An immobilized enzyme producing 3,6-endo-l-galactose
An immobilized enzyme, immobilized enzyme carrier technology, applied in the directions of glycosylase, immobilized on or in an inorganic carrier, enzyme, etc., can solve the obstacles of industrialized preparation of agarose oligosaccharide, high cost of agarase, no L-AHG pure product sales and other issues, to achieve the effects of good thermal stability, increased yield, and reduced production costs
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[0049] 1. Preparation of ferroferromagnetic nanoparticles
[0050] (1) 12.5g FeCl 2 4H 2 O+34.0g FeCl 3 ·6H 2 O+1L ultrapure water at 60°C N 2 Under atmospheric conditions, react for 5 minutes.
[0051] (2) Add 60mL 25.0% NH 3 ·H 2 O, vigorously stirred the reaction for 40min.
[0052] (3) After the color turns black in a large amount, it is separated by a magnet.
[0053] (4) Wash with ultrapure water for 3 times, then wash with absolute ethanol for 3 times, and dry under vacuum at room temperature.
[0054] 2. Silica-coated ferroferric oxide magnetic nanoparticles
[0055] (1) 1.45g Fe 3 o 4 +400mL absolute ethanol, constant N 2 flow, ultrasonic for 40min to suspend and disperse.
[0056] (2) Add 60mL ultrapure water, 30mL 25.0% NH 3 ·H 2 O, after stirring for 10min, add 4mL TEOS, stir at room temperature for 5h.
[0057] (3) Wash 3 times with absolute ethanol, 3 times with ultrapure water, and dry under vacuum at room temperature.
[0058] 3. NH 2 and TCT-...
Embodiment 1
[0070] Example 1: Screening of new agarobiohydrolases with improved thermostability
[0071] 1. Screening of AgWH117A enzyme protein mutant protein
[0072] By analyzing the Gibbs free energy change ΔGqq caused by the charge distribution of each amino acid in the AgWH117A enzyme protein, the influence of the charge distribution of amino acids on the enzyme stability was measured. By calculating the ΔGqq of each amino acid in the AgWH117A enzyme protein, the amino acid sites with smaller free energy were excluded. At the same time, considering that the amino acid change of the active site has a great possibility of causing inactivation, the amino acid of the active site is also excluded. Furthermore, the amino acids on the Loop ring play a key role in protein folding, so the amino acids on the Loop ring are avoided. Finally, the remaining amino acid positions were scanned by alanine to determine the amino acid residue positions for site-directed saturation mutation (Table 1)....
Embodiment 2
[0086] Example 2: Co-immobilization of α-new agarobiohydrolase K134D and β-agarase
[0087] Take by weighing 10.0mg magnetic nanoparticles in a 2mL centrifuge tube, and successively express recombinantly expressed, purified β-agarase AgWH50B (the amino acid sequence is SEQ ID NO: 5, and the nucleotide sequence of a coding gene is SEQ ID NO: 6) and α-new agarobiohydrolase K134D were immobilized on the immobilized material to obtain co-immobilized enzyme K134D AgWH50B-CC-Fe 3 o 4 @SiO 2 .
[0088] First, add 800 μL pH 8.0 phosphate buffer solution of AgWH50B (0.05-283.64 μg) into a 2 mL centrifuge tube containing immobilization materials, mix well, place in a constant temperature shaker at 30°C for 30 min at 180 rpm, and take out , magnetic separation, after buffer washing, then add 800 μL pH 8.0 phosphate buffer containing 200.0 μg K134D, mix well, place in a shaker at 10°C at 180 rpm for 60 min, take it out, magnetic separation, after buffer washing, Enzyme activity to be ...
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