Acid protease bs2688 mutant y282l with improved thermal stability and its gene and application
A technology of acid protease and thermal stability, applied in the field of agricultural biology, can solve the problems of low catalytic efficiency and low enzyme activity of acid protease
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Embodiment 1
[0044] Example 1 Preparation of protease Bs2688 mutant
[0045] 1. Cloning of the protease encoding gene Bs2688
[0046] The fungus Bispora sp. MEY-1 cultured in liquid for 3 days was centrifuged at 12,000 rpm for 10 min. The collected mycelia were added to a mortar that had been sterilized at high temperature. They were quickly ground to powder with liquid nitrogen, and then the ground mycelia were transferred. Put it into a new 50mL centrifuge tube containing 15ml of CTAB lysis solution, mix it up and down gently, and place it in a 65°C water bath for 3 hours. Every 20min, invert it up and down and mix it gently to fully lyse the cells. Centrifuge at 4°C and 12,000 rpm for 10 min, suck the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. The supernatant was taken and then an equal volume of phenol / chloroform was added for extraction and left at room...
Embodiment 2
[0066] Example 2. Verifying the enzymatic properties of the recombinant protease
[0067] The activity of the protease of the present invention was analyzed by the Folin phenol reagent color development method. The specific method is as follows: under the conditions of pH 3.0 and 55 ℃, 1 mL of reaction system includes 500 μL of appropriate diluted enzyme solution and 500 μL of substrate, react for 10 min, and add 1 mL of trichloroacetic acid (0.4 mol / L) to terminate the reaction; The system was centrifuged at 12000 rpm for 3 min, 500 μL supernatant was aspirated, 2.5 mL sodium carbonate (0.4 mol / L) was added, 500 μL Folin phenol reagent was added, and the OD value was measured at 680 nm after color development at 40 °C for 20 min and cooling. Definition of protease activity unit: Under certain conditions, the amount of enzyme required to decompose the substrate casein to generate 1 μmol of tyrosine per minute is 1 activity unit (U).
[0068] 1. Optimum pH and pH Stability of ...
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