Method for carrying out coupling immobilization on coenzyme and coenzyme dependent enzyme
A dependency and coenzyme technology, applied in the direction of multi-enzyme systems, can solve problems such as inability to bind, hinder reaction sites, and large random distances, and achieve controllable catalytic performance, reduce mass transfer steric hindrance, and avoid coenzyme loss Effect
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Embodiment 1
[0036] 1) Each weighed 1g of chitosan carrier, dissolved in a beaker containing 100mL of 1% (w / w) acetic acid solution, added 5mL of coupling agent glutaraldehyde dropwise under stirring, adjusted the pH to 7, and ultrasonicated for 15min. make it fully dispersed;
[0037] 2) Add 1 mmol of reduced coenzyme I (NAD) to the dispersion + ),well mixed;
[0038] 3) Centrifuge the solution obtained in step 2), filter, wash or membrane separation to remove uncoupled coenzyme, retain the affinity carrier, according to the fact that chitosan does not contain elemental phosphorus but coenzyme contains, take part of the affinity carrier for digestion reaction to measure phosphorus content and calculate the coupling rate, take a part of the affinity carrier and grind it, scan the ultraviolet-visible spectrum with a spectrophotometer, and transfer the rest to a beaker.
[0039] 4) Dissolve 100 mg of lactate dehydrogenase in 1 mL of phosphate buffer solution, and slowly add it dropwise int...
Embodiment 2
[0046] 1) Weigh 1g of agarose carrier each, dissolve it in a beaker containing 100mL of deionized water, add 5mL of coupling agent diglycidyl ether dropwise under stirring, adjust the pH to 6, and ultrasonicate for 15min to fully disperse it.
[0047] 2) Add 10 mmol of oxidized coenzyme I to the dispersion and mix well.
[0048] 3) Centrifuge, filter, wash or membrane-separate the solution obtained in step 2) to remove uncoupled coenzyme, retain the affinity carrier, take a part for digestion reaction to measure phosphorus content, and transfer the rest to a beaker.
[0049] 4) Dissolve 50 mg of L-glutamate dehydrogenase and 50 mg of lactate dehydrogenase in 1 mL of PBS buffer solution, and slowly add them dropwise into the beaker with stirring.
[0050] 5) After the solution obtained in step (4) was subjected to a coupling reaction at 4° C. for 24 hours, it was filtered, washed, and freeze-dried to obtain an immobilized enzyme; at the same time, the filtered filtrate and the ...
Embodiment 3
[0054] 1) Each weighed 1g of chitosan carrier, dissolved in a beaker containing 10mL of 1% (w / w) acetic acid solution, added dropwise 5mL of coupling agent glutaraldehyde under stirring, adjusted the pH to 8, and ultrasonicated for 15min to make fully dispersed.
[0055] 2) Add 100 mmol of reduced coenzyme I to the dispersion and mix well.
[0056] 3) Centrifuge the solution obtained in step 2), filter, wash or membrane separation to remove uncoupled coenzyme, retain the affinity carrier, according to the fact that chitosan does not contain elemental phosphorus but coenzyme contains, take part of the affinity carrier for digestion reaction to measure phosphorus content and calculate the coupling rate, and transfer the rest to a beaker.
[0057] 4) 1 mL of phosphate buffer containing 100 mg lactate dehydrogenase and 0.25 mmol / L pyruvate was slowly added dropwise to the beaker, and the process was accompanied by stirring.
[0058] 5) Investigate the coupling effect of lactate ...
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