Immobilized Alkaline Protease Research Articles

Immobilization of cellulase on magnetic nanoparticles for rice bran oil extraction in a magnetic fluidized bed

Abstract This paper presents a method for extracting rice bran oil using magnetic immobilized cellulase (MIC) in a magnetic fluidized bed (MFB). Cellulase was immobilized on Fe3O4/SiO x -g-P (glycydylmethacrylate) with an average grain size of 120 nm. The rice bran was hydrolyzed using MIC combined with magnetic immobilized alkaline protease to extract rice bran oil. Under intermittent conditions, the MIC concentration was 1.6 mg/g, the liquid to material ratio was 4:1, the enzymatic hydrolysis time was 150 min, and the oil yield was as high as 85.6 ± 1.20% at 55 °C. The fluid in the MFB had a magnetic field strength of 0.022 T, a flow velocity of 0.005 m/s, and an oil extraction rate of 90.3%. This provides a theoretical basis for the extraction of rice bran oil using the subsequent MFB hydroenzyme method.

ENCAPSULATION OF PROTEASE ENZYME FOR DOMESTIC APPLICATION

The use of alkaline protease as commercial catalysts has increased significantly in recent years. Often main industrial and commercial uses, like the food fabric and medical diagnostics industries depend heavily on the protease enzyme. The free movement of enzymes is constrained in the cell immobilization technique and a continuous fermentation method can be used. The procedure was usually used with different carriers, such as chitosan, agar and alginate, to create alkaline proteases. In conjunction to the encapsulation of an enzyme using an Encapsulator instrument, we attempted to use this technique to examine the implementation of individual matrices to immobilize the protease enzyme. This enzyme was previously recovered from Bacillus Pseudofirmus Mn6 EU315248. Some physical characteristics of the immobilized enzyme such as activity temperature, pH effect and operational stability were assessed. Results revealed that the maximum pH stability attained throughout the chitosan matrix-entrapped enzyme, when incubated at pH of 10.5 for 15min. is nearly 200%. The protease enzyme immobilized in chitosan, displayed excellent stability when incubated at 50oC for 1h with a local detergent. The enzyme retained its maximal activity even after 1h of incubation with the majority of the tested detergents. Washing performance of the immobilized alkaline protease was also applied on two types of dirty cloth spots, i.e. blood and chocolate spots. Results confirmed that the immobilized protease enzyme must be used as one of the ingredients in the detergents industry.

Study on Preparation of Immobilized Alkaline Protease and Its Performance

The nano-Fe₃O₄ particles were prepared by hydrothermal method. Cellulose-based magnetic Fe₃O₄ was prepared by adding cellulose solution dissolved in sodium hydroxide / thiourea / urea system. The basic protease was used as the catalyst and glutaraldehyde was used as the crosslinking agent, The production of magnetic recyclable immobilized enzyme can greatly reduce the cost of hydrolysis of sludge. The aldehyde groups in the glutaraldehyde and the amino groups in the protein can be stably crosslinked by coupling. The immobilization effect of nanometer Fe₃O₄ was reflected by investigating the activity of immobilized enzyme and immobilization rate of enzyme under different conditions.

Optimal conditions for deproteinizing natural rubber using immobilized alkaline protease

BACKGROUND Natural rubber latex contains allergenic proteins. Therefore, processes for deproteinizing the latex are needed. An immobilized enzyme process for deproteinization is reported. An optimal protocol was first developed for immobilizing a protease on cellulose−chitosan composite beads. The beads were then used in developing an optimal deproteinization treatment. The main effects and the interactions of the factors for the two processes were identified using a two-level full factorial experimental design. RESULTS Under optimal conditions (15% cellulose in beads, immobilizing reaction pH of 9, immobilization period of 24 h), the beads attained the highest specific activity of 1685.3 U g−1 beads. Using these beads, the optimal conditions for deproteinization of the latex were: an enzyme loading of 0.1 parts per hundred of rubber (phr), a sodium dodecyl sulfate concentration of 20 phr, 30°C, and a treatment period of 12 h. The nitrogen content of the rubber was reduced to 0.012% from an initial value of 0.3%. The enzyme beads were operationally stable and could be reused for at least five cycles. CONCLUSIONS The optimized treatment effectively deproteinized the natural rubber. The final product was free of peptides. This deproteinization treatment was more effective than the conventional urea-based treatment performed for comparison. © 2014 Society of Chemical Industry

English

In this study, extracellular alkaline protease producing bacterial isolates EN-2 and EN-3 from the agricultural soil of C.R.C. Pantnagar were identified as Bacillus megaterium strains EN-2 and Bacillus subtilis strain EN-3 on the basis of 16S rDNA gene sequencing. During kinetic characterization, optimum pH for EN-2 and EN-3 protease activity was 10 and 9, respectively. While optimum temperature, for maximum protease activity in both isolates, was 50°C. The crude extracellular alkaline protease from isolates EN-2 and EN-3 were partially purified using ammonium sulphate fractionation and dialysis to 1.50 and 1.42 fold with 53.77% and 42% recovery respectively. The observed values of Vmax and Km for protease from isolate EN-2 were found to be 11.57 U/ml and 17.442 mg/ml, while for EN-3 protease these were 42 U/ml and 10.62 mg/ml, respectively. The partially purified enzyme from both bacterial strains was then immobilized in sodium alginate beads with maximum immobilization efficiency at 3% (w/w) and some change in their kinetic properties. The immobilized alkaline protease from EN-2 and EN-3 showed their maximum protease activity at pH 9 and 10, and temperature 60 and 50°C, respectively. Due to these properties, isolated extracellular alkaline proteases from the two strains are ideal choice for application in detergent formulation, leather and food industries.   Key words: Bacillus megaterium, Bacillus subtilis, alkaline protease, immobilization.

Studies on the properties of graphene oxide–alkaline protease bio-composites

Graphene oxide (GO) nanosheets as functional material have a unique planar structure and intriguing mechanical that have attracted intensive interests recently. A method was developed for the immobilization protease on GO sheets using glutaraldehyde as cross-linking reagent. The results showed that the thermostability and reusability of immobilized protease have been obviously improved compared to the free enzyme. However, there was no significant change in optimum pH value between the free and immobilized protease. The immobilized protease exhibited good operational stability. The apparent Km and Vmax for free and immobilized alkaline protease were determined, and the bio-catalytic activity was not impaired by immobilization.

Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

(3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

Drug synthesis methods and manufacturing technology

Alkaline protease from Bacillus subtilis and lysozyme from chicken egg white have been immobilized for the first time in poly(N-vinylpyrrolidone) modified by polysilicic acid sol. Elastic polymeric hydrogels that are insoluble under physiological conditions are obtained and provide potential wound-healing coatings with high retention of lytic and proteolytic activities (80 – 100%) and prolonged action (24 h) and complex preparations with proteolytic and 1.7-fold increased lytic activity. It is established that immobilized alkaline protease and lysozyme are stable under conditions modeling the wound pH level during storage for one year and upon sterilization by -irradiation.

Preparation and characterization of alkaline protease immobilized on vermiculite

Alkaline protease was immobilized on vermiculite by covalent linkage. When compared to the free enzyme the immobilized form exhibited a lower specific activity towards high molecular weight substrates but a broader pH tolerance and higher thermal stability. The pH optima of the immobilized alkaline protease was shifted towards the alkaline side by one unit. However, there was no change in optimum temperature between the free- and immobilized alkaline proteases. The immobilized alkaline protease exhibited good storage stability and re-usability.

Covalent linking of alkaline protease on trichlorotriazine activated nylon

Alkaline protease was covalently attached to nylon using trichlorotriazine. Enzymatic activity was determined at different trichlorotriazine activation temperature and time. It was observed that, the optimum temperature and pH of the immobilized alkaline protease was comparable to that of the free enzyme. Immobilization improved the thermal stability and pH stability of the enzyme.

Role of .BETA.-casein in milk curdling.

The tension values were determined for milk curds formed by six proteases using a curd tension meter. In curds with relatively high tension values, which were prepared by rennet, Mucor rennin and pepsin (acid proteases), αs- and β-casein were negligibly hydrolyzed during incubation after clotting, whereas both casein fractions in the fragile curds prepared by Alkaline-protease from Bacillus subtilis, trypsin and thermolysin (alkaline proteases) were electrophoretically degraded during further incubation after clotting. This phenomenon was more prominent in the β-casein fraction. However, when alkaline proteases were employed in an immobilized form and removed from the test-milk at the initiation of clotting, the degradation of β-casein was decreased, and curd tension values were increased. Inhibition of the enzymatic degradation of β-casein by addition of sodium chloride (5%) led to an increase in curd tension. When the test-milk treated by each immobilized alkaline protease was fortified with β-casein, c...

APPLICATION OF IMMOBILIZED ALKALINE PROTEASE TO CHEESE‐MAKING

ABSTRACTAn alkaline protease immobilized on Dowex MWA‐1 by glutaraldehyde was applied to a step of cheese‐making process. By reversing the order of the lactic acid fermentation process and the “rennetting” (hydrolysis) process in the traditional cheese‐making method, and by raising pH of milk to 7.0, coagulation of milk in the alkaline protease column was avoided. As a result of these modifications, it became possible to operate a stable hydrolysis process of milk proteins at 50°C. Average degree of hydrolysis was about 3%. Curd was prepared from this milk after lowering pH to 6.2 by lactic acid fermentation and was processed to make Cheddar‐type cheese. After ripening this cheese for 6 months at 10°C, its ripening ratio was similar to that of cheese prepared by the traditional method. It is concluded that the immobilized alkaline protease could be used for cheese‐making.