Preparation method and application of a mutant trehalose synthase
A technology of trehalose synthase and mutants, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low conversion rate of trehalose synthase, etc., and achieve the effects of increased yield, large yield, and simple preparation
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Embodiment 1
[0041]Example 1: Preparation of mutants of trehalose synthase
[0042](1) Single mutation
[0043]Two mutant enzymes V407M and K490L of trehalose synthase derived from Pseudomonas putida P06: According to the gene sequence of trehalose synthase of Pseudomonas putida P06, primers for introducing mutations of V407M and K490L were designed and synthesized. Perform site-directed mutagenesis, determine the DNA coding sequence, and introduce it into E. coli for expression to obtain a single mutant trehalose synthase. Site-directed mutagenesis of single mutation V407M and K490L: Use the Quickchange site-directed mutagenesis kit and use the constructed expression vector pET-15b-TreS plasmid as a template:
[0044]The site-directed mutagenesis primers for introducing the V407M mutation are:
[0045]Forward primer: 5'-aaccatgacgagctgaccatg gagctggtgcacttctgg-3' (mutated bases are underlined)
[0046]Reverse primer: 5'-ccagaagtgcaccagctccat ggtcagctcgtcatggtt-3' (mutated bases are underlined)
[0047]The site-d...
Embodiment 2
[0059]Example 2: Fermentation induction and purification of mutant enzyme
[0060]The mutants were inoculated in 50mL LB liquid medium (containing 100μg / mL ampicillin), cultivated to OD at 37℃, 200r / min600When it reaches 2.5-3.0, add the inducer lactose to a final concentration of 4g / L at 27°C and induce 8h at 200r / min. The fermentation broth was centrifuged at 4°C and 8000r / min for 10min, and the cells were resuspended in 5mL 10mM PBS (pH8.0), and the mutant enzyme was extracted with an ultrasonic disintegrator. The disintegration conditions: 300W, working time 5s, intermittent 5s, and the whole process 15min. The crushed liquid was centrifuged at 8000r / min for 10min at 4℃, the supernatant was collected, and the supernatant was filtered and sterilized with a 0.22μm filter membrane.
[0061]Nickel column affinity chromatography: Pour the collected crude enzyme supernatant into the regenerated nickel column; after the supernatant is drained, rinse with wash buffer (25mM PBS, pH8.0, 100mM N...
Embodiment 3
[0062]Example 3: Determination method of conversion rate of trehalose synthase mutant
[0063]Take 5 mL of the crude enzyme solution to transform the maltose solution (pH 8.0) with a substrate concentration of 300 g / L, react for 8 hours at 25°C, and terminate the reaction by boiling water for 15 minutes. Centrifuge at 13000r / min for 15min at room temperature, take the supernatant and use High Performance Liquid Chromatography (HPLC) to detect the content of trehalose, glucose, and maltose in the conversion system. The same mutant was repeated 3 times, the difference of the measured values was less than 5%, and the average value was taken. The measured results are shown in Table 1.
[0064]Table 1 The content of glucose, maltose and trehalose in the original enzyme and mutant transformation system
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[0066]
[0067]Comparing the mutant enzyme obtained by the expression of the above mutant with the original enzyme, it can be found that the mutant achieves an increase in the conversion rate...
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