[0040] Example 3: Determination of the properties of purified recombinant α-galactosidase AgaAHJ8
[0041] 1. Activity analysis of purified recombinant α-galactosidase AgaAHJ8
[0042] The activity determination method of the purified recombinant α-galactosidase AgaAHJ8 adopts the pNPG method: pNPG is dissolved in 0.1M buffer to make the final concentration 2mM; the reaction system contains 50μL appropriate enzyme solution, 450μL of 2mM substrate; After preheating the substance for 5 minutes at the reaction temperature, add enzyme solution and react for 10 minutes, then add 2mL 1M Na 2 CO 3 The reaction was terminated, and the released pNP was measured at a wavelength of 405nm after cooling to room temperature; 1 unit of enzyme activity (U) was defined as the amount of enzyme required to decompose pNPG to produce 1 μmol pNP per minute. The method for determining the activity of the substrate raffinose and guar gum adopts the DNS method: the substrate is dissolved in 0.1M buffer to make the final concentration 0.5%; the reaction system contains 50μL of enzyme solution and 450μL of substrate; After preheating for 5 minutes at the reaction temperature, add enzyme solution and react for an appropriate time, then add 2.0mL DNS to terminate the reaction, boil for 5 minutes, cool to room temperature and measure the OD value at 540nm wavelength; 1 enzyme activity unit (U) It is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol of reducing sugar (calculated as galactose) per minute under the given conditions. The glucose oxidase method is used to determine the activity of the substrate denbiose: the substrate is dissolved in 0.1M buffer to make the final concentration 0.5% (w/v); the reaction system contains 50μL appropriate amount of enzyme solution, 450μL bottom Substance; After the substrate is preheated at the reaction temperature for 5 minutes, the enzyme solution is added and then reacted for 10 minutes, and then according to the principle of glucose oxidase-peroxidase method, the glucose determination kit (Shanghai Rongsheng Biopharmaceutical Co., Ltd., CAT361500 ) The manual measures the enzyme activity; 1 unit of enzyme activity (U) is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol of glucose per minute under the given conditions.
[0043] 2. Determination of pH activity and pH stability of purified recombinant α-galactosidase AgaAHJ8:
[0044] Determination of the optimal pH of the enzyme: the α-galactosidase AgaAHJ8 was subjected to the enzymatic reaction at 37°C and 0.1M pH3.0–10.0 buffer. Determination of the pH stability of the enzyme: Place the enzyme solution in a 0.1M pH3.0–10.0 buffer solution, treat it at 37°C for 1h, and then perform the enzymatic reaction at pH5.5 and 37°C to use the untreated enzyme As a control. The buffer is: 0.1M McIlvaine buffer (pH3.0–8.0) and 0.1M glycine–NaOH (pH9.0–10.0). Using pNPG as a substrate, react for 10 minutes to determine the enzymatic properties of the purified AgaAHJ8. The results show that the optimum pH of AgaAHJ8 is 5.5, and it maintains more than 72% of the enzyme activity in the range of pH 4.0-8.0 ( figure 2 ); After pH4.0-8.0 buffer solution at 37℃ for 1h, the remaining enzyme activity reaches more than 73% ( image 3 ).
[0045] 3. Determination of thermal activity and thermal stability of purified recombinant α-galactosidase AgaAHJ8:
[0046] Enzyme thermal activity determination: Enzymatic reaction is carried out in a buffer of pH 5.5 at 10–70°C. Enzyme thermostability determination: put the enzyme solution with the same amount of enzyme at 37°C and 50°C respectively, and after treatment for 0-60 minutes, perform the enzymatic reaction at pH 5.5 and 37°C, and use the untreated enzyme solution as a control . Using pNPG as a substrate, react for 10 minutes to determine the enzymatic properties of the purified AgaAHJ8. The results show that the optimum temperature of AgaAHJ8 is 50℃( Figure 4 ); The enzyme is stable at 37°C, with a half-life of less than 5min at 50°C ( Figure 5 ).
[0047] 4. Determination of kinetic parameters of purified recombinant α-galactosidase AgaAHJ8:
[0048] Enzyme kinetic parameters first-level reaction time determination: at pH 5.5 and 50 ℃, with 1 mM pNPG or 14 mM denbiose as a substrate, the reaction is terminated within 1-30 min of the enzymatic reaction and the enzyme activity is determined. Calculate the ratio of enzyme activity to reaction time. If the ratio remains stable for a certain period of time, this time is the first-order reaction time. Use 0.05–2.0mM pNPG or 2.9–36.5mM denbiose as the substrate, and determine K according to the Lineweaver–Burk method at pH 5.5, 50°C and first-order reaction time m , V max And k cat. It has been determined that at 50℃ and pH5.5, AgaAHJ8 has an effect on the K of pNPG m , V max And k cat Respectively 2.0mM -1 , 111.1μmol min -1 mg -1 And 83.2s -1 , The K for mibiose m , V max And k cat 13.2mM respectively -1 , 0.9μmol min -1 mg -1 And 0.7s -1.
[0049] 5. The influence of different metal ions and chemical reagents on the activity of purified recombinant AgaAHJ8:
[0050] Add 1.0mM metal ions and chemical reagents to the enzymatic reaction system to study its effect on enzyme activity. At 37°C and pH5.5, pNPG was used as a substrate to measure enzyme activity. The results (Table 1) show that 1.0mM SDS, HgCl 2 And AgNO 3 It can completely or almost completely inhibit AgaAHJ8; other metal ions and chemical reagents have little effect on AgaAHJ8.
[0051] Table 1. The effect of metal ions and chemical reagents on the activity of purified recombinant α-galactosidase AgaAHJ8
[0052]
[0053] 6. Activity of purified recombinant α-galactosidase AgaAHJ8 in NaCl:
[0054] Determination of enzyme activity in NaCl: Add 3.5-30.0% (w/v) NaCl to the enzymatic reaction system, and perform the enzymatic reaction at pH 5.5 and 37°C. Using pNPG as a substrate, react for 10 minutes to determine the enzymatic properties of the purified AgaAHJ8. The results showed that: AgaAHJ8 has good salt tolerance, adding 3.5-30.0% (w/v) NaCl to the reaction system, the enzyme still has more than 70% enzyme activity ( Image 6 ).
[0055] 7. Determination of protease resistance of purified recombinant α-galactosidase AgaAHJ8:
[0056] Protease resistance of the enzyme: Treat the recombinase with trypsin (pH 7.5) and proteinase K (pH 7.5) equivalent to 45 times (w/w) of the recombinase at 37°C for 1 h, and then at pH 5.5 and 37 The enzymatic reaction was carried out at ℃, and the enzyme solution placed in the pH buffer solution corresponding to the protease but without protease was used as a control. The results showed that after trypsin and proteinase K were treated at 37°C for 1 h, AgaAHJ8 could still maintain 101.1% and 108.9% of the enzyme activity, respectively.
[0057] 8. Degradation of substrate by purified recombinant α-galactosidase AgaAHJ8:
[0058] At pH5.5 and 50℃, the specific activity of this enzyme to 2mM pNPG is 43.5±1.0U mg -1 , The specific activities of 0.5% (w/v) melibiose, raffinose and guar gum are 0.47±0.07, 1.93±0.07 and 0.46±0.06 U mg, respectively -1.