Improvement of cyclodextrin glucanotranferase excretion and cell viability of recombinant Escherichia Coli immobilized on hollow fiber membrane

The excretion of a recombinant enzyme into culture medium presents significant advantages over cytoplasmic expression. However, cell lysis is one of the major drawbacks during the excretion of recombinant enzyme when using Escherichia coli (E. coli) as a host. Cell immobilization is a promising solution for the enhancement of enzyme excretion with reduction of cell lysis. In the present study, a recombinant E. coli was immobilized using hollow fiber membrane to improve the enzyme excretion, cell viability and plasmid stability. The effects of different polymers of hollow fiber membrane and culture conditions on the cyclodextrin glucanotransferase (CGTase) excretion, cell lysis and plasmid stability of immobilized E. coli were investigated. The immobilized cells on a polyvinylidene fluoride polymer exhibited a 2.0–4.5-fold increase in the CGTase excretion with 18-95% reduction of cell lysis and over 100% increment of plasmid stability compared to the free cells. The CGTase excretion was successfully optimized by response surface methodology. Under the optimized conditions [25 ºC of post- induction temperature, 0.011 mM isopropyl β-D-1-thiogalactopyronoside and pH 8.8], the CGTase excretion was 3.8-fold higher with 80% reduction of cell lysis compared to the value before optimization process. The use of low tryptone concentration (5 g/l) reduced the occurrence of cell lysis (90% reduction) and increased the plasmid stability (86% increment) without significant change in CGTase excretion in comparison with initial tryptone concentration (20 g/l). This approach (5 g/l) produced an approximately two times higher CGTase excretion (compared with 20 g/l during) recycle process. The membrane bioreactor also showed 2.5-fold increase in the CGTase excretion (473 x 103 U/ml) with 75% reduction of cell lysis compared to shake flask culture (190 x 103 U/ml of CGTase activity). Hence, the immobilization of E. coli on hollow fiber membrane proved to be valuable for the excretion of recombinant proteins in E. coli with high cell stability

Immobilization of CGTase from Bacillus licheniformis on pineapple waste for production of cyclodextrin

CD is a nonreducing maltooligosaccharides with a hydrophobic inside and hydrophilic surface outside. CD able to form inclusion complexes with many hydrophobic molecules, changing their physical and chemical properties. Ability to reduce of unwanted taste and odour in food and pharmaceutical. CD also approved by Generally Recognized As Safe (GRAS) and Food and Drug Administration (FDA) in food application

Optimization of reaction parameters of cyclodextrin glycosyltransferase from bacillus licheniformis on cyclodextrin production

Cyclodextrin is produced from enzymatic degradation of starch. Improves guest molecule’s chemical and physical properties. Optimization of the reaction parameter to maximize the production of C

Screening of culture conditions for production of xylanase from landfill soil bacteria

Xylan is a major constituent of hemicellulosic polysaccharides found in plant’s cell wall which represent up to 20-30% of the total dry weight in tropical plant biomass [1]. Besides, 9-12% of municipal solid waste are composed of hemicellulose on dry weight basis [2]. Enzymatic method can be used for the degradation of these materials involving the use of microbial enzymes [3] that are less polluting, environmental friendly, energy saving and lower disposal problems [4]. Xylanase is a biocatalyst which specifically degrade xylan into smaller sugars such as xylose and xylobiose [5]. It has been used in many important industrial applications such as pulp, paper, bakery, juice and beer industries [6]. This enzyme has been employed in paper manufacturing to bleach paper pulp and increase the brightness of paper pulp instead of using toxic and expensive chemicals [5]. Xylanase also being used in biofuel production such as ethanol from lignocellulose biomass and used in treatment of barley and wheat to improve the properties of animal diet in animal feed industries [1]. Besides, this enzyme can be applied for conversion of xylan into xylose in agricultural wastewater and to clarify fruit juices in beverage industries [7]. Microbes are prefer by the industries to produce various enzymes, because of high growth rate and large volume of enzymes can be obtained within a short time [8]. Employing microbes such as bacteria, yeast and filamentous fungi, that are known for their ability to secrete extracellular enzymes into the environment, may help to overcome the current challenge in reducing the volume of waste in landfill by biological conversion of municipal solid waste into bioenergy [9]

Effects of Ultrasonic Waves on Vapor-Liquid Equilibrium of Cyclohexane/Benzene

Separation of azeotropic mixtures is one of the most challenging separation processes in chemical industry. Special separation techniques such as azeotropic and extractive distillation are required to separate these mixtures. In this work, an innovative technique which incorporates ultrasonic waves during separation of Cyclohexane (Chx)/ Benzene (Bz) via distillation process was explored. The effects of ultrasonic wave at different intensities at 50, 100, 200 and 250 W/A.cm2 and at a fixed frequency of 40 kHz on vapor-liquid equilibrium (VLE) of Chx/ Bz were investigated. Studies were also done to examine the effects of ultrasonic frequencies on the VLE data at 25 and 68 kHz. It was found that ultrasonic waves at 100 W/A.cm2 intensity and 25 kHz frequency gave the highest relative volatility, α at 2.505 and completely eliminated the azeotropic point. The results revealed that ultrasonic waves had potential to favourably manipulate α, and hence, the VLE of Chx/ Bz

Menhaden fish oil encapsulation by spray drying process: influence of different biopolymer materials, inlet air temperature and emulsion ratios

BACKGROUND: Incorporating fish oils into commonly consumed foods is an emerging technique for increasing the daily intake of omega-3 fatty acid. However, the high vulnerability of fish oil towards oxidative deterioration reduces shelf life stability. Microencapsulation by spray drying with different combinations of biopolymers and other parameters may provide a solution by preventing further oxidation of fish oil and permitting its delivery to food items. This study emphasised the influence of developed biopolymer materials (maltodextrin, maltodextrin + gum arabic, maltodextrin + starch, maltodextrin + whey pro- tein isolates and maltodextrin + sodium caseinate) for emulsion formulation, inlet air temperature (160, 170, 180, 190 and 200 °C) and emulsion ratios (15, 20, 25, 30 and 35%) on the physicochemical properties of powdered menhaden fish oil by spray drying technique. RESULTS: Microencapsulated menhaden fish oil was evaluated for its moisture content, size distribution, microcapsule effi- ciency, peroxide value, free fatty acid, acid value and morphological structure. It was observed that the coating materials of maltodextrin + whey protein isolates gave the lowest moisture content and peroxide value of 4.410% and 4.031 mEq kg−1, respectively. The morphological images showed a smooth surface with no cracks and minimal shrinkage, indicating lower per- meability to gases and effectively protecting the oil against deterioration. CONCLUSION: The spray drying procedure using maltodextrin + whey protein isolates at an inlet air temperature 190 °C and emulsion ratio of 30% successfully produced fish oil microcapsules with improved properties

Effect of the Cross Linkers’ Concentration on the Immobilization of Recombinant Escherichia Coli Cells on Hollow Fiber Membrane for Excretion of Cyclodextrin Glucanotransferase (CGTase)

The excretion of recombinant proteins into the culture medium is a preferred approach over cytoplasmic expression because of the high specific activity and ease of purification. However, cell lysis is one of a major problem during the excretion of recombinant protein due to the pressure build up in the periplasmic space through overproduction of the expressed recombinant protein. Cell immobilization is a promising solution for the improvement of protein excretion with reduction in cell lysis. In this study, the effect of cross linkers concentrations on the process of cell immobilization for the cyclodextringlucanotransferase (CGTase) excretion and cell viability were investigated. The hollow fiber membrane was treated using different concentrations (0.3, 0.67 and 1%) of cross linkers namely glutaraldehyde (GA), poly-L-lysine (PLL) and polyethylenimine (PEI). The optimum concentration of cross linkers was found to be 0.3% with the high excretion of CGTase. Interestingly, the immobilized cell on the non-treated hollow fiber membrane showed up to 15% increment of CGTase excretion with 55% reduction of cell lysis, as compared to the immobilized cells on the treated hollow fiber membrane. It could be concluded that, the low concentration of cross linkers exhibited the highest CGTase excretion. Moreover, the non-treated hollow fiber membrane is a promising approach for attachment of cells to the membrane without requiring treatment with any chemicals for the production of recombinant enzyme

Optimization of culture conditions of immobilized cells for enzyme excretion and cell lysis

Recombinant enzyme excretion is essential due to stable and active protein production. However, enzyme excretion, especially in the Escherichia coli system, may increase the occurrence of cell lysis. Thus, cell immobilization was used, and optimization of culture conditions was conducted to increase enzyme excretion and limit cell lysis. The culture conditions were optimized using a response surface methodology. CGTase excretion and cell lysis were analyzed by CGTase and β-galactosidase assays, respectively. High CGTase excretion with low cell lysis was achieved after the optimization process. The results suggest that optimization of the cell immobilization system increased CGTase excretion with low cell lysis and made it more valuable for industrial applications

Hollow Fiber Membrane as a Carrier for Enzyme Immobilization

Cyclodextrin (CD) is produced from starch by an enzymatic conversion catalyzed by cyclodextrin glucanotransferase (CGTase). CD has been used in a wide range of industries, especially in food, cosmetic, pharmaceutical, and agrochemical industries, due to its ability to improve the physicochemical properties of organic molecules, conferring greater chemical resistance to environmental factors, higher solubility, and reduced volatility (Ching et al. 2022). The commercialization of CGTase for industrial purposes is highly challenging due to the instability of the CGTase, sensitivity to the process condition, and high cost of isolation and purification (Guzik, Hupert-Kocurek, and Wojcieszyńska 2014; Mohamad et al. 2015). The instability of CGTase during the reaction process results in low yield CD. Therefore, enzyme immobilization has been applied to improve CGTase stability and achieve higher CD yields

Microwave assisted demulsification of water-in-crude oil emulsions

Emulsions are of considerable practical significance both in processing industry and in the consumer sector. In the petroleum industry emulsions phenomena are particularly aware because it can lead to upstream and downstream production upset. A recent report stated that about 80% of exploited crude oil exists in an emulsion state, all over the world (Xia et al., 2004)