Neoagarobiose hydrolase with improved thermostability
A new technology of agarobiose and thermal stability, applied in the field of enzyme transformation, can solve the problems of poor thermal stability and achieve the effect of enhanced thermal stability
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Embodiment 1
[0032] Example 1: Screening of new agarobiohydrolases with improved thermostability
[0033] 1. Screening of AgWH117A enzyme protein mutant protein
[0034] 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
[0043] Embodiment 2: Comparison of AgWH117A and K134D enzymatic properties
[0044] On the basis of screening the mutant K134D, the optimum reaction temperature and optimum reaction pH were determined, as shown in Figure 4 and 5 As shown; the optimum reaction temperature of the mutant K134D is about 3°C higher than that of the original enzyme, from the initial 27°C to 30°C. When the temperature is below 35°C, the enzyme activity changes slowly and the curve tends to be gentle. The optimal pH of K134D and the original enzyme is about 6.0, and the pH adaptability range is relatively wide, and the enzyme activity can be maintained above 80% between pH 5.0 and 8.0.
[0045] After determining the optimal reaction conditions of the enzyme, some kinetic parameters of K134D were detected. By fixing the amount of enzyme, the concentration of the substrate new agarobiose was continuously increased until the concentration of the substrate was further increased and the enzyme activi...
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