cost of products.

Screening of Nutrients for Enrichment of Extracellular Pullulanase Production by Isolated Bacillus cereus KKSJ1981 Using Plackett–Burman Design

Screening of Nutrients for Enrichment of Extracellular Pullulanase Production by Isolated Bacillus cereus KKSJ1981 Using Plackett–Burman Design

Autoinduction AND Gate Inhibits Cell Lysis to Enhance Protein Production in Bacillus subtilis Controlled by Population Density and Cell Physiological State.

The extracellular protease-deficient strain Bacillus subtilis WB600 is commonly used as a chassis cell for the production of industrial proteins. However, B.subtilis WB600 exhibits an increased susceptibility to cell lysis and a reduction in biomass. Inhibition of cell lysis by knocking out lytic genes will impair physiological function. Here, we dynamically inhibited cell lysis in B.subtilis WB600 to balance the impairment of physiological function with the accumulation of biomass. First, the inducible protein degradation systems (IPDSs) were constructed and used to investigate the effects of inhibiting cell lysis on biomass, cell morphology, and protein production at different times (using pullulanase as a test). The highest pullulanase activity was obtained at 20 h of inhibiting cell lysis, 184.8 U/mL, which was 44% higher than the activity of B.subtilis WB600. Then, to avoid addition of inducers, we introduced orthogonal quorum sensing and constructed autoinduction protein degradation systems (AIPDSs). The optimized AIPDS showed similar pullulanase activity to the optimal IPDS (20 h), 181.3 U/mL. Next, we constructed dual-signal input autoinduction protein degradation systems (DSI-AIPDSs) via AND gate to further address two deficiencies of AIPDS, one-time activation and damage to new cells. These DSI-AIPDSs were controlled by quorum sensing and stationary phase promoters that respond to population density and single-cell physiological state, respectively. Finally, the OD600 and pullulanase activity of the strain with optimal DSI-AIPDS were 51% and 115% higher than those of B.subtilis WB600 in pullulanase production, respectively. We provided a B.subtilis chassis strain with considerable potential for biomass accumulation and enhanced protein production.

Advances in heterologous expression, structural elucidation and molecular modification of pullulanase

Starch is composed of glucose units linked by α-1, 4-glucoside bond and α-1, 6-glucoside bond. It is the main component of foods and the primary raw material for starch processing industry. Pullulanase can effectively hydrolyze the α-1, 6-glucoside bond in starch molecules. Combined with other starch processing enzymes, it can effectively improve the starch utilization rate. Therefore, it has been widely used in the starch processing industry. This paper summarized the screening of pullulanase-producing strain and its encoding genes. In addition, the effects of expression elements and fermentation conditions on the production of pullulanase were summarized. Moreover, the progress in crystal structure elucidation and molecular modification of pullulanase was discussed. Lastly, future perspectives on pullulanase research were proposed.

Production and Optimization of Pullulanase by Stenotrophomonas maltophilia under Submerged Fermentation Using Response Surface Methodology (RSM)

Pullulanase is a starch debranching enzyme, that cleaves the α-1,6, glucosidic linkage. The research investigates the screening, production, and optimization of pullulanase from bacterial isolates. Five different soil samples were collected from Choba (River State) and Oshodi (Lagos State). The total culturable heterotrophic bacterial counts ranged from 2.21x10⁶ to 3.2 x 10⁶Cfu/ml. A total of eight bacterial isolates exhibited the potential to degrade pullulan which produced a clear halo zone at 70% around the colonies. The isolate with the highest pullulanase degradation was further investigated for production using wheat bran as substrate under submerged fermentation (SmF) applying RSM. Identification and characterization of the isolate were done using both conventional and molecular techniques. This isolate was identified molecularly as Stenotrophomonas maltophilia. The 16S rRNA sequence was deposited in GenBank under accession number MH225448.1. The optimum production of pullulanase obtained using One-factor-at-a-time (OFAT) was achieved with wheat bran (2.469U/ml) at 48h, pH 7 and temperature of 37⁰C. Optimization using RSM revealed the following optimum conditions; temperature 36ºC, pH 7.5, incubation period of 36h, and pullulan concentration of 1.5%. The result gathered from this study showed that the bacterial isolate can successfully utilize agro-waste materials for production of pullulanase. This poses great potential in ameliorating the environmental pollution arising from their poor disposal as well as encouraging the production of the enzyme at a low cost. The data from this study also makes the isolate a good candidate for the production of pullulanase for industrial purposes.

Properties of a neutral, thermally stable and surfactant-tolerant pullulanase from worker termite gut-dwelling Bacillus safensis as potential for industrial applications

The gut of termite has been observed to host communities of bacteria which exhibited pullulan-degrading ability. Bacillus safensis displayed maximum pullulanase (a debranching enzyme) activity and it was therefore selected for production, purification and characterization of pullulanase which was the aim of the study. The crude enzyme obtained from the pullulanase production medium was subjected to ammonium sulphate precipitation, ion exchange and gel-filtration chromatography and the physicochemical properties of the purified was thereafter characterized. A purified pullulanase with the yield of 13% and 24-fold purification was obtained and its homogeneity was established by molecular weight of 42 kDa. The optimum pH 7 and 60 °C were obtained while the enzyme was stable between 40-60 °C and pH 4–5 and 7–8 respectively with significant amount of residual activities recorded. The purified pullulanase was stimulated in the presence of Ca2+, urea and SDS while Al3+, Fe2+, Co2+, Cu2+, Mg2+ and chelating agent, EDTA mildly inhibited the activity of the enzyme in a concentration-dependent manner. The Km and Vmax were found to be 0.324 μmol/ml/min and 6.85 mg/ml respectively. The exceptional physicochemical properties of B. safensis pullulanase could find application in several industrial processes.

Agro-waste as a substrate for the production of pullulanase by Penicillium viridicatum under solid-state fermentation

One of the key enzymes utilized in the food industry is pullulanase. But its major drawbacks are its low yield and high production costs. In this regard, the current research aims to screen agro-waste substrates for optimal pullulanase production in solid-state fermentation. Of various agro-wastes used as a substrate, the maximum enzymic activity (9.74 U/gds) was observed in a medium based on 5 g of green gram husk and incubated for 3 days at 30 °C. The effects of 16 different nutrients on the yield of pullulanase production were studied using the Plackett–Burman experimental design. The incorporation of FeSO4, MnSO4, and MgSO4 into the pullulanase production medium significantly increased the yield and showed a 5.7-fold increase (56.25 U/gds) in comparison with the unoptimized media. The Box-Behnken experimental design was used to study the effect of interactions between Fe2+, Mg2+, and Mn2+ on the production of pullulanase. Box-Behnken showed a 1.1-fold increase (62.1 U/gds) in pullulanase production. The total increase in yield after all optimization was 6.37-fold. The present study reports for the first time the applicability of green gram husk as a potent substrate for pullulanase production by Penicillium viridicatum.

Detection of Disinfectant property of purified Amylopullulanase from Citrobacter freundii SW

There are varieties of marketable detergents and disinfectants purchased; in order to choose an effective one; it is very necessary to ensure its decontamination proficiency. So that a screening of pullulanase production from many isolates of Citrobacter freundii have been done using dried water cabbage as inducer of pullulanase production in the culture medium.Pullulanase producing bacteria was isolated and strain SW was designated. Morphological and biochemical tests in addition theanalysis of 16s rRNA observed that the strain SW revealed a 100% sequence identity withsimilarity to that of Citrobacter freundii. The pullulanase was purified by using two-step purification procedure consisting of ammonium sulphate precipitation, ion exchange chromatography followed by gel filtration chromatography with a yield of 29.7%. The pullulanase has an important role in augmentation of disinfectant efficiency. Depending on the current results, the combination between pullulanase and commercial disinfectant resulted to increase the disinfectant activity to greater levels against fungi more than bacteria.

PRODUCTION AND UTILIZATION OF PULLULANASE FROM BACILLUS CLAUSII FOR ENHANCED RESISTANT STARCH PRODUCTION IN GARRI

Resistant starch is the portion of starch which escapes digestion in the small intestine and passes into the large intestine where it is fermented by gut microflora. It is considered a functional component of food due to the health benefits it confers following its consumption. The aim of this research was to produce garri with high resistant starch using pullulanase enzyme from Bacillus clausii. The organism was isolated from different cassava processing sites in Awka and identified based on phenotypic, biochemical and molecular characteristics. Pullulanase assay was carried out using standard procedures at 540 nm. Optimum conditions for pullulanase production was determined, after which the pullulalanse enzyme was used to modify garri. The test organism with the highest pullulanase activity was identified as Bacillus clausii. Optimum pullulalanse activity was observed with an innoculum size of 3.0ml, pH 6, an incubation temperature of 35o C and incubation time of 48 hours. The resistant starch content was found to be higher for enzyme-treated garri samples (13.81%) than for the untreated garri samples with (2.69%). This research shows that pullulanase enzyme from Bacillus clausii is a very useful industrial raw material for the increased production of resistant starch in foods.

Optimization of pullulanase production by Aspergillus flavus under solid-state fermentation

Pullulanase is a promising candidate for industrial applications. But less yield and high cost limited its application. Hence, the present study aims to optimize nutrients for increasing the yield of fungal pullulanase to resolve these limitations. The findings of Plackett Burman Design showed that only 5 of 19 nutrients influenced the yield of pullulanase significantly. Box-Behnken Design (BBD) was implemented to establish the optimal concentration of the significant nutrients. After statistical optimization for KH2PO4, yeast extract, peptone, and MnSO4, supplemented in the substrate wheat bran, the overall increase in yield was 1.63 fold. In all the flasks (500 to 5000 mL capacity containing 10–100 g dry substrate), almost the same yield has been produced (647.00 ± 25.139 U/gds) suggesting that the production of pullulanase can readily be scaled up under optimized conditions. This is the first nutrient optimization study of pullulanase production by Aspergillus flavus in solid state fermentation using wheat bran.

Enhancing Extracellular Pullulanase Production in Bacillus subtilis Through dltB Disruption and Signal Peptide Optimization.

Bacillus subtilis has many attributes that make it a popular host for recombinant protein production. Although its protein production ability has been enhanced through protease gene disruption, residual proteases like quality control HtrA and HtrB can limit protein yield by degrading inadequately folded proteins present during overexpression. In this study, two strategies were employed to increase production of industrial enzyme pullulanase: enhancing extracellular pullulanase folding and optimizing its signal peptide. The hypothesis was that disruption of dltB gene of expression host B. subtilis WS9 would enhance recombinant extracellular folding by increasing cation binding to the cell's outer envelope. Consistent with this hypothesis, disrupting dltB enhanced pullulanase production by 49% in shake-flask cultures. The disruption also enhanced extracellular α-CGTase and β-CGTase production by 25% and 35%, respectively. Then, more effective signal peptide for pullulanase production was identified through high-throughput screening of 173 unique B. subtilis signal peptides. Replacing the native signal peptide of pullulanase with that encoded by ywtF increased extracellular pullulanase activity by an additional 12%. Three-liter fermenter scale-up production yielded the highest extracellular pullulanase activity reported to date: 8037.91 U·mL-1. This study highlights the usefulness of dltB deletion and signal peptide optimization in enhancing extracellular protein production.

A Novel Culture Medium Using Lettuce Plant (Lactuca Sativa) For Detection of Pullulanase Production by Paenibacillus Macerans Isolated from Agricultural Wastes

Pullulinase is an enzyme catalyzing the hydrolysis of pullulin forming maltotriose, panose and maltooligosaccharides that have a great prominence owing to their prospective use in medication, drink, diet and fine chemical industries. Because of the production of this enzyme is too expensive and characterized with low yield, there is a growing need to create a substrate to make the total process cheaper and more creative. In this study, the authors have reported pullulanase production from Paenibacillus macerans. Six isolates of Paenibacillus macerans were isolated from soil and agricultural wastes samples and screened for higher pullulanase production, Paenibacillus macerans isolated from second sample of wheat husk was gave superior inhibition zone around the colony as compared with other tested isolates. The activity of pullulanase was raised to 9.83U/ml when this strain was grown under the optimal conditions including using basal medium containing 1% (w/v) dried leaves of lettuce and 2% (w/v) peptone at pH 6, temperature of 50°C, inoculums size of 1.5 ml for 72 hour. The pullulanase exhibited highest activity (6.80U/ml) and (6.43U/ml) in presence of lettuce. Lettuce has aptitude as an efficient and economically suitable as compared with traditionally used substrates as pure pullulin and other types of saccharides.

Improve Production of Pullulanase of Bacillus subtilis in Batch and Fed-Batch Cultures.

Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m3 bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m3 based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.

Production of a Thermostable Pullulanase in Bacillus subtilis by Optimization of the Expression Elements

AbstractPullulanase is a debranching enzyme commonly used in the starch processing industry. Production of a thermostable pullulanase with high activity is desired due to the application of the enzyme to starch processing at high temperature. In this study, a thermostable pullulanase with high activity is overproduced in Bacillus subtilis. Via improvement of the plasmid backbones, promoters, and ribosome‐binding‐site (RBS) sequences, an optimal recombinant strain B. subtilis WB800/RBS7 is obtained with an extracellular pullulanase activity of 154.9 U mL−1 in a shake flask, which is 136.8 times higher than that of the wild type. When the strain is cultured in a 5‐L bioreactor, the pullulanase activity is significantly improved to 269.1 U mL−1 (2.74 mg mL−1). These results are expected to aid pullulanase production in industrial starch debranching applications.

Optimizing the DO-stat protocol for enhanced production of thermostable pullulanase in Escherichia coli by using oxygen control strategies.

Production of a thermostable pullulanase by DO-stat fed-batch fermentation of recombinant Escherichia coli BL 21 was investigated in a 5 L of fermentor. The effect of three oxygen control strategies, glucose feedback, shifting fermentor pressure, and adding oxygen-enriched air, on cell growth and pullulanase expression were examined. The oxygen-transfer capacity was found to be enhanced with increasing fermentor pressure and oxygen ratio in oxygen-enriched air, but the cell growth and pullulanase production were restrained under high fermentor pressure. The highest cell density and pullulanase activity reached 55.1g/L and 412 U/mL, respectively, in the case by adding oxygen-enriched air, which was suggested as an effective approach to enhance both cell growth and pullulanase production. PRACTICAL APPLICATIONS: This thermostable pullulanase displayed optimal activity at 90°C and pH 5.4, which could be applied for one-step saccharification of starch biomass. The optimization of the DO-stat fed-batch fermentation in high cell density level would provide a research basis for its industrialization.

Enhancement of pullulanase production from recombinant Bacillus subtilis by optimization of feeding strategy and fermentation conditions

Pullulanase is an important starch-debranching enzyme mostly used in starch processing-related food industries. However, the levels of pullulanase produced from recombinant Bacillus subtilis, a Generally Recognized as Safe (GRAS) host, are generally limited. To enhance the activity of pullulanase, batch fermentation and fed-batch fermentation were systematically investigated. The overall purpose is to improve the fermentation yield by optimizing the feeding strategy in the fermentation process, thereby increasing the enzyme activity of pullulanase. Therefore, in this study, the feeding methods, the feeding ingredients, the feeding concentration, and pH values were studied in detail. The optimized fermentation conditions for pullulanase production from recombinant B. subtilis were determined as following: inoculum volume 7%, pH 6.5, the dissolved oxygen level 30%, and constant-rate feeding of 100 mL glucose solution (400 g L−1) in late logarithmic growth. The OD600 of recombinant B. subtilis and enzyme activity were 84.54 and 102.75 U mL−1, which were respectively 141% and 144% higher than that before optimization. These findings provided a prerequisite for further amplification of the fermentation system to obtain higher enzyme activity.

Screening of agro-industrial waste and physical factors for the optimum production of pullulanase in solid-state fermentation from endophytic Aspergillus sp.

Abstract The selection of a suitable agricultural residue, its availability and the cost are critical factors to be considered while selecting a raw material in solid-state fermentation (SSF). In the case of SSF parameters like temperature, moisture content and spore concentration also play a significant role in enzyme production. In this study, endophytic fungi were isolated and screened for their pullulanase activity. The most promising isolate identified as Aspergillus sp. was evaluated for the pullulanase production using agricultural residual substrates like wheat bran, rice bran, sugarcane bagasse, orange peel, mosambi peel, and banana peel. Among all the substrates tested by the classical approach, the wheat bran was found to be the most effective substrate. The highest yield was 65.33 ± 2.08U/gds, while in case of rice bran the maximum yield was 44.66 ± 1.527U/gds. The comparatively lower yield was obtained in sugarcane bagasse, orange peel, mosambi peel, and banana peel. Maximum Pullulanase activity (67.00 ± 01.00 U/gds) was reached on 3rd day; thereafter the enzyme activity declined. Experiments conducted at the optimum physical conditions (temperature 28.62 °C; moisture 69.92%; inoculum size 6.42 log) demonstrated that the pullulanase yield (396.2 ± 1.33U/gds) was closer to the predicted value (394.5U/gds). There was an improvement of yield by 6.064 fold relative to that obtained from un-optimized physical parameters. The good correlation between predicted and experimental values after optimization justified the validity of the response model and the existence of an optimum point. This is the first report of pullulanase production from Aspergillus species in solid-state fermentation.

Enhancement of the production of Bacillus naganoensis pullulanase in recombinant Bacillus subtilis by integrative expression

Pullulanase is widely used in the starch processing industry as a debranching enzyme. However, extracellular production of pullulanase from recombinant Bacillus subtilis is limited and the loss of plasmids during fermentation of B. subtilis recombinants seriously affects the expression of the foreign protein, especially in large-scale production. In this study, a universal integrated plasmid was conducted harboring the pul cassette that included the pul gene encoding Bacillus naganoensis pullulanase (PUL), a constitutive promoter, PHpaII, and an extracellular signaling peptide, LipA. This cassette was inserted into the genomes of B. subtilis WB800 and B. subtilis WB600 by double homologous recombination. The pullulanase activity of up to 30.32 U/ml and 18.83 U/ml was achieved for B. subtilis WB800-PHpaII-pul and B. subtilis WB600-PHpaII-pul, respectively, under primary conditions. To further enhance the yield of PUL, the effects of four important factors (inoculum size, incubation temperature, shaking speed, and initial pH) on the expression of PUL in shake flask fermentation were evaluated by “one-factor-at-a-time” technique for B. subtilis WB800-PHpaII-pul. Consequently, the extracellular production of PUL was significantly enhanced, resulting in an activity of 60.85 U/ml.