A low-temperature β-xylosidase mutant with improved thermostability and specific activity, its coding gene and application
A technology of xylosidase and thermal stability, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low temperature xylosidase thermal stability, low specific activity, unfavorable application of wild-type low temperature β-xylosidase, etc., to overcome Poor thermal stability, overcoming low specific activity, wide application prospects
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Embodiment 1
[0090] Cloning of embodiment 1 mutant enzyme and wild enzyme expression gene
[0091] The present invention uses the low-temperature xylosidase AX543 as the parent, adopts the method of Overlap-PCR, uses the primers in Table 1 to mutate and express the low-temperature xylosidase AX543, and its gene sequence is as shown in SEQ ID NO.4 / SEQ ID NO.5 / SEQ ID NO.6 shown.
[0092] Table 1. PCR-specific primers for β-xylosidase mutants G110S, Q201R and loop2
[0093]
Embodiment 2
[0094] Expression and purification of embodiment 2 mutant enzyme and wild enzyme in Pichia pastoris
[0095] The PCR products of β-xylosidase mutants G110S, Q201R, and loop2 and the expression vector pPIC9 were subjected to double enzyme digestion (EcoRI+XholⅠ), and the cut gene fragments were connected to the vector to obtain a low-temperature protein with high thermal stability and specific activity. The recombinant plasmids of β-xylosidase mutants G110S, Q201R and loop2 were linearized and transformed into Pichia pastoris GS115 competent cells by electric shock to obtain recombinant yeast strains GS115 / G110S, GS115 / Q201R and GS115 / loop2.
[0096] Pick the monoclonal colony on the MD plate, and use a toothpick stained with bacteria to spot it on another numbered MD plate and a 96-well plate containing 1mL of BMGY medium, respectively. While culturing the MD plate at 30°C, Cultivate a 96-well plate containing 1mL of BMGY medium at 30°C and 220r / min for 48h; after 48h of cultu...
Embodiment 3
[0098] Activity Analysis of Embodiment 3 Mutant Enzyme and Wild Enzyme
[0099] One enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate pNPX to produce 1 μmol p-nitrophenol (pNP) per minute under certain reaction conditions.
[0100] Determination of β-xylosidase activity: Add 150 μL citric acid-disodium hydrogen phosphate buffer solution to 250 μL substrate pNPX, add 100 μL enzyme solution (2U / ml) after preheating at 20°C for 2 minutes, react at 20°C for 10 minutes, add 1.5mL, 1mol / L Na 2 CO 3 Solution termination reaction, OD 405 Measure its absorbance.
[0101] Optimum temperature for β-xylosidase: 2mM pNPX as substrate, 150μL 0.2mol / L citric acid-disodium hydrogen phosphate buffer (pH6.0) was added to 250μL substrate, and the substrate and buffer The mixture was preheated in a water bath at 0°C-50°C for 2 minutes, then 100 μL of enzyme solution (2U / ml) was added, reacted at 0°C-50°C for 10 minutes, and 1.5 mL of 1M Na 2 CO ...
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