Oxidation-resistant amylase mutant and preparation method and application thereof
A technology of oxidation resistance and amylase, applied in biochemical equipment and methods, botanical equipment and methods, applications, etc., can solve the problems of difficulty in obtaining target strains and large blindness, and achieve shortened transformation time and efficient degradation. , the effect of broad application prospects
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Embodiment 1
[0021] Example 1: Analysis and method of site-directed mutation of amylase oxidation resistance
[0022] By analyzing the 3D spatial structure of amylase ( figure 2 ) analysis to identify oxidation-labile Met residues (M135, M204, M219, M237) within the catalytic region. At the same time, the analysis of other Met residues on the surface of the enzyme structure molecule around the active site found that M307 is on the surface of the amylase molecule and is easily oxidized by oxidants, resulting in reduced or inactivated amylase activity.
[0023] According to the amylase sequence of Bacillus alcalophilus, it was fully synthesized by chemical total synthesis and then cloned into the plasmid pET-22b(+) to construct the recombinant plasmid pAAQ( figure 1 ).
[0024] For site-directed mutagenesis of different Met sites, corresponding site-directed mutagenesis primers were designed (Table 1). Using site-directed mutagenesis primers, amylase was used for site-directed mutagenesi...
Embodiment 2
[0027] Embodiment 2: amylase in different concentration H 2 o 2 Determination of residual enzyme activity after treatment under conditions
[0028] Determination of Alkaline Amylase Activity by DNS Method
[0029] 1) Configuration of DNS reagent: Weigh 2.5g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of water, add 0.5g of phenol, then dissolve 0.075g of sodium sulfite, 2.5g of sodium hydroxide, and 50g of sodium potassium tartrate. Transfer to a 500mL volumetric flask, shake well and make up to volume, store in a brown bottle and place it in a refrigerator at 4°C until use.
[0030] 2) Preparation of maltose standard curve: prepare maltose solutions with different concentrations of 0.2g / L-1.0g / L. Take 1mL of different concentrations of maltose and mix it with the same volume of DNS solution, put it in a boiling water bath, and keep the water bath for 10min. Cool with cold water, dilute to 10mL, A 540 Measure the absorbance. Take the concentration of ma...
Embodiment 3
[0035] Embodiment 3: amylase is in 500mM H 2 o 2 Determination of residual enzyme activity under different conditions
[0036] In 500mM H 2 o 2 Under the condition of 40 ℃ for different time. Use catalase (1200U / mL) to degrade the residual H 2 o 2 . Adopt pH 10.0 glycine-sodium hydroxide buffer solution, buffer solution is mixed with soluble starch, adopt 3) method to measure residual enzyme activity after amylase treatment (see Figure 4 ). After M135, M204, M219, M237, M307 were mutated into Ser, the concentration of 500mM H 2 o 2 Treat at 40°C for 30min. After 5 hours of treatment, the remaining enzyme activities were 63%, 70%, 54%, 46%, and 56% of those before treatment. The oxidation resistance of the amylase is greatly improved, and the amylase can efficiently degrade starch in a strong oxidizing environment.
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