272 recognizes six uronic acid residues (both guluronate and mannuronate oligomers) ( Fig. The kinetic analysis suggests that the enzyme from Pseudoalteromonas sp. Analysis using purified alginate oligomers with defined DP revealed that the enzyme did not recognize trimeric guluronate and mannuronate, but efficiently acted on oligomers longer than tetramers. Since the enzyme caused immediate decrease in the viscosity of sodium alginate solution, this enzyme is considered to have an endo-type action manner. Although the exact reason for this is still unclear, Tris might act as detrimental factor for this enzyme. In terms of heat stability, the enzyme was more stable in phosphate buffer (pH 7.0) than in Tris–HCl buffer (pH 7.0). The optimal pH and stable pH range of the enzyme were 7.5–8.0 and 5–11, respectively. As a conformational feature of the enzyme, far-ultraviolet circular dichroic spectrum of this enzyme suggested the presence of β-sheet structure. Hence, the enzyme is relatively acidic protein. The isoelectric point of bifunctional alginate lyase from Pseudoalteromonas sp. Oda, in Advances in Food and Nutrition Research, 2016 2.3.2 Characterization For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins rather than the target protein. The greatest disadvantage of isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. Mineral acids, such as hydrochloric and sulfuric acids, are used as precipitants. The pI of most proteins is in the pH range of 4 to 7. However, at the pI, the negative and positive charges are balanced, reducing repulsive electrostatic forces, and the attraction forces predominate, causing aggregation and precipitation. Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged, and repulsion between proteins occurs. At solution pH that is above the pI, the surface of the protein is predominantly negatively charged, and therefore like-charged molecules will exhibit repulsive forces. The isoelectric point (pI) is the pH of a solution at which the net charge of a protein becomes zero. Havlíček, in Proteomic Profiling and Analytical Chemistry (Second Edition), 2016 4.6 Isoelectric Point Precipitation Thus, the pH value may require adjustment when the isoelectric point is chosen after the addition of metal ions to the solution. For example, the isoelectric point of insulin is 5.3, but it increases to 6.2 when combined with Zn 2+. The isoelectric point of a protein will shift if the protein combined with metal ions. If the isoelectric points of the different proteins were similar, then the proteins would be precipitated at the same time, which can seriously influence the sedimentation effects of the proteins during precipitation. 2.3.4.2 Types of Proteinsĭifferent protein types have different isoelectric points. HCl is commonly used to regulate the pH value during the extraction of sweet potato protein. The effects of different acidity regulators on protein precipitation are different. However, this method sometimes leads to partial proteolysis or degeneration. In addition, fermentation can produce some acidic components to reduce the pH value of the solution and adjust it to near the protein isoelectric point. 2.3.4.1 Type of Acidity Regulatorįor the isoelectric point precipitation method, acidity regulators, including HCl, sulfuric acid, acetic acid, phosphoric acid, citric acid, and lactic acid, are commonly used to maintain a pH level. There are many factors that affect the precipitation of proteins. Cheng Wang, in Sweet Potato Processing Technology, 2017 2.3.4 Factors Influencing Isoelectric Point Precipitation

Calculate pi isoelectric point