Glucosa Isomerasa

Glucose Isomerase Activity
Scope
This procedure is designed for the determination of glucose isomerase preparations derived fromActinoplanes missouriensis, Arthrobacter globiformis, Bacillus coagulans, Streptomyces olivaceus, Streptomyces olivochromogenes, and Streptomyces rubiginosus.
Principle
The assay is based on measurement of the rate of conversion of glucose to fructose in a packed bedreactor.
The procedure as outlined approximates an initial velocity assay method. Specific conditions are: glucose concentration, 45% w/w; pH (inlet) measured at room temperature in the 7.0 to 8.5 range, as specified; temperature, 60.0°; and magnesium concentration, 4 x 10-3 M. The optimum conditions for enzymes from different microbial sources and methods of preparation may vary; therefore, ifdifferent pH conditions, buffering systems, or methods of sample preparation are recommended by the manufacturer, such variations in the instructions given herein should be used.
Reagents and Solutions
Glucose substrate: Dissolve 539 g of anhydrous glucose and 1.0 g of magnesium sulfate, MgSO4.7H2O, in 700 ml of water or the manufacturer's recommended buffer, previously heated to 50° to 60°.Cool the solution to room temperature, and adjust the pH as specified by the enzyme manufacturer. Transfer the solution to a 1,000-ml volumetric flask, dilute to volume with water or the specified buffer, and mix. Transfer to a vacuum flask, and de-aerate for 30 min.
Magnesium sulfate solution: Dissolve 1.0 g of magnesium sulfate, MgSO4.7H2O, in 700 ml of water. Adjust the pH to 7.5 to 8.0 asspecified by the manufacturer, using 1 N sodium hydroxide, dilute to 1,000 ml with water and mix.
Note: Glucose isomerase activity of the commercial enzyme is usually determined on the enzyme that has been immobilized by binding with a polymer matrix or other suitable material. This method is designed for use with such preparations.
Column Assembly and Apparatus
The column assembly is shown in Figure1 below.
Note: Make all connections with inert tubing, glass or plastic as appropriate.
Use a 2.5 x 40-cm glass column provided with a coarse sintered glass bottom and a water jacket connected to a constant-temperature water bath, maintained at 60.0° by means of a circulating pump. Connect the top of the column to a variable-speed peristaltic pump having a maximum flow rate of 800 ml per h. Thediameter of the tubing with which the peristaltic pump is fitted should permit variation of the pumping volume from 60 to 150 ml per h. Connect the outlet of the column with a collecting vessel.
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Figure 1. Diagram of a column assembly for assay of Immobilized Glucose Isomerase

Sample Preparation
Transfer to a 500-ml vacuum flask an amount of the sample, accurately weighed in gor measured in ml, as appropriate, sufficient to obtain 2,000 to 8,000 glucose isomerase units (GIc U). Add 200 ml of Glucose Substrate, stir gently for 15 sec and repeat the stirring every 5 min for 40 min. De-aerate by vacuum for 30 min.
Column Preparation
Quantitatively transfer the Sample Preparation to the column with the aid of Magnesium Sulfate Solution as necessary. Allow the enzymegranules to settle, and then place a porous disk so that it is even with, and in contact with, the top of the enzyme bed. All of the air should be displaced from the disk. Place a cotton plug about 1 or 2 cm above the disk. (This plug acts as a filter. It ensures proper heating of the solution and traps dissolved gases that may be present in the Glucose Substrate.) Connect the tubing from theperistaltic pump with the top of the column, and seal the connection by suitable means in order to protect the column contents from the atmosphere. Place the inlet tube of the peristaltic pump into the Glucose Substrate solution, and begin a downward flow of the Glucose Substrate into the column at a rate of at least 80 ml per h. Maintain the flow rate for 1 h at room temperature.
Procedure
Adjust...