Medium-temperature bacterium for producing alkali-resistant metal ion-resistant organic solvent-resistant ester hydrolase, and application thereof
A technology resistant to organic solvents and metal ions, applied in the field of microorganisms, can solve problems such as difficult to meet actual application requirements, limit practical applications, and inhibit enzyme activity, and achieve the effects of increased enzyme activity, high catalytic activity, and high catalytic activity
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Embodiment 1
[0047] Example 1: Activity detection of ester hydrolase Aln10 produced by mesophilic bacteria SIOC 00170
[0048] The activity of purified ester hydrolase Aln10 was determined by p-nitrophenol hexanoate method. Specific operation: 1 ml reaction system including 1 mM p-nitrophenol hexanoate, 100 mM CHES-NaOH buffer (pH 9.5) and 0.36 μg pure enzyme protein, using a UV-visible spectrophotometer (Beckman DU800, USA) The absorbance value A405 was continuously measured for 2 min at 50 °C, and the inactivated enzyme solution was used as a control for zero adjustment. One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 µmol p-nitrophenol from p-nitrophenol esters per minute. The measured esterase activity was 13553.39 U / mg.
Embodiment 2
[0049] Embodiment 2: Ester hydrolase Aln10 substrate specificity analysis
[0050] The substrate specificity analysis of ester hydrolase Aln10 uses the system (1 ml): 100 mM CHES-NaOH buffer (pH 9.5), 1 mM substrate, adding 0.36 μg of pure enzyme protein, and continuously measuring the absorbance at 50°C A405 2 min. The substrates used in the determination are: p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8) , p-nitrophenol caprate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol myristate (C14), p-nitrophenol palmitate (C16). It has been determined that Aln10 has higher catalytic activity for p-nitrophenol esters with shorter acyl carbon chains (C2, C4, C6 and C8), and the highest catalytic activity is when the substrate is p-nitrophenol hexanoate (C6). ( figure 1 ). The results showed that the hydrolase Aln10 had better catalytic activity on lipids with shorter acyl carbon chains, and its hydrolysi...
Embodiment 3
[0051] Embodiment 3: Analysis of optimal reaction conditions of ester hydrolase Aln10
[0052] The optimal reaction pH of ester hydrolase Aln10 was determined in the range of 4.0 to 11.0. The specific operation is: add 1 mM p-nitrophenol hexanoate and 0.36 μg pure enzyme protein to different pH buffers, and continuously measure the absorbance value A348 at 50°C for 2 minutes. The buffer used for the determination is: 100 mM citric acid-sodium citrate buffer (pH 3.0~6.0), 100 mM potassium dihydrogen phosphate-sodium hydroxide buffer (pH 6.0~8.0), 100 mM Tris hydrochloric acid buffer ( pH 7.5~9.0) and 50 mM 2-cyclohexylaminoethanesulfonic acid-sodium hydroxide buffer (pH 9.0~11.0). The results showed that the optimal reaction pH of Aln10 was 9.5, and it was active in the range of pH 6.0-11.0 ( figure 2 ).
[0053] The optimal reaction temperature of ester hydrolase Aln10 was determined in the range of 15-70 degrees Celsius. The specific operation is: in 1 ml reaction system...
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